SP.

01

What we Have Learned from Structures of the Ribosome

Venki Ramakrishnan

MRC, Cambridge, United Kingdom

Ever since its discovery in the 1950s, the ribosome has been the object of study by labs
world-wide, because of its central role in the translation of genetic information into proteins.
However, its large size meant a long delay in the determination of its structure, despite the
fact that the crystals of the ribosome were first obtained around 1980. I shall discuss our
determination of the structure of the 30S subunit, and more recent work on high-resolution
functional complexes of the entire ribosome, as well as insights into ribosome function from
these structures. In particular, the structural basis of decoding, in which the tRNA
corresponding to a codon on mRNA is accurately selected, will be discussed.
AW.01

Crystallography - is the Gold Standard Getting Tarnished?

David Watkin

University of Oxford, Oxford, United Kingdom

X-rays were discovered by Roentgen in 1895, diffraction of X-rays by von Laue in 1912, and
the crystal structure of sodium chloride was elucidated by Bragg in 1913. Initially structures
were determined by trail and error methods, but in 1936 Patterson published his interpretation
2
of the F synthesis - the Patterson function. After that, there was no looking back, and the
determination of molecular structures using X-ray diffraction developed rapidly. By the end of
the 1960's most of the theoretical background which we now use had been worked out. The
technique had been almost completely explored in just 30 years, so that many of the senior
practitioners had experienced all the stages in its development. Because they had done
much of the work by hand, from estimating intensities through to laborious calculations, they
appreciated the significance of all the different kinds of data they were working with. They
understood the risks in taking short cuts, and were able to make informed decisions about the
advantages and disadvantages in alternative procedures. Because these people really
understood what they were doing, their results (within the limits of the instrumentation
available) were soundly based and secure - they were The Gold Standard of analytical
techniques.

I trained as a student in the 1960's and so had a reasonably formal education in

crystallography. I experienced the transition from cameras to analogue diffractometers, to
serial diffractometers, TV and image plate diffractometers, through to ccd and now pixel array
detectors. Computers have reduced calculation times by perhaps four orders of magnitude
(one could now do in one year what would have taken 10,000 years). Most of the theories
developed by the pioneers are routinely available in brilliant programs. While crystallography
in the broad sense still has many exciting frontiers, much molecular structure analysis has
become routine, and some would say of a deteriorating quality. The IUCr, in trying to
maintain standards, has the unenviable tasks of trying to distinguish good work on
poor samples from poor work on good samples.
02.01.1

How Low Can You Go: Investigation of the Effects of Redundancy on Absorption
Correction

Michael Takase

Massachusetts Institute of Technology, Cambridge, MA, United States

Semi-empirical absorption correction using the multi-scan approach [1] is based on the
comparison of equivalent reflections. It can be expected that this method would work best if
the number of equivalent reflections in a given dataset is high. Similarly, semi-empirical
absorption correction should not work very well for datasets with low or very low
redundancies. In an attempt to determine the critical value of minimum redundancy (if, in fact,
there is such a value) below which effective semi-empirical absorption correction is no longer
possible, we are analyzing a number of datasets from a variety of different samples.
Variables to be examined are crystal size, presence or absence of heavy atoms, Laue
symmetry and wavelength of X-radiation

[1] R.H. Blessing, Acta Crystallogr., Sect A 1995, 51, 33-38.

02.01.2

How are we doing? A Review of Small Molecule Crystallography based upon Data
Mined from the Cambridge Structural Database.

Joseph Reibenspies

Texas A & M University, College Station, Texas, United States

It is human nature for one to stand back from time to time and ask themselves: “how am I
doing?” The question is of course very subjective; however it is fair and reasonable to ask
such a question of a group as a whole. To measure progress and thus provide an answer to
the title question one can examine a representative instrument, such as the Cambridge
1
Structural Database . The database consists of crystallographic information from 1923 to the
present and encompasses organic and inorganic molecular compounds. Information such as
2
structure counts, residual factors, volumes, percent errors, total disorder and bond distance
precision provides statistical data that can be used to measure progress in terms of quantity,
quality and complexity. With this information in hand one can also be so bold as to predict
the direction the science of crystallography is taking and what the future holds. At the end of
the discussion we may ask ourselves “How are we doing?” and the answer may come as a bit
of a surprise.

1
Allen, F. R. (2002). Acta Cryst. B58, 380–388.
2
Flippen-Anderson, J. L., J. R. Deschamps, Gilarid, R.D., George, C. (2001) Crystal
Engineering 4, 131-139.
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The validation of crystal structures via the checkCIF software began in late 1997. Before then,
it was up to the practitioner to check carefully that all was well with each structure
determination; a manageable task when only a few dozen or less structures were determined
per year and a wise professional crystallographer was at hand for guidance. Nowadays,
diffractometers, in some cases in do-it-yourself labs, have such high throughput that mere
mortals are overwhelmed by the amount of data being accumulated. To cope with the number
of structures being determined, intelligent tools to automate the routine parts of the
experiment are welcome and new automatic structure determination software is appearing.
Can we now sit back, push a button and enjoy seeing a finished (routine) structure appear
before our eyes together with a clean validation report, then assume all is okay? There may
be aspects in any structure determination that the validation tools cannot evaluate or give
appropriate feedback about before it is too late. It is still necessary to keep a watchful eye on
the entire experiment and to think carefully and critically about the results. Every crystal has
its own peculiarities, some of which may need special treatment, so even at the data
collection stage one should be watchful of what is unfolding and be prepared to take
appropriate action.

A recent addition to the checkCIF suite is the ability to validate the structure factor listing
against the corresponding CIF. This enables users to confirm that their archived structure
factor file corresponds with the refinement run used to generate the CIF. The tests may also
give feedback about, among other things, overlooked twinning and other inconsistencies
within the CIF that might arise, for example, from incorrect editing of an existing CIF after a
new refinement. Access to the service is at:

http://journals.iucr.org/services/cif/checking/checkcifhkl.html
02.01.4

High Pressure Cryocooling at MacCHESS

1 1 1 1 1
Chae Un Kim , Irina Kriksunov , William A. Miller , Mike Cook , Doletha M. E. Szebenyi , Sol
2,3
M. Gruner
1 2
MacCHESS (Macromolecular diffraction at CHESS), Ithaca, NY, United States, Cornell High
3
Energy Synchrotron Source, Ithaca, NY, United States, Physics Department, Cornell
University, Ithaca, NY, United States

A novel high-pressure cryocooling technique for macromolecular crystallography has been

developed and explored at the Macromolecular Diffraction Facility at the Cornell High Energy
Synchrotron Source (MacCHESS) [1]. The method involves cooling macromolecular crystals
to cryogenic temperatures (~ 100 K) in high-pressure (up to 200 MPa) helium gas.
Applications include successful cryocooling with little or no penetrating cryoprotectant. The
method has been extended to Kr/Xe single-wavelength anomalous dispersion (SAD) phasing,
native sulfur SAD phasing, and preparation of cryocooled crystal samples in capillaries. A
mechanism involving high-density amorphous (HDA) ice is used to explain why the method
works. Surprising results include visualization of ligands which could not be seen using other
methods [2, 3], and insight into the phases of water in a protein crystal [4]. The high pressure
cryocooling method is available to researchers with suitable crystals:

See
[http://www.macchess.cornell.edu/MacCHESS/about_macchess.html#Pressur
e].

References

[1] C. U. Kim, R. Kapfer, and S. M. Gruner (2005), Acta Cryst. D61, 881-890.

[2] R. A. Albright, J. -L. V. Ibar, C. U. Kim, S. M. Gruner, and J. H. Morais-Cabral (2006), Cell
126, 1147-1159.

[3] J. F. Domsic, B.S. Avvaru, C. U. Kim, S. M. Gruner, M. Agbandje-McKenna, D. N.

Silverman, and R. McKenna (2008), J. Biol. Chem. 283, 30766-30771.

[4] C. U. Kim, B. Barstow, M. W. Tate, and S. M. Gruner (2009), Proc. Natl. Acad. Sci., 106,
4596-4600.
02.01.5

Progress in Using Short Wavelength Radiation for Chemical Crystallography

1 1 2 2 2
Juergen Graf , Bernd Hasse , Francesca Fabbiani , Thomas Schulz , Dietmar Stalke , Holger
3 1
Ott , Carsten Michaelsen
1 2
Incoatec GmbH, Geesthacht, Germany, University of Goettingen, Goettingen, Germany,
3
Bruker AXS GmbH, Karlsruhe, Germany

Combining synthetic multilayer mirrors with microfocus X-ray sources (rotating or stationary
target) has become a standard with in-house X-ray sources for single crystal diffraction as
well as a number of applications in powder diffraction. The maximum angle of incidence at
which a multilayer mirror reflects is significantly smaller for higher energy radiation, such as
Mo-Kα or Ag-Kα radiation than it is for Cu-Kα radiation. This is why synthetic multilayer mirrors
traditionally have been used for Cu-Kα radiation or softer wavelengths. Modern deposition
technology, however, allows for the reproducible production of high quality multilayer mirrors
with smaller d-spacing. In consequence these mirrors reflect higher energy radiation at larger
angles of incidence. Combined with the latest generation of microfocus sealed tubes this
provides new high-performance low-power X-ray sources for shorter wavelengths.

We will present selected results on the use of these low-power consumption, high-
performance sources in small molecule and high-pressure crystallography.
02.01.6

A Hybrid Pixel Detector in the Home Laboratory: Prospects for Better Data

Joseph Ferrara, Colin Acheson, Angela Criswell, Pierre Le Magueres, James Pflugrath,
Katsunari Sasaki

Rigaku Americas Corp, The Woodlands, TX, United States

We have begun using a hybrid pixel detector (HPD), specifically the Dectris Pilatus 100K, in
home lab single crystal X-ray diffraction experiments. In order to assess the utility of such a
device for the home lab, we have studied the performance of this device for both small
molecule and protein data collection experiments with copper radiation. We will present
results comparing HPD data collection to conventional CCD data collection as well as results
comparing conventional data collection to “shutterless” data collection in terms of data quality
and increased throughput.
07.01.1

First Results of Femtosecond Protein Nanocrystallography

1 1 1 1 1 2
Mark Hunter , Petra Fromme , Rick Kirian , Uwe Weierstall , Bruce Doak , Henry Chapman ,
1
John Spence
1 2
Arizona State University, Tempe, AZ, United States, CFEL/University of Hamburg,
Hamburg, Germany

Serial crystallography has been used to show the first proof-of-principle for femtosecond
nanocrystallography at the Linac Coherent Light Source, a 2keV pulsed X-ray laser at SLAC
-15
which provided 3-300 femtosecond (10 s) pulses. The intensity of the X-ray pulses exceeds
rd
3 generation sources by 12-orders of magnitude, yet the pulses are so short that X-ray
diffraction data are collected before the sample is destroyed. In serial crystallography, X-ray
diffraction is collected from a stream of fully hydrated protein nanocrystals in their mother
liquor. The jet introduced nanocrystals of Photosystem I, a complex membrane protein with a
mass of 1056000 Da consisting of 36 protein subunits and 381 cofactors, to the femtosecond
X-ray beam produced at the LCLS. Individual diffraction patterns, read out at 30 Hz, could
then be indexed and assembled into a working data set. Over six million diffraction patterns
from Photosystem I nanocrystals were collected, and diffraction was recorded to the detector-
limited resolution of 9Å. The experiments indicate that in this diffract-and-destroy mode, even
a 70 fs pulse may terminate before detectable radiation damage or spot fading occur, to the
available resolution. The impact and potential of the LCLS for future structural determinations
of membrane proteins will be discussed. This project is a large international collaboration,
involving the CAMP group from three Max Plank Institutes and ASU physics. PIs include H.
Chapman, P. Fromme, I. Schlichting, B. Doak, U. Weierstall, J. Uhlich, A. Barty, L. Struder, D.
Rolles, the LCLS staff and the ASG team.
07.01.2

Crystal structure of the membrane fusion protein CusB from Escherichia coli

Edward Yu

Iowa State Universiry, Ames, United States

Gram-negative bacteria, such as Escherichia coli, frequently utilize tripartite efflux complexes
belonging to the resistance-nodulation-division family to expel diverse toxic compounds from
the cell. These systems contain a periplasmic membrane fusion protein that is critical for
substrate transport. We here present the x-ray structures of the CusB membrane fusion
protein from the copper/silver efflux system of E. coli. This is the first structure of any
membrane fusion proteins associated with heavy-metal efflux transporters. CusB bridges the
inner membrane efflux pump CusA and outer membrane channel CusC to mediate resistance
+ +
to Cu and Ag ions. Two distinct structures of the elongated molecules of CusB were found
in the asymmetric unit of a single crystal, which suggests the flexible nature of this protein.
Each protomer of CusB can be divided into four different domains, whereby the first three
domains are mostly -strands and the last domain adopts an entirely helical architecture.
Unlike other known structures of membrane fusion proteins, the -helical domain of CusB is
folded into a three-helix bundle. This three-helix bundle presumably interacts with the
periplasmic domain of CusC. The N and C-termini of CusB form the first -strand domain,
which is found to interact with the periplasmic domain of the CusA efflux pump. Atomic
+ +
details of how this efflux protein binds Cu and Ag were revealed by the crystals of the CusB-
Cu(I) and CusB-Ag(I) complexes. The structures indicate that CusB consists of multiple
binding sites for these metal ions. These findings reveal novel structural features of a
membrane fusion protein in the resistance-nodulation-division efflux system, and provide
evidence that this protein specifically interacts with transported substrates.
07.01.3

Crystal structure of the ectodomain complex of the CGRP receptor, a Class-B GPCR,
reveals the site of drug antagonism

Ernst ter Haar, Christopher Koth, Norzehan Abdul-Manan, Lora Swenson, Joyce Coll, Judith
Lippke, Christopher Lepre, Miguel Garcia-Guzman, Jonathan Moore

Vertex Pharmaceuticals Incorporated, Cambridge, MA, United States

The calcitonin gene-related peptide (CGRP) is a potent vasodilator directly implicated in the
pathogenesis of migraine. Its receptor (CGRP-R) is a heterodimer containing the calcitonin
receptor-like receptor (CLR), a class B GPCR, and RAMP1, a receptor activity-modifying
protein. We have solved the crystal structure of the CLR/RAMP1 N-terminal ectodomain
heterodimer, revealing how RAMPs bind to and modulate the activities of the CLR GPCR
subfamily. We have also determined the structures of CLR/RAMP1 in complex with
antagonists olcegepant (BIBN4096BS) and telcagepant (MK0974). Both drugs act by blocking
access to the CGRP binding cleft at the interface of CLR and RAMP1. These structures
reveal how small molecules bind to and modulate the activity of a class B GPCR, and
highlight the challenges of designing potent receptor antagonists for the treatment of migraine
and other class B GPCR-related diseases.
07.01.4

Resolving the Structures of Membrane Pores Formed by Antimicrobial Peptides

Huey Huang

Rice University, Houston, Texas, United States

Antimicrobial peptides (AMPs) are ubiquitous components of the innate immune systems
found in all plants and animals. Soon after their discovery in the 80’s, they were found to kill
microbes by forming pores in the microbial membranes. Since the conventional antibiotics
have been facing the serious issue of resistance, this new type of antimicrobials has attracted
a great deal of interest. However their molecular mechanisms as well as the structures of their
pores have been controversial. In this talk I will describe how we used neutron scattering and
X-ray diffraction to resolve the structural and mechanism issues. In particular we have
developed a new MAD procedure to resolve the phase problem of diffraction.
07.01.5

Interaction of Lipid Monolayers and Single Supported Bilayers with Cholera Toxin: X-ray
and Neutron Reflectometry and Grazing Incidence X-Ray Diffraction Studies
1 2 2,1 1,2
Jaroslaw Majewski , Tonya Kuhl , Chad Miller , Erik Watkins
1 2
Los Alamos National Laboratory, Los Alamos, NM, United States, University of California
Davis, Davis, CA, United States

Biological membranes are critical components of functioning cells and many bacterial
toxins bind to and gain entrance to target cells through specific interactions with
membrane components. Using surface sensitive neutron and x-ray reflectometry and
grazing-incidence diffraction we were able to follow the process of cholera toxin attack
on a model lipid mono- and bi-layer. In-plane and out-of-plane changes in 2-D packing
of cholera toxin molecules and the lipid membrane were investigated. We followed the
process of the toxins assault on the monolayer in time. A firm understanding of the
molecular mechanisms by which cholera toxin penetrates and translocates across a
membrane will stimulate the design of possible interventional therapies to prevent
infections that use the same mechanism to enter the cell. Furthermore, a similar
mechanism could be employed to transfect cells with a desired therapy.
07.01.6

Lipid membrane-mediated 2D assembly of proteins and viruses at liquid interfaces

1 1 1 1 2
Masafumi Fukuto , Suntao Wang , Sumit Kewalramani , Matthew Lohr , Zhongwei Niu ,
2 2 1
Giang Nguyen , Qian Wang , Lin Yang
1 2
Brookhaven National Laboratory, Upton, NY, United States, University of South Carolina,
Columbia, SC, United States

Biomolecular nanoparticles (BNPs), such as proteins and viruses, are ideal nanoscale building blocks
because of the intrinsic monodispersity in their size, shape, and surface properties. In particular, BNPs
bound to a lipid monolayer at a solution-vapor or solution-substrate interface are well suited for
investigating ordered 2D assembly of nanoscale objects. Using in situ grazing-incidence x-ray
scattering, we have recently studied density-driven 2D crystallization of BNPs for two types of BNP-
membrane interactions, one based on specific ligand binding and the other based on electrostatic
interactions. For the first system, the 2D assembly of the protein streptavidin (SA) on a biotin-
bearing lipid monolayer was studied as a function of the surface density of biotin, a protein-binding
ligand. The results of detailed x-ray scattering and optical Brewster-angle microscopy measurements
reveal that the 2D crystallization of the lipid-bound SA occurs as a density-driven first-order phase
transition. Significantly, the threshold biotin density for inducing the 2D crystallization is found to be
roughly equal to the density of the ligand-binding sites in the SA crystal. Moreover, the extracted
protein adsorption isotherm indicates that the fully bound state of SA, corresponding to two biotin-
lipids per protein, is achieved already below the threshold biotin density. These results demonstrate that
in addition to a well-defined molecular orientation, high lateral packing density is essential to the 2D
crystallization of proteins. For the second system, the electrostatic 2D assembly of cowpea mosaic
virus (CPMV) on a mixed cationic-zwitterionic (DMTAP/DMPC) lipid monolayer was studied as a
function of the subphase pH and the membrane charge density. GISAXS data show that 2D crystals of
CPMV are formed above a threshold membrane charge density and only in a narrow pH range just
above CPMV's isoelectric point, where the charge on CPMV is expected to be weakly negative. The
particle density for the 2D crystals is similar to that for the densest lattice plane in the 3D crystals of
CPMV. The results demonstrate that the 2D crystallization is achieved in the part of the phase space
where the electrostatic interactions are expected to maximize the adsorption of CPMV onto the lipid
membrane.
01.01.1

LOUIS DELBAERE and the Early Days of Crystallography in Edmonton, Alberta.

Title:

Michael James

University of Alberta, Edmonton, Alberta, Canada

Louis Delbaere made many seminal contributions to the advancement of Canadian

Crystallography during his days in Edmonton. He solved several structures of
monosaccharides while he was a post doctoral fellow in Ray Lemieux’s laboratory. He also
made major contributions to the understanding of the structures of the blood-group
determinants. Louis also made major contributions to protein crystallography and to solving
the first protein structure done in my laboratory in Edmonton. He was one of the main
motivating forces in solving the crystal structures of the A and B peptidases from
Streptomyces griseus. He also collaborated with Gary Brayer on the structure of the alpha-
lytic protease. He worked with I-Nan Hsu and Theo Hofmann on the structure of the first
aspartic peptidase to be solved, that of Penicillopepsin.
01.01.2

Louis Delbaere: Friend and Colleague

J. Wilson Quail

Department of Chemistry, University of Saskatchewan, Saskatoon, Saskatchewan, Canada

For years to come, many Canadians who never knew Louis will benefit from his efforts. He
left a mark on many people and organizations.
01.01.3

Louis Delbaere and the International Union of Crystallography

Sine Larsen
1 2
International Union of Crystallography, Chester, United Kingdom, University of Copenhagen,
Copenhagen, Denmark

When Louis too early passed away in 2009 he had served as a member of the IUCr Executive
Committee (EC) for 14 months. Both his election to the EC and the selection of Montreal as
the venue for the 2014 IUCr congress demonstrated the respect and appreciation that the
international crystallographic community held for Louis. Having known Louis from the time we
both started our crystallographic career it was a personal pleasure to work with him in the EC
the short time we had together. This presentation will focus on the role Louis played in the
international crystallographic society and the impact of his work for the IUCr.
01.01.5

Surface Constrained Protein Nanotubes for Bionanoelectronics

Gerald Audette, Stephanie Lombardo, Agnesa Shala

York University, Toronto, ON, Canada

Powering the next generation of implantable devices will rely systems that are more
biologically accessible. To achieve this, we must harness the power of proteins and interface
them with scaffolds, creating unique bio-nanosystems. Bionanoelectronics are interface a
protein system to a conducting surface, thereby connecting them to micro fuel sources such
as biofuel cells. One system we are currently investigating is the type IV pilus from
Pseudomonas aeruginosa, a nanofibre composed of multiple copies of a single protein
subunit, the type IV pilin. In the presence of a hydrophobic surface or solution, engineered
pilin monomers oligomerize into soluble, high molecular weight structures – protein nanotubes
(PNTs). P. aeruginosa pilins, pili and pilin-derived PNTs have been shown to bind both biotic
surfaces (cells) as well as abiotic surfaces such as stainless steel. We have recently
examined the oligomerization of PNTs onto alkylthiol functionalized gold surfaces. PNTs
oligomerized from surface constrained alkylthiols have been observed to be several
micrometers in length with an average diameter of 36 ± 3 nm. In comparison with reported
values for the diameter of native type IV pili and PNTs in solution (~6 nm), the average
observed diameter of surface oligomerized PNTs suggests a multiple PNT clustered filament
on the gold surface. Current research targets the directed engineering of the pilin monomer
to facilitate metal ion binding for bionanowire development and patterned PNT oligomerization
on gold surfaces via differential functionalization of the gold surface with a variety of
alkylthiols.
01.01.6

AlgK and AlgE form the outer membrane secretin of a novel bacterial expolysaccharide
secretion system
1,2 1,2 1,2 1 1
Lynne Howell , John Whitney , Carrie Keiski , Maria Amaya , Mirela Neculai , Patrick
1 1 1 1 1 4
Yip , Laura Riley , Joel Weadge , Francis Wolfram , Yura Lobsanov , Howard Robinson , Lori
3 5
Burrows , Dennis Ohman
1 2
The Hospital for Sick Children, Toronto, ON, Canada, University of Toronto, Toronto, ON,
3 4
Canada, McMaster University, Hamilton, ON, Canada, Brookhaven National Laboratory,
5
Upton, NY, United States, Virginia Commonwealth University Medical Center, Richmond, VA,
United States

Pseudomonas aeruginosa is the predominant pathogen associated with chronic lung

infections in Cystic Fibrosis patients. During colonization of the lung, P. aeruginosa converts
to a mucoid phenotype characterized by the overproduction of the exo-polysaccharide
alginate. Ten proteins, encoded by the algD operon, have been implicated in alginate
biosynthesis. Since polymerization requires protein components in both the inner and outer
membrane, these proteins are believed to form a large multi-protein complex that spans the
cell envelope and facilitates export of the polymer. To understand how alginate is exported we
have undertaken structure-function studies on two outer membrane components of this
system, the lipoprotein, AlgK, and the -barrel porin, AlgE, both of which are essential for the
production of high molecular weight alginate. Complementation and subcellular fractionation
studies have shown that AlgE is required for alginate secretion, and that AlgK plays an
important role in localizing AlgE to the outer membrane; a finding that suggests that the two
proteins may interact directly. Structures of AlgK and AlgE have been determined at 2.5 and
2.3Å resolution, respectively, and reveal that AlgK is an all -helical solenoid protein with at
least 9.5 tetratricopeptide-like (TPR) protein-protein interaction motifs. This topology suggests
that AlgK may play a role in assembly of the alginate biosynthetic complex. AlgE is a
monomeric 18-stranded -barrel that shows remarkable structural similarity to members of the
substrate-specific OprD-family of outer membrane channels. The occlusion of AlgE’ s
electropositive conduit by extracellular loop L2 and periplasmic loop PL8, suggests that,
unlike in other channels, conformational changes may be required to facilitate alginate export.
Bioinformatic analyses have also revealed that the proteins BcsA, PgaA and PelB, involved in
the production and export of cellulose, poly- -1,6-N-Acetyl-D-glucosamine and Pel
exopolysaccharide, respectively, share the same topology as AlgK/E. Together our data
provide a model for alginate export and suggest that AlgK/E represent the periplasmic and
outer membrane components of a new type of outer membrane secretin that differs from
canonical bacterial capsular polysaccharide secretion systems.
01.01.7

Dynamics of Siderophore Reception at the Staphylococcus aureus Cell Surface

1 2 2 2 1
Jason Grigg , John Cooper , Johnson Cheung , David Heinrichs , Michael Murphy
1 2
University of British Columbia, Vancouver, BC, Canada, University of Western Ontario,
London, ON, Canada

Staphylococcus aureus is a devastating bacterial pathogen. It thrives in the human body by

scavenging iron with secreted small molecule chelators called siderophores. S. aureus
synthesizes two polycarboxylate-type siderophores, staphyloferrin A (SA) and staphyloferrin B
(SB). Siderophore-iron complexes are bound at the cell surface by lipoprotein receptors (HtsA
and SirA) and shuttled to membrane permease for import. We have determined the crystal
structures of both receptors in their apo and siderophore-bound complexes. The SA receptor,
HtsA, binds a single SA-Fe complex with nM affinity. Structures have been determined of
apo-HtsA (1.35 Å resolution), HtsA-SA-Fe in an open conformation (1.30 Å resolution) and
closed conformation (2.2 Å resolution). The closed ternary complex structure was obtained
from a twinned crystal (P21 with operator l,-k,h). Together these structures reveal that instead
of more typical domain movement, significant local conformational changes isolated to three
loops in the C-terminal domain coincide with SA-Fe binding. A ~12 Å C shift of Tyr239
brings it into H-bonding distance of SA, enclosing the SA-Fe complex in the pocket. The
conformational change also results in a ~ 2.8 Å shift of Glu250, located at the apex of the
domain where it likely forms a salt-bridge with the permease, allowing discrimination between
apo and holo receptors without large scale domain movement. Importantly, the structure of
SA-Fe is also well defined in the binding pocket allowing assignment of chiral centers, one of
which differs from previous predictions based on model compound properties. The apo-SirA
(2.2 Å resolution) and SirA-SB-Fe ternary complex (1.7 Å resolution) structures have been
determined, again allowing definition of the SB-Fe structure. SirA belongs to the same
structural family as HtsA and also undergoes localized conformational changes following
ligand binding. However, the large structural change occurs on the opposite side of the
binding pocket to HtsA, highlighted by a ~13 Å shift of Asp263 C to enclose SB-Fe.
Localized conformational changes bring two arginine residues in H-bonding distance of SB as
well as a reorientation of Glu245 by ~1.6 Å, which analogous HtsA is predicted to allow
discrimination of ligand bound and apo-SirA.
07.02.1

Ultrafast synchrotron and laboratory in situ powder diffraction studies on the

synthesis of functional materials

John Evans

Durham University, Durham, United Kingdom

Traditionally the vast majority of functional inorganic materials have been synthesised under
conditions of thermodynamic control - the "heat and beat" method of solid state synthesis.
Such methods are, of course, unsuitable for the preparation of metastable materials. In this
presentation I'll discuss how in-situ powder diffraction methods have been crucial in allowing
the preparation of several metastable inorganic framework materials, and in suggesting the
topotactic mechanisms that allow their isolation. Examples will include the preparation of new
molybdate and phosphate materials followed using laboratory based techniques.[1]

I'll also discuss new ultrafast powder diffraction experiments performed at beamline ID11 at
the ESRF in which full quantitative Rietveld refinement is possible on reacting ceramic
systems with a time resolution down to 0.1 seconds. These experiments have allowed us to
identify conditions under which we can prepare the negative thermal expansion material
ZrMo2O8 directly from its constituent oxides. The ability to rapidly scan temperature and
composition space on a reaction that is complete in time periods of ~10 seconds has allowed
us to discover a small temperature window at around 1500 K in which this material is
thermodynamically stable, and to optimise conditions for its synthesis.

I'll also discuss data analysis methodologies which allow one to extract the maximum
information from large bodies of diffraction data collected during in-situ experiments.

[1] Lister, S.E., Evans, J.S.O., unpublished results.

[2] Readman, J.E., S.E. Lister, L. Peters, J. Wright, and J.S.O. Evans, Direct Synthesis of
Cubic ZrMo2O8 Followed by Ultrafast In Situ Powder Diffraction. Journal of the American
Chemical Society, 2009. 131(48): p. 17560-17562.
07.02.2
3+
Structural behaviour of (Mg,Fe )(AlSi)O3 perovskite at pressures of the Earth’ s lower
mantle
1 1 1 2
Tiziana Boffa Ballaran , Alexander Kurnosov , Konstantin Glazyrin , Marco Merlini , Daniel J.
1
Frost
1 2
Bayerisches Geoinstitut, Bayreuth, Germany, Dept. Earth Science, University of Milano,
Milano, Italy

Throughout the bulk of the Earth’s lower mantle, MgSiO3-perovskite is expected to contain
significant proportions of both Al and Fe, with Fe potentially in both 2 and 3+ oxidation states.
Several studies have reported that ferrous iron exists in an intermediate spin state throughout
the Earth’s lower mantle whereas ferric Fe may be involved in a high-spin low-spin transition.
Changes in spin states in silicate perovskite can be expected to affect the coordination
polyhedra around the iron atoms and influence therefore the elastic properties of this mineral.
In order to quantify the effect of such spin transitions on the structural behaviour of perovskite,
we have synthesised at the Bayerisches Geoinstitut single-crystals of a very Fe,Al-rich
perovskite using a multi-anvil technique. The crystals are of an excellent quality as indicated
by their sharp diffraction profiles and lack of twinning. High-pressure single-crystal X-ray
diffraction has been performed at the beam line ID09 at the European Synchrotron Radiation
3+
Facility (ESRF). A single crystal of (Mg,Fe )(AlSi)O3 (longest dimension 25 microns) has
been loaded in a diamond anvil cell with ruby as pressure standard and He as pressure
transmitting medium. Intensity data have been collected at different pressures up to 75 GPa
and isotropic structural refinements always converged with discrepancy factors smaller than
4%. Octahedral and dodecahedral sites have similar compressibility and the orthorhombic
distortion only sligthly increases with pressure. However, the octahedral tilting along the c axis
shows a change in behaviour above 50 GPa.
07.02.3

Probing the high-pressure behaviour of H2SO4 and MgSO4 hydrates with neutrons
1,2 1,2 3
A. Dominic Fortes , Ian G. Wood , Matthew G. Tucker
1 2
Department of Earth Sciences, University College London, London, United Kingdom, Centre
3
for Planetary Sciences at UCL/Birkbeck, London, United Kingdom, ISIS Facility, Rutherford
Appleton Laboratory, Chilton, Oxfordshire, United Kingdom

Hydrates of sulfuric acid, and of magnesium sulfate, have been reported on the surfaces of
the Galilean satellites of Jupiter [1], and models suggest that these hydrates will be abundant
in their deep interiors [2], consequently experiencing modest hydrostatic pressures.
Investigation of their high-pressure behaviour is important since there are likely to be changes
in both the hydrogen bond network as well as possible changes in ion speciation, and
pressure-induced dehydration. Such phase changes may influence heat transport inside icy
planetary bodies, and hence control their overall structure and evolution. Using neutron
powder diffraction, we have carried out studies upon a range of hydrates relevant to the
internal structure and dynamics of icy satellites, including deuterated isotopologues of sulfuric
acid 8-, 6½-, and 4-hydrate, and magnesium sulfate 11-, and 7-hydrate, at pressures up to 4
GPa in the Paris-Edinburgh press. Much of our earlier work is summarised in reference [3].
In sulfuric acid tetrahydrate, we have identified two monoclinic high-pressure polymorphs,
SAT-II and SAT-III, in addition to the low-pressure tetragonal phase. SAT-II is formed by
warming SAT-I above 235 K at 550 MPa, and this has been successfully recovered to
atmospheric pressure at 50 K. SAT-III has been observed over the range 1.6—3.9 GPa,
melting at 380 K at 3.9 GPa. However, sulfuric acid tetrahydrate is extremely difficult to
crystallise at high-pressure, requiring deep undercooling.
In the MgSO4-hydrates we have now observed a reproducible sequence of phase transitions,
in agreement with ultrasonic wave-velocity observations reported elsewhere [4], in which
pressure-induced dehydration occurs. At 295 K, MgSO4· 7D2O (synthetic epsomite) undergoes
its first transition at 1.2 GPa to a lower hydrate + aqueous solution, this phase being stable
over only a narrow pressure range (0.2 GPa), before the onset of the next phase transition
and the growth of ice VII. Similarly, MgSO4· 11D2O (synthetic meridianiite), when compressed
at 240 K, breaks down to a lower hydrate + ice VI at ~ 0.9 GPa.
We report the status of our work to interpret the high-pressure behaviour of these materials,
including recent attempts to recover the products of various phase transitions to ambient
pressure for better characterisation.

References

[1] Orlando et al. (2005) Icarus 177, 528-533: McCord et al. (2001) Science 292, 1523-1525.

[2] Kargel (1991) Icarus 94, 369-390.

[3] Fortes & Choukroun (2010) Space Sci. Rev. 10.1007/s11214-010-9633-3.

[4] Gromnitskaya et al. (2010) High Press. Res. 30, 51-54.

07.02.4

Crystallographic studies of medical gases adsorbed in metal-organic frameworks

Russell Morris

University of St Andrews, St Andrews, United Kingdom

Metal-organic frameworks comprise one of the most exciting classes of solids in current
science. These highly porous solids have been of particular interest as gas sorbent materials.
Many of these studies have concentrated on adsorbing hydrogen, carbon dioxide, methane
and other gases of interest for energy and environmental applications. However, metal-
organic frameworks are also exceptional materials for the adsorption, storage and delivery of
medically important gases, such as nitric oxide (NO). In this presentation I will describe our
crystallographic studies of NO adsorbed into several different framework solids, and how this
information helps us to understand the particular properties of the materials in question.

To complete these studies we primarily use single crystal X-ray diffraction studies at
synchrotron sources (Daresbury, UK and the ALS, USA). The experiments are completed
using a specially designed environmental gas cell that allows the single crystals to be
thermally activated and then loaded with gas while it is on the diffractometer.

References.

1. Xiao B et al. Chemically blockable transformation and ultraselective low-pressure gas

adsorption in a non-porous metal organic framework Nature Chemistry 289-294 (2009).

2. McKinlay AC et al. Exceptional behavior over the whole adsorption-storage-delivery

cycle for NO in porous metal organic frameworks J. Am. Chem. Soc. 130, 10440-10444
(2008).

3. Warren JE et al A prototype environmental gas cell for in situ small-molecule X-ray

diffraction J. Appl. Crystallogr. 42, 457-460 (2009).
07.02.6

High pressure behavior of a family of perovskite-related metal formates,

2+ 2+ 2+ 2+
[DMA][M(CHO2)3], M = Mg , Co , Mn and Zn
1 2 3 1,4
Lauren A. Borkowski , Hyunsoo Park , Stephen Moggach , John B. Parise
1
Mineral Physics Institute, Stony Brook University, Stony Brook, NY, United States,
2 3
Department of Chemistry, Indiana University, Bloomington, IN, United States, School of
4
Chemistry, The University of Edinburgh, Edinburgh, Scotland, United Kingdom, Departments
of Chemistry and Geosciences, Stony Brook University, Stony Brook, NY, United States
2+
The M formates crystallize in a perovskite-related structure, ABX3, where the A cation is
dimethylamine (DMA), and some display multi-ferroic behavior at low temperatures. The
inorganic perovskites display remarkable structural flexibility, often dominated by polyhedral
distortion and tilting. We are interested in exploring the high pressure behavior of this new
class of materials and comparing this to known perovskite compression mechanisms.
Previous work at low temperature shows [DMA][Mg(CHO2)3] transforms from R-3c to C2/c.
2+ 2+
Single crystal and powder high pressure experiments on the Mg and Co analogs show
that the high pressure behavior of these materials consists of tilting of the metal octahedra as
well as a partial ordering of the DMA molecules on the A site.
07.02.5

The XIPHOS diffraction facility for extreme sample environments: In-house

experiments at ultra low temperatures
1 1 1 1 2
Craig Robertson , Michael Probert , Jonathan Coome , Andres Goeta , Brian Michell , Judith
1
Howard
1 2
Durham University, Durham, United Kingdom, Bruker AXS Inc., Madison, WI, United States

The XIPHOS (X-ray – Interface for Photo-Induced High pressure lOw temperature Structural
studies) diffraction facility has been developed to collect diffraction data within a range of
1
sample environments. The system couples a Bruker direct drive Mo rotating anode
generator operating at 5.4 kW with the latest Helios focusing optics. This source is mounted
on a four circle Huber goniometer equipped with an APEX II CCD detector. To reach ultra
rd
low temperatures, an APD 202E Displex cryogenic refrigerator with an additional 3 Joule-
Thompson stage has been installed, allowing for temperatures as low as 2 K to be
maintained. This new diffraction facility will be presented in detail; furthermore, low
temperature calibration, crystal centring and the results of recent experiments will be
presented.

References: 1) Probert, M. R.; Robertson, C. M.; Coome, J. A.; Howard, J. A. K.; Michell, B.
C.; Goeta, A. E. Submitted, J. Appl. Cryst., February 2010 (hx5107).
07.02.7

'The Big Squeeze on Porous Materials.'

Stephen Moggach

The University of Edinburgh, Edinburgh, United Kingdom

Recent interest in gas storage materials has led to a plethora of papers on the synthesis of
[1, 2]
metal organic framework materials (MOFs). Structural variation in MOFs can be achieved
through chemical modification, with accompanying changes in pore size and shape (and
therefore internal surface area) giving rise to an increasingly diverse array of sorption
properties. Such sorption measurements are performed at pressures up to 0.01GPa, though
what effect higher pressures have on the framework is relatively unknown. A sub-family of
MOFs are the so called zeolitic imidazolate framework (ZIF) materials. ZIFs, related to
zeolites through the 145˚ angle subtended at the bridging imidazolate ligand, are of increasing
interest. Their tuneable pore size, chemical robustness and thermal stability combine the
most desirable features of conventional MOF and zeolite structures, making them ideal
candidates for gas storage applications. Over the last 10 years, developments in high-
pressure single-crystal diffraction techniques have allowed us to study much larger
[3]
compounds than was previously possible. The scope for pressure to change material
properties has been demonstrated in previous work on amino acids and molecular magnets.
This work focussed on tuning hydrogen bonding interactions or magnetism respectively.
Here, we present the effect of pressure on porous molecular materials, in particular ZIF-8
(Zn(MeIM)2, MeIM = 2-methylimidazolate). On increasing pressure, we were not only able to
[4]
tune the pore size, but also the pore content of this material through ‘pressure’ modification.

[1]R. Banerjee, A. Phan, B. Wang, C. Knobler, H. Furukawa, M. O’Keeffe, O. M. Yaghi,

Science 2008, 319, 939.

[2]R. Kitaura, G. Onoyama, H. Sakamoto, R. Matsuda, S.-i. Noro, S. Kitagawa, Angew.

Chem., Int. Ed. 2004, 43, 2684.

[3]S. A. Moggach, D. R. Allan, S. Parsons, J. E. Warren, J. Appl. Crystallogr. 2008, 41, 249.

[4]S. A. Moggach, T. D. Bennett, A. K. Cheetham, Angew. Chem., Int. Ed. 2009, 48, 7087.
07.03.1

Data Collection, Reduction and Semi-automatic Structure Solution with HKL-3000

1 1 1 2 1
Wladek Minor , M. Cymborowski , M. Chruszcz , Z. Otwinowski , D. Borek
1 2
Univ. of Virginia, Charlottesville, VA, United States, UT Southwestern Medical Center,
Dallas, TX, United States

HKL-3000 integrates data collection, data reduction, phasing, and model building to
significantly accelerate the process of structure determination, and on average, minimize the
number of data sets and crystals required for structure solution. Execution of the package
merges several modules and software applications into the structure determination pipeline.
There are modules for experimental control of some beamlines and home instruments, data
reduction, phasing by SAD/MAD or molecular replacement, fast model building, and initial
refinement. The system is being developed and tested in the high-throughput environment of
the Midwest Center for Structural Genomics (MCSG) and Center for Structural Genomics of
Infectious Diseases (CSGID). The robustness of HKL-3000 has improved considerably over
time and currently over 1000 structures have been determined with it.
The continuous advancement of the decision-making procedures within HKL-3000 have made
it the system of choice for MCSG and CSGID projects. Transforming raw images into a solved
structure (with 70% of the model built) in 10-15 minutes is no longer a surprise, but a routine
operation for crystals that diffract to 2.5 or better. Our experience with the determination of
hundreds of structures by experimental phasing methods helped us to establish rules for the
best approaches when the available data fall into three categories: unsolvable with current
data, borderline and easy. Current work concentrates on improving the approach to borderline
cases of structure determination rather than optimizing intermediate calculations for easy
cases, thus shifting borderline cases into the easy category and unsolvable into borderline.
An important implication is that simple experimental protocols are sufficient in most cases and
may even be optimal for the most challenging ones. Feedback from fast preliminary structure
solution proved to be one of the critical components of success.
References
Minor W, Cymborowski M, Otwinowski Z, Chruszcz M (2006) Acta Crystallographica Section
D: Biological Crystallography62:859-66.
Kirillova O, Chruszcz M, Shumilin IA, Skarina T, Gorodichtchenskaia E, Cymborowski M,
Savchenko A, Edwards A, Minor W (2007) Acta Crystallographica Section D: Biological
Crystallography63:348-54.
Otwinowski Z, Borek D, Majewski W, Minor W (2003) Acta Crystallographica. Section A:
Foundations of Crystallography59:228-34.
Zheng H, Chruszcz M, Lasota P, Lebioda L, Minor W (2008) Journal of Inorganic
Biochemistry102(9):1765-76.
Chruszcz M, Wlodawer A, Minor W (2008) Biophysical Journal95(1):1-9.
Wlodawer A, Minor W, Dauter Z, Jaskolski M (2008) Febs Journal275:1-21.
Otwinowski Z, Minor W (1997) Methods in Enzymology275:307-326.
07.03.2

CMDDENZO and CMDXDS: Single-Line-Command-Driven User Interfaces for

Automated Data Processing at SER-CAT
1,2 1,2 3 1,2 1,2
Zheng-Qing Fu , Zhongmin Jin , Andy Howard , John Chrzas , Jim Fait , Unmesh
1,2 1,2 1,2 1,2 1,2
Chinte , John Gonczy , Rod Salazar , John Rose , Bi-Cheng Wang
1
Department of Biochemistry & Molecular Biology, University of Georgia, Athens, United
2 3
States, SER-CAT, APS, Argonne National Lab, Argonne, United States, BCPS, Illinois
Institute of Technology, Chicago, United States

As part of ongoing SER-CAT efforts to monitor data quality on-the-fly we have developed
command-line-driven user interfaces (UI's) CMDDENZO and CMDXDS, which exploit
functions in some of the widely-used data reduction packages such as
1) 2) 3) 4) 5)
DENZO/SCALEPACK , D*TREK , SPGR4D, 3DSCALE , XDS , X-GEN for X-ray single
crystal diffraction data reduction. These non-graphical UIs are not intended to match the
expert use of a particular program, but to provide a means to automatically process and
characterize a data set, which includes determining Space Group, Resolution Cutoff, Rmerge,
Completeness, Redundancy, I/SigI etc. They also provide a set of handy diagnostic tools to
quickly identify problems, if any, which should be helpful for remote and/or quick data
collection at synchrotron beamlines. Details of the various UI's and their application to real
data will be presented.

Work supported by the SER-CAT Member Institutions.

References:

1). Otwinowski, Z. & Minor, W. (1997), Met. Enz. 276:307-326.

2). Pflugrath, J.W. (1999), Acta Cryst. D55:1718-1725.

3). Fu, Z.Q. (2005), Acta Cryst. D61:1643-1648.

4). Kabsch, W. (1988), J. Appl. Cryst. 21:916-924.

5). Howard, A.J. (1996), Proc.Macromol. Cryst. Comput. Sch., Oxford University
Press,Oxford, UK.
07.03.3

DrugSite: A Web-based Platform for Sharing Overlaid Protein:Ligand Complex

Structures

Barry Finzel, Ramprasad Akavaram, Aravind Ragipindi

University of Minnesota College of Pharmacy, Minneapolis, MN, United States

The popular drug design techniques of scaffold hopping and target hopping rely heavily on
the accurate overlay of experimentally derived protein:ligand complex structures to
emphasize both the differences and similarities in diverse examples of ligand binding. We
have long believed that optimal superposition of such complexes results from an overlay of
select protein substructures surrounding the binding site. The chosen substructure should
include key structural elements contributing to complex stabilization, but not include elements
subject to conformational change when different ligands bind. Suitable substructures are
empirically identified, and the selection can evolve over the course of a project as different
classes of ligands are added into the ensemble.

Modern software has not evolved to simplify this approach. Rather, the emphasis has been
on ready access to algorithms employing sequence and secondary structure matching that
seeks to empower users to overlay progressively more divergent protein family members.
While these algorithms have their uses, they do not result in the most useful alignments of
ligands for drug design applications.

We have been working to develop a database of “overlay methods” that captures optimized
procedures for aligning important targets in drug-design. A web-based interface provides
access to computational tools to apply these methods to either user-contributed complex
structures or structures from the PDB, and allows users to share aligned structures with
collaborators. The resulting platform provides an excellent means for communicating ligand
structural data in a most useable form – already aligned on relevant homolog structures – for
use by chemist and biochemist collaborators who might not otherwise have the expertise to
devise an optimal superposition of the complexes.
07.03.4

Auto-Rickshaw: A tool for online validation of X-ray diffraction experiment and model
completion

Santosh Panjikar, Venkataraman Parthasarathy, Manfred Weiss, Victor Lamzin, Paul Tucker

EMBL-Hamburg Outstation, Hamburg, Germany

The Auto-Rickshaw is an automated structure determination software pipeline and it is based

on several distinct computer coded crystallographic decision-makers, which invoke a variety
of macromolecular crystallographic programmes/ programme packages during the structure
determination process [1]. The primary aim of the pipeline is to validate the crystallographic
experiment at the synchrotron site while the crystal is still at, or near, the beamline. The
system is optimized for speed, so that structure determination can proceed within minutes
after integrated and scaled diffraction data are available. Typically within a few minutes the
answer is provided whether the collected data will be of sufficient quality to allow successful
structure determination. Currently, the platform offers S/MAD, SIRAS, RIP and MR phasing
protocols as well as combination of MR with various experimental phasing methods [2]. The
result is an improvement of poor MR, or poor experimental phases and the determination of a
larger percentage of the model, from X-ray data better than 2.9 Å resolution, in an automated
manner.

The platform has been installed on a Linux cluster at EMBL-Hamburg and is remotely
accessible to the beamline users via a web-server [3]. It is accessible from most Internet
browsers and allows beamline users to validate their X-ray diffraction experiments and model
completion. Since 2008, Auto-Rickshaw web server [3] has been made accessible to the
worldwide scientific community.

References

[1] Panjikar et al., (2005). Acta Cryst. D61, 449-457.

[2] Panjikar et al., (2009). Acta Cryst. D65, 1089-1097

[3] http://www.embl-hamburg.de/Auto-Rickshaw/
07.03.5

The CCP4 Software Suite - Current Status and Future Developments

1 2 1 1 1
Ronan Keegan , Martyn Winn , Eugene Krissinel , Charles Ballard , Natalie Zhao , George
1
Pelios
1 2
STFC Rutherford Appleton Laboratory, Oxfordshire, United Kingdom, STFC Daresbury
Laboratory, Cheshire, United Kingdom

CCP4 exists to produce and support a world-leading, integrated suite of programs that allows
researchers to determine macromolecular structures by X-ray crystallography. CCP4 aims to
develop and support the development of cutting edge approaches to experimental
determination and analysis of protein structure, and integrate these approaches into the suite.
The current CCP4 software suite is on release series 6.1.x. A particular focus of these
releases is the automation of significant parts of the structure solution process, including XIA2
for data processing, Crank for experimental phasing, MrBUMP and Balbes for Molecular
Replacement, and Buccaneer for model building. There are also a number of new programs,
including Pointless for Laue group and spacegroup determination, the new iMosflm interface,
Parrot for density modification, and PISA for identification of protein-protein interfaces. We will
give an overview of the additions to the CCP4 suite, as well as an update on established
programs.
A major overhaul of the CCP4 suite is under development. A new graphical front-end will
provide easier control of the suite, and considerable help with interpreting and evaluating the
results. At the core, there will be in-built support for automation, making straightforward
structures simple to solve, while continuing support for more challenging projects. Finally,
usage of the suite will be underpinned by better data management, with support for database
back-ends.
CCP4 also aims to enhance its functionality related to the maintenance and use of data on
small molecules (ligands). Firstly, a considerably larger library of chemical compounds will be
provided with the Suite. Extended search functions will be provided to allow for efficient
retrieval of known compounds or their close analogs. Secondly, existing functions for
generating restraint data for new ligands will be enhanced by the inclusion of relevant
software, such as ProDRG, into the Suite, as well as by the development of new methods for
structure reconstruction on the basis of partial similarity to structures in the library.
Functionality will be available through a graphical front-end application, jLigand.
07.03.6

How good is “good enough”? Predicting the success or failure of structure solution
from first principles.

James Holton
1 2
University of California, San Francisco, CA, United States, Lawrence Berkeley Laboratory,
Berkeley, CA, United States

How much x-ray exposure is required to solve a structure? Multiplicity is “good” but
how much will add “too much” read-out noise? What about a better detector? What about a
perfect detector? Answering these questions requires that damage, noise and signal be
placed on a common, absolute scale. To this end, a quantitative simulator of the entire
diffraction experiment called "MLFSOM" (MOSFLM in reverse) was created. The input to the
simulator is a protein data bank (PDB) file and parameters such as photon flux, crystal size
and detector performance characteristics entered in conventional units such as photons/s and
millimeters. MLFSOM was used to produce images in SMV format that were subsequently
processed with ELVES. The general result of these trials was that one and only one of the
many sources of noise in the diffraction experiment will dominate a given data set, and the
optimal strategies for MAD/SAD and high-angle data collection are mutually exclusive. Faint,
high-angle spots are best collected with exposures long enough to “bury” the detector read
out noise under the background-photon noise (but no longer), but the optimal strategy for
MAD/SAD was collecting a large number of very brief exposures, or “dose slicing”.
07.04.1

Radiation damage in macromolecular crystallography: current challenges.

1 2
Elspeth Garman , Ian Carmichael
1 2
University of Oxford, Oxford, United Kingdom, Notre Dame Radiation Laboratory, Notre
Dame, United States

For protein crystals at room temperature, radiation damage during the diffraction experiment
is rapid even on a laboratory X-ray source. In the past, the required data had to be collected
from several different crystals and merged together. The intense X-ray beams produced by
third generation synchrotrons can destroy crystalline order in a matter of seconds. Over the
last 20 years, the use of cryo-cooling techniques which allow X-ray data to be collected with
the sample held in a stream of cooled nitrogen gas at 100K, has become the norm [1, 2]; at
100K crystals can withstand many times the dose (J/kg=Gy) [3] compared with room
temperature (depending on the dose rate [4]), and the necessary data can usually be
obtained from a single crystal.

However, observations of degradation of crystal diffraction with increasing radiation dose at

100K have now become commonplace at third generation synchrotrons. Researchers are
trying to understand the physical and chemical processes involved in this damage (reviewed
in [5,6]), which manifests itself in a number of different ways, including: changes in crystal
colour, decreasing diffraction power with dose, a small but measurable linear increase in unit
cell volume, and specific structural damage to covalent bonds in the amino acids of the
protein molecules [7]. Enzyme active sites seem particularly sensitive to damage, so this
phenomenon can lead to incorrect conclusions on biological mechanisms being drawn. Thus
the issue of radiation damage during diffraction experiments has recently come to the fore as
a concern for all structural biologists.

Current issues being addressed and the challenges of research into this area will be
outlined, informed by the material presented at the Sixth International Workshop on Radiation
Damage to Biological Crystalline Samples held at SSRL in March 2010.

References:

[1] Teng, T-Y (1990) J Appl. Cryst. 23, 387-391

[2] Garman, E.F. & Schneider, TR (1997) J Appl. Cryst. (1997) 30, 211-237.

[3] Owen, RL, Rudiño-Piñera, E and Garman, EF (2006) PNAS (2006) 103, 4912-4917.

[4] Southworth-Davies, RJ.,Medina, MA.,Carmichael, I, & Garman, EF. Structure (2007) 15,
1341.

[5] Ravelli, RGB & Garman, EF (2006) Current Opinion of Structural Biology (2006) 16, 624.

[6] Garman, EF (2010) Acta Cryst. D66, 339-351.

[7] Weik, M et al. (2000) PNAS 97, 623-628.

[8] Burmeister, W.P. (2000) Acta Cryst. D56, 328-341.

[9] Ravelli, R.G.B. & McSweeney, S. (2000) Structure 8, 315-328.

07.04.2

Quality Versus Quantity: the Role of Carefully Planned Diffraction Experiments in High-
throughput Crystallography
Tobias Krojer, Frank von Delft

Structural Genomics Consortium, Oxford, United Kingdom

Our experience at SGC Oxford shows that the rate of success in a high-throughput
environment does not only depend on the number of proteins going into crystallization, but on
careful planning of data collection experiments. SGC-Oxford, is part of a world-wide structural
genomics initiative and currently the Oxford site deposits four novel, human structures per
month. Because our target list is fixed and the proteins consistently challenging, the emphasis
in the crystallography group is on ensuring success even for marginal experiments through
best practice data collection, which dramatically lowers the workload on the upstream
pipeline. A vital ingredient in this philosophy is frequent access to high-quality beamlines, in
our case at SLS and DIAMOND. Although these facilities provide equipment of
unprecedented quality, we note that successful data collection still depends equally on
experimenters' experience and skills.
Here we present results from the numerous data sets that we have collected over the last six
years at synchrotron beamlines and come up with suggestions for future software
developments. We conclude that (i) beam sizes smaller than the diffracting volume of a
crystal are of little benefit, and that (ii) exploratory datasets are of great value for predicting
crystal lifetimes but hard to interpret for marginal diffraction. Thus, we still lack tools for
routine experiments, specifically for characterizing the intersection of beam and crystal, as
well as robust, real-time metrics for monitoring crystal decay.
07.04.3

The minimum crystal size needed to solve a structure.

1,2 1
James Holton , Kenneth Frankel
1 2
University of California, San Francisco, CA, United States, Lawrence Berkeley Laboratory,
Berkeley, CA, United States

The total amount of photons scattered into diffraction spots by a cryo-cooled protein
crystal before it is “dead” is fixed because radiation damage and accumulated scattered
intensity (photons/spot) are both proportional to fluence (incident photons/area). This means
that damage-limited data quality is independent of data collection time, and therefore also
independent of flux (photons/s). We calculated the damage-limited spot intensity from a
protein crystal at a desired resolution given the molecular weight, crystal volume, solvent
content, Wilson B factor and X-ray wavelength using classic scattering formulae and a simple
spot-fading model. Theoretically, a perfect lysozyme crystal 1.2 micron in diameter should be
sufficient for a complete data set (4 photons/hkl at 2 Å), but background scattering on
contemporary equipment pushes this “minimum lysozyme” size up to 8 microns. An easy-to-
use calculator for other cases is available at http://bl831.als.lbl.gov/xtalsize.html
07.04.4

Matthew Warkentin, Robert Thorne

Cornell University, Ithaca, NY, United States

We report the temperature dependence of global radiation damage to thaumatin crystals

between T=300 and 100 K. The amount of damage for a given dose decreases sharply as
the temperature decreases from 300 K to 220 K, and then decreases much more gradually on
further cooling below the protein-solvent glass transition.

We observe two regimes of temperature-activated behaviour. At temperatures above ~200 K,

the activation energy of 4.3 kcal/mol indicates that radiation damage is dominated by diffusive
motions: Diffusion of radicals through solvent channels, the diffusive relaxation of protein
hydration water, and diffusive motions of loose side chains all have activation energies in this
range.

At temperatures below ~200 K the activation energy is only 0.24 kcal/mol, on the order of the
thermal energy. Similar activation energies describe the temperature dependence of
radiation damage to a large variety of small-molecule organic crystals over the temperature
range between T=300 K and 80 K. These systems have atomic vibrational spectra and
energies that are similar to those of proteins. This suggests that the temperature dependence
of radiation damage below T=200 K is associated with the thermal occupation of the first few
excited atomic vibrational states, and that diffusive processes do not contribute significantly to
global damage. Below ~80 K, vibrational excitations are frozen out, zero point motions
dominate, and global radiation damage becomes temperature independent.

Using the radiation damage model of Blake and Phillips (1962), we show that radiation
damage proceeds sequentially, with native protein first becoming disordered and then
amorphous at all temperatures. The ratio of the amorphization rate to the disordering rate is
constant below T~200 K but grows above it. Large scale conformational and molecular
motions are frozen out below T=200 K, but become increasingly prevalent and make an
increasing contribution to overall damage at higher temperatures.

Blake, C., and Phillips, D.C. (1962). Effects of X-irradiation on single crystals of myoglobin. In
Proceedings of the Symposium on the Biological Effects of Ionising Radiation at the Molecular
Level (Vienna: International Atomic Energy Agency), pp. 183–191.
07.04.5

Spatial dependence and mitigation of MX radiation damage by focusing

1 1 2 3 3
Edward Stern , Yanhui Zou , Yizhak Yacoby , Andrzej Joachimiak , Randy Alkire , Kenneth
4
Evans-Lutterodt
1 2
Univ. of Washington, Seattle, WA 98195, United States, Racah Institute of Physics, Hebrew
3
Univ., Jerusalem, Israel, Argonne National Laboratory, Argonne, IL 60439, United States,
4
Brookhaven National Lab, Brookhaven, NY, United States

Recently, strategies to reduce primary radiation damage have been proposed which depend
on focusing x-rays to dimension smaller than the penetration depth of excited photoelectrons
(PE’s). For a line focus as used here the penetration depth is the maximum distance from the
irradiated region along the x-ray polarization direction that the PE’s penetrate. Reported here
are measurements to determine the penetration depth and magnitude of PE damage excited
by 18.6keV photons in a lysozyme crystal. It is found that the x-ray dose has a significant
contribution from the crystal’s 9 w% solvent NaCl atoms. The 15.8 keV PE’s of the Cl atoms
and their accompanying 2.8 keV localized dose from the decay of the resulting excited atoms
more than doubles the dose deposited in the focused region because of a much greater cross
section and higher energy of the excited atom, degrading the mitigation of radiation damage.
Eliminating heavier atoms from the solvent will significantly improve the mitigation of damage
by focusing. The experimental results showed the penetration depth of ~17 keV PE’s is
1.36+/- 0.2 µm, well below previous theory estimates. Such a small penetration depth raises
challenging technical issues to mitigate damage by focusing because the optimum
requirements are gaussian line focused beams with sigma of 0.15 µm and distance between
lines of 1.8 µm to reduced damage by a factor of 2.
07.04.6

Reduced Radiation Damage In Protein Crystals With Micron-Sized X-Ray Beams

1 1 1 1 1,2
Robert F. Fischetti , Ruslan Sanishvili , Derek Yoder , Sudhir Pothineni , Janet L. Smith ,
3 1 1 1
Gerold Rosenbaum , Shenglan Xu , Oleg Makarov , Sergey Stepanov , Venugopalan
1 1
Nagarajan , Stefan Vogt
1
gm/Ca-Cat, Biosciences Division, Argonne National Laboratory, Argonne, Il, United States,
2
life Scineces Inst., Dept. Of Biological Chemistry, U. Of Michigan, Ann Arbor, Mi, United
3
States, dept. Of Biochemistry And Molecular Biology, U. Of Georgia, Athens, Ga, United
4
States, experimentl Facilities Division, Aps, Argonne National Laboratory, Argonne, Il, United
States

Cryo-cooling of protein crystals significantly reduces X-ray induced radiation damage, but
does not eliminate it. The predominant mechanism of interaction of an X-ray with an atom in
the crystal is the emission of a photoelectron carrying most of the energy of the incident X-ray
and causing damage as it deposits that energy in the crystal. When a photoelectron interacts
with an atom, it loses energy slowly at first and then more rapidly as its energy decreases.
Thus, if the beam size is small compared to the distance the photoelectron travels from its
point of emission, then deposition of photoelectron energy outside the beam footprint may
reduce radiation damage inside the beam footprint. Monte-Carlo simulations predict that a
photoelectron of typical energy could travel 4 – 5 m from the point of emission before being
absorbed. We studied radiation damage to lysozyme crystals by monitoring the diffracted
intensity of 18.5-keV X-rays as a function of dose and beam size (0.86 – 20 m) at beamline
23-ID-B at the Advanced Photon Source. We observed a 3-fold reduction of damage per
dose absorbed within the footprint of the smallest compared to the largest beam. In addition,
the spatial extent of radiation damage was mapped using both 15.1- and 18.5-keV X-rays and
a ~1- m beam. The damage profiles displayed spatial anisotropy with greater damage
occurring along the direction of the X-ray polarization, as expected. The spatial extent of the
damage was limited to about 4 m.

GM/CA CAT is supported by the NIH National Institute of General Medical Sciences and
National Cancer Institute. The APS is supported by the US Department of Energy.
07.05.1

Probing reactions in real time using pair distribution function analysis

Karena Chapman

X-ray Science Division, Argonne, IL, United States

The pair distribution function (PDF) method provides valuable insights into the local atomic
structure in materials independent of crystallinity, heterogeneity or particle size. Recent
advances in experimental methods and the advent of dedicated X-ray PDF beamlines, such
as 11-ID-B at the Advanced Photon Source, have led to rapid growth in both PDF studies and
the associated user community. This growth has occurred in parallel with the increasing
interest in nanoscale and disordered materials, for which conventional Bragg crystallographic
methods offer limited insight.

Current state-of-the-art PDF set ups (with optimized beam intensity, sample environments
and detectors) now allow total scattering data suitable for PDF analysis to be collected at up
to 30 Hz. This allows for the structural changes during reactions to be probed in-situ to reveal
changes in bonding during catalytic reactions and particle nucleation and growth—from the
earliest X-ray amorphous multi-atom clusters to nanoparticles and beyond. The insights
gained into the reaction kinetics and mechanism can ultimately lead to greater control of
structure and functional behavior.
07.05.2

STRUCTURE OF CRYSTALLOGRAPHICALLY CHALLENGED HYDROGEN

STORAGE MATERIALS

Hyunjeong Kim

Los Alamos National Laboratory, Los Alamos, NM, United States

Hydrogen is considered a promising alternative fuel for transportation, provided we

can find a way to store a large amount of it in a compact way. The realization of such
a storage system can be achieved by developing materials that can easily absorb,
safely store, and rapidly release hydrogen. However, there is currently no material to
meet all the requirements for on board storage. Great efforts have been made to
look for a way to improve properties or to prepare new materials. One of widely
adopted ways to prepare new hydrogen storage materials is mechanical alloying or
ball milling. Materials prepared by this method are often nano- or amorphous-phases
or mixture of both and they exhibit interesting hydrogen storing properties [1].
Alternatively, packing hydrogen storage materials into porous materials [2] leads to
great improvement in their properties; such nano-confinement allows materials to
release high purity hydrogen at lower temperatures without a significantly long
induction period. Despite of favorable changes in properties, little is known about the
structure of both types of systems. This is partly because amorphous or nano-sized
nature limits the use of conventional crystallographic analysis and, therefore,
structural determination becomes very challenging. In this talk, I will present our
local structural studies on such crystallographically challenged hydrogen storage
materials by using the atomic pair distribution function analysis [3] on total scattering
data. The systems of interest are MgxCo100-x alloys prepared by ball milling [4] and
nano-phase ammonia borane (NH3BH3) confined in pores of mesoporous silica
MCM-41 [5].

[1] S. Orimo and H. Fujii, Appl. Phys. A 72, 167-186 (2001).

[2] A. Gutowska et al., Angew. Chem. Int. Ed. 44, 3578-3582 (2005); R. K. Bhakta et
al., J. Am. Chem Soc. 131, 13198-13199 (2009).

[3] T. Egami & S. J. L. Billinge, Underneath the Bragg Peaks: Structural Analysis of
Complex Materials, Pergamon Press Elsevier, Oxford, England, 2003; Th. Proffen &
H. J. Kim, J. Mater. Chem. 19, 5078-5088 (2009).

[4] Y. Zhang et al., J. Alloys Compd. 393, 147-153 (2005); H. Shao et al., Scripta
Materialia 60, 818-821 (2009); J. Matsuda et al., Nanotechnology 20, 204015 (2009).

[5] H. J. Kim et al., J. Am. Chem. Soc. 131, 13749-13755 (2009).

07.05.3

Local Structure Effects in Magnetoresistance Materials

1 2 2 2 1
Efrain Rodriguez , Anna Llobet , Thomas Proffen , Katharine Page , Mark Green
1 2
NIST Center for Neutron Research, Gaithersburg, MD, United States, Lujan Neutron Center,
LANL, Los Alamos, NM, United States

The magnetoresistance (MR) effect is a technologically important property employed in

magnetic hard disks and sensors. In efforts to find new materials with promising MR
properties, researchers have focused on the mixed valence manganites Ln1-xAxMnO3 where
Ln is a trivalent lanthanide and A is a divalent alkaline earth metal. Another set of materials
showing promise in this area is the series of solid solutions with the stoichiometries Zn1-
xCuxCr2Se4. This talk will focus on how we employed neutron and X-ray powder diffraction to
obtain the local and long-range structure of both materials to have a better understanding of
the microscopic interactions leading to the MR effect. In particular, we present the case of
La0.5Ca0.5MnO3 below the charge-ordering temperature. By combining Rietveld and pair
distribution function (PDF) analysis with the total neutron scattering data, we examined two
competing models describing the low temperature, charge-ordered/orbital-ordered (CO-OO)
3+/ 4+
phase: 1) the Mn Mn checkerboard model and 2) the Mn-Mn dimer model or so-called
Zener polaron model. In the case of the selenides, we use PDF analysis of X-ray data to find
how the local environment of the Cu and Cr cations lead to the observed magnetic and
transport properties.
07.05.4

Coupling total scattering and density functional theory computations to solve the
structure of complex disordered aluminosilicates
1 1 2 1 1
Claire White , John Provis , Thomas Proffen , Daniel Riley , Jannie van Deventer
1 2
University of Melbourne, Victoria, Australia, Los Alamos National Laboratory, Los Alamos,
NM, United States

Understanding the atomic structure of complex metastable materials is of great importance in

research and industry, however, such structures resist solution by most standard techniques.
Here, a novel synergy between total scattering and density functional modelling is presented
to solve the structure of the metastable aluminosilicate material metakaolin. Metakaolin is
obtained by calcination of kaolinite, and is used in quantities of millions of tonnes per annum
in blending with Portland cement for concretes, as well as being a useful geopolymer
precursor, and is a key intermediate in processing of many fired ceramics. The structure is
elucidated by two independent methods, both based on the combination of total
scattering/pair distribution function analysis (PDF) and density functional theory (DFT).

In the first method, the structure is obtained by iteration between least-squares real-space
refinement using neutron PDF data, and geometry optimisation using DFT. The resulting
structural representation is both energetically feasible and in excellent agreement with
experimental data. In the second, the process of kaolinite dehydroxylation is modeled using
DFT and a step-wise methodology, where several water molecules at a time are removed
from the original kaolinite structure, geometry optimization is carried out, and the process is
repeated until the dehydroxylated structure is reached. The structures generated during the
dehydroxylation process are then validated by comparison with X-ray and neutron PDF data.

This study provides new insight into the local environment of the aluminum atoms in
metakaolin, including evidence of the existence of tri-coordinated aluminum. By the
availability of this detailed atomic description of its structure, there exists the opportunity to
tailor chemical and mechanical processes involving metakaolin and other complex metastable
materials at the atomic level to obtain optimal performance at the macro-scale.
07.05.5

Phase progression of alumina nanoparticle catalyst supports as a function of synthetic

temperature
1,2 1 2 3 2
Stacey Smith , Branton Campbell , Baiyu Huang , Calvin Bartholomew , Brian Woodfield ,
2 4 4 5
Juliana Boerio-Goates , Katherine Page , Hyunjeong Kim , Karena Chapman
1 2
Brigham Young University, Physics & Astronomy, Provo, UT, United States, Brigham Young
3
University, Chemistry & Biochemistry, Provo, UT, United States, Brigham Young University,
4
Chemical Engineering, Provo, UT, United States, Los Alamos National Laboratory, Los
5
Alamos Neutron Science Center, Los Alamos, NM, United States, Argonne National
Laboratory, Advanced Photon Source, Chicago, IL, United States

We have developed a simple and uniquely cost-effective synthetic method for producing -
Al2O3 nanoparticles of exceptional size (3-5 nm) and purity. The product shows promise as an
improved industrial catalyst support due its enhanced surface area and the mesoporous
character of its agglomerates. To establish the temperature range through which we can
produce the catalytically-active gamma phase, we must determine the phase progression of
our samples as a function of synthetic temperature. This is challenging because the alumina
phase diagram includes many closely-related phases that are not readily distinguished from
powder-diffraction data due to the extremely particle-size broadened Q-space peaks. In
these cases, PDF analysis was able to resolve the distinct local structures of the candidate
phases. We will demonstrate that a combination of PDF and Rietveld refinements best
resolves our alumina nanoparticle phase progression pathway.
07.05.6

Ferroelectric-relaxor crossover in Ba(Ti1 xZrx)O3 studied using

neutron total scattering measurements and reverse Monte Carlo

modeling

Ilkyoung Jeong, C. Y. Park, J. S. Ahn, S. Park, D. J. Kim

Pusan National University, Busan, Korea, Republic of

Comprehensive structural studies on normal ferroelectric to relaxor crossover in Ba(Ti1-xZrx)O3

(BTZ) are performed using neutron total scattering measurements analyzed by reverse Monte
Carlo modeling. In BTZ solid solution, we estimated the degree of the displacement
correlation between Ti ions and found that it is stronger and extends much longer for
ferroelectric state than relaxor state. In addition, we present evidence that the overall o_-
centering behavior of Ti ion changes from directional to random displacements between
ferroelectric and relaxor phases, and thus provide atomistic picture for ferroelectric-relaxor
crossover with increasing Zr concentration.
07.05.7

Applications of Single Crystal Diffuse X-ray Scattering for Studies of Polymorphism in

Pharmaceuticals.

Eric Chan, Darren Goossens, Aidan Heerdegen, Richard Welberry

Australian National University, Canberra, Australia

Due to the recent advancements in modern computing power, the analysis and interpretation
of single crystal X-ray diffuse scattering for molecular crystals now involves the construction
of a computer model of a dynamic crystal. The method allows inclusion of structural features
on a local level that may be tested against their effect on the observed diffuse scattering. This
gives great insight into the dynamic behavior of organic molecules in the solid state and the
models can also be used to explain the structural nature of packing defects or lattice strain. In
this paper we discuss the analysis of three polymorphic systems; namely, benzocaine,
paracetamol and aspirin.

For benzocaine, a low temperature phase transition occurs whereby the orthorhombic phase
(form II) transforms to a twinned monoclinic phase (form III). The low temperature twinned
crystal displays many 'well-defined' Bragg peaks. For the room temperature form II, diffuse
scattering features are observed in the absence of 'low temperature' Bragg peaks which,
when modeled, show that at a local level the form II crystal has a structure which exhibits
precursor effects of the incipient phase transition.

All the simulations use Hooke's law springs associated with intermolecular connections to
approximate the normal modes of vibration in a molecular crystal. For benzocaine a simplified
set of important connections were used and force constants needed to be determined through
trial and error. The work on the monoclinic and orthorhombic forms of paracetamol
demonstrates that much trial and error is no longer necessary and the force constants in a
model can be approximated from knowledge of Van der Waals radii provided that all
intermolecular interactions within a certain distance threshold are taken into consideration.
The approximation works well and its effect on the simulation is shown quantitatively using a
least squares refinement.

Results from modeling diffuse data collected from form II of aspirin suggests that the crystal
has undergone layer dislocations, during or after the crystallization, that resemble the form I
packing. Because these layer dislocations are not perfect within the crystal, a resultant lattice
strain is also observed in the diffuse scattering. This strain should affect the solid-state
physical properties of the form II crystal.
07.06.1

Precise Absolute Structure Refinement Using Quotient Restraints

1 2 3 4 1
Simon Parsons , Howard Flack , Oliver Presley , Trixie Wagner , Paul McGovern
1 2
University of Edinburgh, Edinburgh, Scotland, United Kingdom, University of Geneva,
3 4
Geneva, Switzerland, Oxford Diffraction, Yarnton, Oxfordshire, United Kingdom, Novartis,
Basel, Switzerland

In an absolute structure determination one absolute structure is refined competitively against

the inverted alternative. The result is expressed by the Flack parameter x(u), which for
absolute configuration determination can be interpreted as the mole fraction of the alternative
enantiomer in the crystal. The physical range of x is 0 to 1 and even if the bulk material is
known to be enantiopure the standard uncertainty (u) should be less than 0.1 before any firm
conclusions regarding the absolute structure can be drawn. This criterion has proved to be
extremely demanding for light-atom structures.

We have developed a method for absolute structure determination based on the quantity

I (h) - I (-h) F 2 (h) - F 2 (-h)

D(h) (1- 2 x )
I (h) I (-h) F 2 (h) F 2 (-h)
The term based on I(h) and I(-h) and its standard uncertainty can be calculated from a single
2 2
crystal X-ray diffraction data set. The term based on F (h) and F (-h) can be calculated from
the model. It is therefore possible to write out a set of restraints based on observed and
calculated values of D(h) and apply these in an absolute structure refinement. Systematic
errors in the intensities, such as absorption, tend to cancel out (in an average way) so that
measured values of D(h) should be more accurate that the values of the measured
intensities.

We have found that this method yields significantly more precise values of the Flack
parameter than conventional refinement. For example when a data set was collected for L-
alanine with Cu-K radiation at 100 K, conventional refinement yielded a Flack parameter
equal to 0.12(21), whereas the restrained refinement yielded a value of 0.00(8). The method
also carries the advantage that the Flack parameter is allowed to refine along with all the
other parameters, so that its standard uncertainty reflects correlations present in the
refinement.
07.06.2

Experimental conditions for absolute structure determination using Bayesian statistics

1 2
Martin Lutz , Rob W. W. Hooft
1 2
Utrecht University, Utrecht, Netherlands, Netherlands Bioinformatics Centre, Nijmegen,
Netherlands

The first absolute structure determination of an organic molecule was performed by Bijvoet
and coworkers based on intensity differences of 15 pairs of reflections. This approach was
followed by many similar studies, often with a slightly different way to select the reflection
pairs or with different weighting of the selected pairs. In the 1980’s the original procedure of
examining a subset of Bijvoet pairs was superseded by the inclusion of an absolute structure
parameter in the least-squares refinement. A renaissance of the Bijvoet method appeared by
a contribution of Hooft, Straver and Spek (2008), where likelihood calculations in combination
with Bayesian statistics are applied. In contrast to the original Bijvoet method, where only a
subset of reflection pairs is considered, the Hooft method takes all Bijvoet pairs into account.
The Hooft method appears to be very successful, even if only weak anomalous scatterers
(e.g. oxygen) are present.

This paper will deal with the experimental conditions, which are necessary for a reliable
absolute structure determination. Special emphasis will be on the standard uncertainties of
the experimental intensities. Outlier handling will be discussed on the assumption of a normal
error distribution. In case the error distribution is non-Gaussian, use is made of the Student t-
distribution to increase the robustness of the method. Example data are taken from our own
laboratory as well as from Acta Crystallographica, where reflection data are deposited as
supplementary material.
07.06.3

Improved Light-Atom Absolute Configuration Determination Using Microfocus

Sources.
1 2 1 1 1
Michael Ruf , Holger Ott , Matthew M. Benning , Bruce C. Noll , Charles F. Campana
1 2
Bruker AXS Inc., Madison, Wisconsin, United States, Bruker AXS GmbH, Karlsuhe,
Germany

Determination of absolute configuration for light-atom structures has become central to

research in pharmaceuticals and natural products synthesis. In the absence of elements
heavier than silicon, it is often problematic to make a significant assignment of absolute
configuration. Traditionally, a heavy-atom derivative has been prepared, but this is not always
feasible. Making these assignments has become somewhat easier with the advent of high-
intensity microfocus sources, as the increased flux density can improve the anomalous signal
from these samples through improvements in counting statistics. The improvement in data
quality from a high-intensity microfocus source will be demonstrated in comparison to data
from a conventional sealed-tube source.
07.06.4

Absolute Structure Determination - Interpreting the Flack Parameter

Amber L. Thompson, David J. Watkin

Chemical Crystallography, Oxford, United Kingdom

It is now well established that the different enantiomers of a chiral material can have
significantly different physiological properties - for example d and l-limonene. As a
consequence of this, drug manufacturers and drug authorisation authorities are increasingly
concerned about the absolute configuration of active pharmaceutical ingredients. In
appropriate cases, X-ray structure analysis can give very reliable results.

The first absolute structure determination, of sodium rubidium tartrate, was carried out in 1951
by Bijvoet, Peerdeman and van Bommel. Until Rogers introduced his eta parameter into the
least-squares refinement (1981), direct comparisons of Bijvoet pairs, or the application of the
Hamilton R-factor ratio test were the principal crystallographic techniques used to assign
absolute structures. Rogers eta parameter was quickly superseded by the Flack "x"
parameter, a least-squares parameter which treated the crystal as a mixture of the original
enantiomer and its twin by inversion. Flack pointed out that where as the Rogers parameter
(which varied between +1 and -1) had no physical meaning as it approached the mid point,
zero, the Flack parameter had a physical meaning over its entire range (from 0 to 1). A Flack
parameter somewhere near the middle of the range, and with a suitable small e.s.d., indicated
that the sample was twinned by inversion.

The incorporation of this parameter into most refinement programs, its ease of use, and its
apparent robustness to less-than-ideal data collection strategies contributed to its rapid
acceptance, and to misunderstandings about its interpretation. In 2000, almost 20 years after
the Flack parameter was first described, Flack and Bernardinelli described the statistical
interpretation of the parameter. In spite of this, there continued to be a hunch amongst
practical crystallographers that Flack's own interpretation of his parameter was unduly
pessimistic. The publication of the derivation of the Hooft, Straver and Spek parameter in
2008 encouraged us to carry out a critical analysis of 150 samples of known absolute
configuration, only light atoms and measured with molybdenum radiation.
07.06.5

Absolute Configuration of CHON Organics from Mo Radiation: Can You Believe It?

Frank Fronczek

Dept. of Chemistry, Louisiana State University, Baton Rouge, LA, United States

Analysis of Bijvoet pairs using the method of Hooft et al. (J. Appl. Cryst. (2008). 41, 96-103)
has greatly enhanced the sensitivity of determining absolute structure from compounds with
only light atoms. For oxygen-containing crystals of good quality with Cu radiation, it is quite
reliable and relatively easy. For excellent crystals of oxygen-rich compounds, absolute
configuration determination appears to be possible using the Hooft method even with Mo
radiation. This author (ACA Toronto, P-T076) reported about a dozen such absolute
configuration determinations with MoKð, all of which agreed with the known configurations.
That poster contained the statement “With data from modern instrumentation, I have never
seen a Flack parameter near zero with standard uncertainty 0.4 or less fail to yield the correct
(known) absolute configuration.” Experiences in the quest to find a counterexample will be
described, including several interesting case histories. Oxygen-rich chiral crystals of high
quality frequently yield Flack x near zero with standard uncertainties ~ 0.3 - 0.5 and Hooft
P2(true) ~ 1.0, if care is taken in data acquisition. Datasets which maximize the probability of
success are collected at low temperature to high resolution, with high redundancy and a high
completeness of Bijvoet pairs. It also appears that larger molecules (within reason) are easier
than smaller ones, presumably because more Bijvoet pairs are available.
07.06.6

Absolute Structure without Heavy Atoms: Experimental Tests

Raymond P. Scaringe, John D. DiMarco, Mary F. Malley, Michael A. Galella, Marta Dabros

. Bristol-Myers Squibb, Princeton, NJ, United States

The determination of absolute structure by Flack parameter analysis, although requiring some
1 nd
care , is well established for structures containing 2 row or heavier atoms. However, even
2,3
for structures that lack atoms heavier than oxygen, recent work suggests that a Bayesian
analysis of the Bijvoet differences can provide a reliable route to the determination of absolute
structure. The practical implication of this finding is that a “light atom” material, already
suitable for single crystal analysis, may yield an absolute structure without the need of
preparing heavy atom salts or solvates. Since salt screening, solvent screening, and crystal
growth can be labor and material intensive activities, the method is of considerable practical
interest. In comparison to heavy atom materials, the number of examples of light atom
absolute structure determinations based on resonance scattering methods is much smaller.
In this work we present the results of attempted absolute structure determinations for a
number of compounds containing no atoms heavier than oxygen. Results based on Flack
2
parameter refinement will be compared to those based on Bayesian indicators.
1
Flack, H.D. & Bernardinelli, G. (2008) Chirality 20, 681-690.
2
Hooft, R.W.W., Straver, L.H., Spek, A.L. (2008) J. Appl. Cryst. 41, 96-103.
3
Hooft, R.W.W., Straver, L.H., Spek, A.L. (2009) Acta Cryst. A65, 319–321
07.06.7

Absolute Configuration of Epoxyresibufogenin

1 2 2 2 2
Jeffrey Deschamps , Terrence Boos , Kejun Cheng , Arthur Jacobson , Kenner Rice
1 2
Naval Research Laboratory, Washington, DC, United States, Chemical Biology Research
Branch, National Institute on Drug Abuse and the National, Bethesda, MD, United States

Epoxyresibufogenin is a C-20,21-epoxy relative of the well-known resibufogenin.

Resibufogenin is a cytotoxic steroid first isolated in 1952 from the Chinese drug Chan su.
Chan su (also refered to as "senso" in Japanese) is a traditional Chinese medicine derived
from the venom of various toad species, especially Duttaphrynus melanostictus and Bufo
gargarizans. Chan su acts as a stimulant to the central nervous system and exerts
cardiopulmonary effects, thus components of Chan su have been lead compounds in
numerous drug studies. In this study we report on the absolute configuration of a derivative of
resibufogenin, the C20,20 epoxy-resibufogenin, as determined using the method of Hooft et
al. (2008). This configuration was verified by formation of a p-bromobenzoate of the hydroxyl
on C3.

HO
(S)

(S) O
(R) O
(R)
O
(R)
H (S)
(S)
(Z)

O
(R)
S-001

Structure function studies of vaccinia virus host-range protein K1 reveal a novel

functional surface for ankyrin-repeat proteins
1 2 2 1
Junpeng Deng , Xiangzhi Meng , Yan Xiang , Yongchao Li
1 2
Oklahoma State University, stillwater, United States, University of Texas Health Science
Center at San Antonio, San Antonio, United States

Poxvirus host tropism at the cellular level is regulated by virus-encoded host-range proteins
acting downstream of virus entry. The functioning mechanisms of most host-range proteins
are unclear, but many contain multiple ankyrin (ANK) repeats, a motif that is known for ligand
interaction through a concave surface. Here, we report the crystal structure of one of the
ANK-repeat-containing host-range proteins, the vaccinia virus K1 protein. The structure, at a
resolution of 2.3Å, showed that K1 consists entirely of ANK-repeats, including 7 complete
ones and two incomplete ones; one each at the N and C-terminus. Interestingly, Phe82 and
Ser83, which were previously shown to be critical for K1’ s function, are solvent exposed and
locate on a convex surface, opposite to the consensus ANK interaction surface. The
importance of this convex surface was further supported by our additional mutagenesis
studies. We found that K1’ s host-range function was negatively affected by substitution of
either Asn51 or Cys47 and completely abolished by substitution of both residues. Cys47 and
Asn51 are also exposed on the convex surface, spatially adjacent to Phe82 and Ser83.
Altogether, our data showed that K1 residues on a continuous convex ANK-repeat surface
are critical for the host-range function, suggesting that K1 functions through ligand interaction
and does so with a novel ANK interaction surface.
S-004

Crystal Structures of the Catalytic and Carbohydrate Binding Domains of

Endoglucanase D in Complex with Cellotriose from Clostridium cellulovorans
1,2 1 1 1
Christopher Bianchetti , Robert Smith , Thomas Rutkoski1 , George Phillips, Jr
1 2
Department of Biochemistry, University of Wisconsin, Madison, WI, United States, Graduate
Program in Biophysics, University of Wisconsin, Madison, WI, United States

The enzymatic degradation of cellulose by cellulases is a critical step in the conversion of

plant biomass into an abundant renewable energy source. An understanding of the structural
features that cellulases utilize to bind crystalline cellulose, extract a single cellulose strand,
and hydrolyze the -1,4-glycosidic bonds of cellulose to produce fermentable sugars would
greatly facilitate the engineering of improved cellulases for the large-scale conversion of plant
biomass. Endoglucanase D (EngD) from Clostridium cellulovorans is a modular enzyme
composed of a N-terminal catalytic domain and a C-terminal carbohydrate-binding module
(CBM) that are attached via a flexible proline/threonine-rich linker. Here, we present the 2.1 Å
resolution crystal structures of full-length EngD with and without bound cellotriose. The EngD
CBM, a member of the Carbohydrate-Binding Module Family 2, adopts a β -sandwich fold that
contains a planer strip of aromatic residues that are involved in the binding and localization of
the catalytic domain to the surface of crystalline cellulose. The catalytic domain belongs to the
Glycoside Hydrolase family 5 (GH5) and adopts a (β /α )8 fold. An extended active site cleft
runs along one face of the catalytic domain and is partially enclosed by Trp162 and Tyr232
near the +1 and +2 glucose binding subsites. Two molecules of cellotriose are observed in
the EngD cellotriose-bound structure. One molecule is bound in each monomer at the -3, -2,
and -1 subsites while another cellotriose molecule is bound between two non-crystallographic
symmetry related monomers at the +3, and +4 subsites of the catalytic domain. The residues
located on the active site cleft, in particular the surface-exposed Trp162 and Tyr232, which
interact act with the cellotriose molecules, hints at the possible role these residues play in the
initial attachment and strand extraction of cellulose. The cellotriose-bound EngD structure
presented here describes the residues that form the glucose binding subsites and provides a
structurally based mechanism for the binding of the catalytic domain to crystalline cellulose.
S-007

Crystal structure of the gH/gL complex – a conserved herpesvirus fusion regulator

1 2 2 2
Tirumala Kumar Chowdary , Tina M Cairns , Doina Atanasiu , Gary H Cohen , Roselyn J
2 1
Eisenberg , Ekaterina E Heldwein
1 2
Tufts University School of Medicine, Boston, MA, United States, University of Pennsylvania,
Philadelphia, PA, United States

Herpesviruses enter cells by fusing their viral envelope with the cellular membrane. Unlike
most other enveloped viruses, herpesviruses require not one but three surface glycoproteins
for fusion. These are the conserved glycoproteins B (gB) and the heterodimeric gH/gL, plus
other, non-conserved glycoproteins. gB is a class III viral fusogen that normally functions only
in the presence of gH/gL, the role of which is poorly understood. To gain insight into its role in
herpesvirus-mediated membrane fusion, we determined the crystal structure of the gH/gL
complex from herpes simplex virus 2, at 3-Å resolution. The structure was solved using a
single-wavelength anomalous dispersion method and a selenomethionine derivative. The
structure revealed an unusually tight complex of a novel architecture that, contrary to previous
ideas, does not resemble any known viral fusogen. Instead, we hypothesize that gH/gL
activates gB for fusion by binding it directly. A neutralizing monoclonal antibody inhibited
interaction of gH/gL with gB and thus membrane fusion. Thus, we propose that a putative gB-
binding site on the gH/gL surface overlaps the epitope of the neutralizing monoclonal
antibody.
S-010

Fast and automated functional classification with MED-SuMo: An application on

purine-binding proteins
1 1 1 2
Olivia Doppelt-Azeroual , Francois Delfaud , Fabrice Moriaud , Alexandre G. de Brevern ,
3
Stephane Richard
1 2 3
MEDIT SA, Palaiseau, France, INSERM, Paris, France, MEDIT USA, San Diego, CA,
United States

Ligand–protein interactions are essential for biological processes, and precise

characterization of protein binding sites is crucial to understand protein functions. MED-SuMo
is a powerful technology to localize similar local regions on protein surfaces. Its heuristic is
based on a 3D representation of macromolecules using specific surface chemical features
associating chemical characteristics with geometrical properties. MED-SMA is an automated
and fast method to classify binding sites. It is based on MED-SuMo technology, which builds
a similarity graph, and it uses the Markov Clustering algorithm. Purine binding sites are well
studied as drug targets. Here, purine binding sites of the Protein DataBank (PDB) are
classified. Proteins potentially inhibited or activated through the same mechanism are
gathered. Results are analyzed according to PROSITE annotations and to carefully refined
functional annotations extracted from the PDB. As expected, binding sites associated with
related mechanisms are gathered, for example, the Small GTPases. Nevertheless, protein
kinases from different Kinome families are also found together, for example, Aurora-A and
CDK2 proteins which are inhibited by the same drugs. Representative examples of different
clusters are presented. The effectiveness of the MED-SMA approach is demonstrated as it
gathers binding sites of proteins with similar structure-activity relationships. Moreover, an
efficient new protocol associates structures absent of cocrystallized ligands to the purine
clusters enabling those structures to be associated with a specific binding mechanism.
Applications of this classification by binding mode similarity include target-based drug design
and prediction of cross-reactivity and therefore potential toxic side effects.
S-013

Use of halide quick soaking method for structure solution of a major pilin, SpaA of
gram-positive bacteria group B streptococcus
1 2 1
Vengadesan Krishnan , Hung Ton-That , Sthanam V.L. Narayana
1 2
CBSE, University of Alabama at Birmingham, Birmingham, AL, United States, Department
of Microbiology & Molecular Genetics, University of Texas-Houston Medical School, Houston,
Texas, United States

Many pathogens use their cell wall anchored pili to initiate adherence to host cells,
which is the key initial step for bacterial colonization. The presence of pili in Gram-positive
bacteria is relatively a recent discovery, where the Streptococcus agalactiae or Group B
streptococcus (GBS) pili were discovered in 1997. The Gram-negative pili assembly is well
understood, however the same is not true for Gram-positive bacteria. It is been speculated,
mainly from well studied Corynebacterium diphtheria pili model, that their pili are assembled
through covalent linkage of individual protein subunits (pilins) in contrast to Gram-negative
pili, where they are held together by non-covalent and hydrophobic interactions. A
transpeptidase sortase, conserved all across Gram-positive bacteria, is implicated for such
covalent linkage between pilin subunits.

The GBS causes pneumonia, septicaemia and meningitis in neonates and is responsible
for significant morbidity and mortality in the United States and Europe. Three pilins; SpaA
(GBS80), SpaB (GBS52) and SpaC (GBS104) constitute the pili of GBS. It is hypothesized
that the major pilin SpaA forms pilus shaft, while minor pilins, SpaB decorated along the shaft
and SpaC sitting at the tip of the pili, are essential for adhesive function. We have crystallized
35 kDa fragment of major pilin SpaA containing middle and C-terminal domains. The crystals
diffracted to 1.8Å on a Rigaku R-axis IV imaging-plate detector using CuK
radiation at home
source. The structure was solved by SAD using NaI halide quick soaking method on home
source diffractometer. The structure reveals two IgG-like folds with an iso-peptide bond.
S-016

Structural studies of propionyl-coenzyme A carboxylase

1 1 1 3 2,3
Christine Huang , Kianoush Sadre-Bazzaz , Yang Shen , Binbin Deng , Z. Hong Zhou ,
1
Liang Tong
1 2
Columbia University, New York, NY, United States, Univ. of California, Los Angeles, Los
3
Angeles, CA, United States, Univ. of Texas Medical School at Houston, Houston, TX, United
States

Propionyl-CoA carboxylase (PCC) is a highly conserved, biotin-dependent enzyme of 750

kDa that is required for the conversion of propionyl-CoA to yield D-methylmalonyl-CoA, a
necessary step following the beta-oxidation of odd-carbon fatty acids. In addition to its key
role in the catabolism of fatty acids, cholesterol, and several amino acids, PCC is an essential
metabolic enzyme whose abrogated activities result in propionic acidemia, a genetic disorder
with an incidence rate of 1:100,000 births in the US and results in developmental delays,
neurological deficits, and severe immune deficiencies.

The PCC holoenzyme exists as an α 6β 6 dodecamer, with the α and β subunits harbouring
active sites for carboxyl group tethering and transfer, respectively. We have solved the crystal
structure of a bacterial PCC holoenzyme at 3.2 Å resolution, and present it alongside cryo-EM
data at 15 Å to support a similar structure for human PCC. The structures reveal a number of
new findings regarding the inner workings of the enzyme, including novel subunit
arrangements and active site architecture, and provide a foundation for understanding the
molecular basis of disease mutations associated with propionic acidemia. In addition, the
structures also provide insight into the activities of other biotin-dependent carboxylases, many
of which are fundamental metabolic enzymes.
S-019

Structural insights into inositol pyrophosphate synthase regulation and activity

Gayane Machkalyan, Gregory J. Miller

McGill University, Montreal, QC, Canada

Inositol phosphates (IPs) are small molecules that regulate a variety of cellular signaling
2+
pathways. The best characterized IP, inositol-1,4,5-triphosphate (IP3), triggers Ca release
from intracellular reservoirs; however, there are >30 differently phosphorylated IPs that
perform diverse signaling roles. Diphosphoinositol polyphosphates (diIPs) are the most highly
phosphorylated IPs and are structurally distinct from IPs due to pyrophosphate moieties on 1
or more positions of the inositol ring. Of paramount importance for the advancement of our
understanding of the signaling roles of diIPs is the exploration of the mechanisms through
which diIPs are produced and how their synthesis is controlled. Two classes of IP kinases
produce diIPs: inositol pyrophosphate synthetase (IPS) and inositol hexakisphosphate
kinases (IP6Ks). IPS is a dual-domain protein: at its N-terminus is a kinase domain belonging
to the ATP-grasp family, and at its C-terminus is a histidine phosphatase-like domain. The
substrate for the histidine-phosphatase domain has not yet been determined, and in fact, its
sequence suggests it may not retain catalytic activity at all, but it may function as a ligand- or
protein-binding module. There are no available structures for either the kinase or the histidine
phosphatase-like domains, which would reveal the mechanism for the production of specific
diIP isomers and would provide clues to the functional roles of the C-terminal domain. Using
combinations of X-ray crystallography and solution methods, we explore the structure and
mechanism of IPS to answer key functional and mechanistic questions, including: (1) What is
the structural basis for the production of distinct diIPs? and (2) How is IPS activity regulated?
S-022

Structural and functional analysis of the motor domain regions of the heterodimeric
Kar3/Vik1-like kinesin from Candida glabrata

Da Duan, Michelle Chan, Darlene Davis, John Allingham

Queen's University, Kingston, Ontario, Canada

All animal and plant cells rely on nanometre-sized protein motors called kinesins to segregate
chromosomes between dividing cells and haul vital cargo-containing sacks to where they are needed.
Many kinesins operate as a complex of identical pairs of molecules that cooperate to move along
microtubules and perform a single cellular function. However, a few kinesins in certain species and
cell types mix-and-match different molecules in ways that allow the motor protein to perform multiple
functions. The budding yeast kinesin Kar3, for example, forms heterodimers with two non-catalytic
kinesin-like proteins named Vik1 and Cik1 that influence the cellular localization and function of Kar3
during yeast mating and division. The way in which Kar3 and Vik1, or Kar3 and Cik1, operate at a
molecular level as a motor complex is not yet known. However, our recent determination of the X-ray
crystal structure of the motor domain region of a Vik1 ortholog from Candida glabrata has revealed
structurally dynamic regions in this protein that sheds new light on how this protein may work
at the atomic level with Kar3. Specifically, our crystals of CgVik1 contained two molecules in the
asymmetric unit that exhibit two very different conformations of an alpha-helical segment that is
analogous to the ‘neck’ found in the Drosophila kinesin Ncd. The intramolecular interactions of
the CgVik1 neck and motor domain core differ in each conformation and are accompanied by subtle
movements in elements of the motor domain core that are analogous to the P-loop and part of the
microtubule binding surface of catalytic kinesins. In order to better understand the importance of these
interactions and displacements, the effects of mutating residues that form conformation-dependent
interactions between the neck and motor core were structurally and functionally evaluated.
S-025

The Crystal Structure of the RNA helicase Mtr4 reveals a unique and novel arch domain
that is required for nuclear 5.8 S rRNA processing

Ryan Jackson, Sean Johnson

Utah State University, Logan, UT, United States

The cell must extensively monitor and correctly process a myriad of RNA species in order to
maintain the proper regulation and expression of genes. Exonucleolytic decay is utilized by
the cell as a quality control mechanism that eliminates unneeded or erroneous RNA, as well
as a tool that processes RNA to proper maturity. Mtr4 is an essential and conserved RNA
helicase that is central to the processing and degradation of RNA in the nucleus. Mtr4
activates the multi-subunit nuclear exosome which processes or completely degrades RNA
substrates. Many of the molecular details of how Mtr4 recognizes and delivers appropriate
RNA substrates to the exosome are currently unknown due to a complete lack of structural
data. To enhance the understanding of these mechanisms we have determined the crystal
structure of Mtr4. The structure reveals a novel arch-like domain that is unique to Mtr4 and
Ski2 (the cytosolic homolog of Mtr4). In vivo and in vitro analyses demonstrate that the Mtr4
arch domain is required for proper 5.8 S rRNA processing, and suggest that the arch
functions independently of canonical helicase activity. Additionally, extensive conservation
along the face of the putative RNA exit site highlights a potential interface with the exosome.
These studies provide a molecular framework for understanding fundamental aspects of
helicase function in exosome activation, and more broadly define the molecular architecture
of Ski2-like helicases.
S-028

Structural characterization of ATPase/Response Regulator pairs involved in type IV

pilus biology
1,2 1,3 1,3
Aditya Gupta , Ana M. Misic , Katrina T. Forest
1
Department of Bacteriology, University of Wisconsin-Madison, Madison, WI, United States,
2
Biophysics Graduate Training Program, University of Wisconsin-Madison, Madison, WI,
3
United States, Department of Biomolecular Chemistry, University of Wisconsin-Madison,
Madison, WI, United States

Type IV pili (Tfp) are evolutionarily conserved bacterial appendages utilized for a variety of
functions such as host cell adhesion, DNA uptake, and twitching motility. In Pseudomonas
aeruginosa, over 40 proteins are involved in the biogenesis and retraction of Tfp. Among
these are PilB and PilT/PilU, hexameric P-loop ATPases that play a role in powering pilus
assembly and retraction, respectively. PilG and PilH are two single domain response
regulators (CheY homologs) that control twitching motility. Recent genetic results indicate that
PilG acts upstream of PilB and PilH acts upstream of the pilus retraction process (Bertrand,
West, and Engel 2010), although the biochemical pathway for these control processes has
not yet been elucidated. We will present progress on overexpression, purification, and
crystallization of these response regulator proteins, both alone and as PilG/B and PilT/U/H
complexes. We anticipate that this work will shed light on the molecular mechanisms
involved in the complex process of P. aeruginosa pilus assembly, retraction, and modulation.
S-033

A Uniquely Open Conformation Revealed in the Structure of a Novel Protein Arginine

Methyltransferase

Yuan Cheng, Monica Frazier, Matthew Redinbo

University of North Carolina at Chapel Hill, Chapel Hill, NC, United States

Protein arginine methyltransferase 10 (PRMT10) is a type-I arginine methyltransferase that

plays a critical role in the regulation of flowering-related genes in Arabidposis thaliana.
PRMT10 is only active in the dimeric state and displays unique substrate specificity. In this
work, X-ray crystallography is combined with molecular dynamics simulations and
biochemical studies to explore the mechanism underlying these functional features. The 2.6 Å
resolution crystal structure of PRMT10 reveals a large set of structural features that are
unique to PRMT10, including the surface charge distribution, the shape of the substrate-
binding groove, the active sites’ accessibility, and the mode of dimerization. This information
provides substantial clues to understanding the mechanism governing the substrate
specificity of PRMT10. In addition, structure-based molecular dynamics (MD) simulations
were conducted to understand why dimerization is essential for the enzymatic activity of
PRMT10. Upon dimerization, the motion of the cofactor-binding domain shifted from non-
correlated to highly-correlated, which likely facilitates the binding of cofactor and the
subsequent catalytic reaction. Furthermore, dimerization was shown to help the formation of a
major substrate-binding groove in PRMT10 by reducing the local fluctuation. Similar results
were also observed in MD analyses we conducted on mammalian PRMT1 and PRMT3
structures. Together, these results demonstrate the importance of dynamic motion in the
regulation of PRMT activity, and suggest a general model to explain why dimerization is
essential for the PRMT family of enzymes.
S-035

Crystal Structure of LL-DAP-AT from Chlamydia trachomatis: The open conformation

and its implication on the broad substrate specificity
1 2 2 2
Nobuhiko Watanabe , Chenguang Fan , Matthew D. Clay , Marco J. van Belkum , John C.
2 1
Vederas , Michael N. G. James
1 2
Department of Biochemistry, University of Alberta, Edmonton, Canada, Department of
Chemistry, University of Alberta, Edmonton, Canada

The lysine biosynthetic pathway is an attractive target for the development of novel antibiotics
because it is absent in humans. Previously, three different lysine biosynthetic pathways have
been characterized in bacteria. However, none of the previous bacterial lysine biosynthetic
pathways were found in Chlamydia or in plants. Recently, LL-DAP-AT was discovered to be
the missing piece of the lysine biosynthetic pathways in Chlamydia, plants, and some
archaea. Due to the absence of this enzyme in humans, LL-DAP-AT is an attractive target for
anti-Chlamydial drugs. Previously, we have determined the crystal structure of Arabidopsis
LL-DAP-AT (AtDAP-AT) and elucidated its substrate recognition mechanism. Although the
Chlamydial LL-DAP-AT (CtDAP-AT) is 41% identical to AtDAP-AT in its primary sequence, it
has a much broader substrate specificity than AtDAP-AT does. In order to understand the
differences in the substrate specificity and to assist in the development of novel antibiotics
against Chlamydial infections, we have determined the crystal structure of the pyridoxal-5’-
phosphate-bound CtDAP-AT to 2.3 Å resolution. Unlike our previously determined crystal
structures of AtDAP-AT, the small domain (Ala297-Met388) and the N-terminus arm (Met1-
Gln48) of CtDAP-AT have moved away from the active site significantly with a maximum
displacement of 8.9 Å. Now, the active site is exposed to the solvent and the loops lining the
active site (A: Gly41-Gln48, C: Thr67-Pro73) are completely disordered. From our previous
AtDAP-AT structures, the loop A is determined to be important for substrate entry and
binding. Given that the active site residues are almost completely conserved between the two
species, and that the active site residues of the “closed” conformation of AtDAP-AT makes
very tight interactions with L-Glu and LL-DAP, the “open” conformation with its highly flexible
loops may account for the broad substrate specificity of CtDAP-AT.
S-038

Removal of a dynamic Ω -loop: Structural and enzymatic consequences

Troy Johnson, Todd Holyoak

University of Kansas Medical Center, Kansas City, Kansas, United States

Many studies have shown that dynamic motions of individual protein segments can play a role
in enzyme function. Structural studies on the metabolic enzyme phosphoenolpyruvate
carboxykinase (PEPCK) have revealed a dynamic element in the form of a 10-residue Ω -loop
domain which acts as an active site lid adopting an ordered, closed conformation upon the
formation of complexes that mimic the Michaelis and enolate intermediate states. Based upon
these structural studies and our recent work on the dynamic nature of this loop we have
proposed a model for the mechanism of PEPCK catalysis in which the closed conformation of
the mobile lid-domain is necessary for correct substrate positioning, sequestering of the
reaction intermediate, and protection of the intermediate from alternate chemistries.
Furthermore, the ability of the lid to occupy the closed conformation involves a fine energetic
balance between the entropic penalty for lid closure and the free energy of ligand binding. To
further test this model the flexible Ω -loop was removed and replaced with 1, 2, or 3 glycine
residues. Structural and kinetic characterization was carried out on all three of the lid deletion
mutants. The kinetic experiments were carried out utilizing multiple steady state assays and
the results were as expected: removal of this loop significantly reduces or eliminates the
catalytic activity and efficiency of all three mutant PEPCKs. To further investigate the cause of
this catalytic deficiency the structures of each mutant were solved in complex with either the
physiological substrates or the respective analogs. The structural data revealed an
unexpected backbone shift, which resulted in the Cα of R87 (a catalytically important residue
for OAA binding) being displaced by 6 Å and the side chain being flipped out occupying space
where the Ω -loop would normally reside when in the closed conformation. This suggests that
R87 may play a role in the energetic balance of lid dynamics, acting as a conduit for the
transfer of free energy from binding to offset the entropic penalty of lid closure. The data
presented here supports our model for the role of the Ω -loop lid domain in correctly
positioning substrates, sequestering and protecting the intermediate species, and gives
further insight into how the transfer of free energy could be mediated through the protein
structure to allow the energetically costly lid closed conformation to form so catalysis can
occur.
S-041

1 2 3 3 1
Brandon Goblirsch , Amanda Lee , Bennett Streit , Jennifer DuBois , Carrie Wilmot
1 2
University of Minnesota, Minneapolis, MN, United States, Purdue University, West
3
Lafayette, IN, United States, University of Notre Dame, South Bend, IN, United States

Chlorite dismutase (Cld) is a heme enzyme which rapidly and selectively decomposes chlorite
to Cl¯ and O2. The ability of Cld to promote O2 formation from chlorite (ClO2¯ ) is unusual.
Heme enzymes generally utilize chlorite as an oxidant for reactions such as oxygen atom
transfer to a second substrate. The X-ray crystal structure of Dechloromonas aromatica Cld
co-crystallized with the substrate analogue nitrite (NO2¯ ) was determined to investigate
features responsible for this novel reactivity. The enzyme active site contains a single b-type
heme coordinated by a proximal histidine residue. Structural analysis identified a glutamate
residue hydrogen bonded to the heme proximal histidine that may stabilize reactive heme
species. A solvent exposed arginine residue likely gates substrate entry to a tightly confined
distal pocket. Based on the proposed mechanism of Cld, initial reaction of ClO2¯ within the
distal pocket generates hypochlorite (ClO¯ ) and a compound I intermediate. The sterically
restrictive distal pocket probably facilitates the rapid rebound of hypochlorite with compound I
forming the Cl¯ and O2 products. Common to other heme enzymes, Cld is inactivated after a
finite number of turnovers, potentially via the observed formation of an off-pathway
tryptophanyl radical species through electron migration to compound I. Three tryptophan
residues of Cld have been identified as candidates for this off-pathway radical. Finally, a
juxtaposition of hydrophobic residues between the distal pocket and the enzyme surface
suggests O2 may have a preferential direction for exiting the active site.
S-044

1.75 Å Structure of a Fungal Type III Polyketide Synthase, a Potential Biosynthetic Tool
for "Unnatural" Natural Products

Anna Lytle, Jia Zeng, Jixun Zhan, Sean Johnson

Utah State University, Logan, UT, United States

Polyketides, a class of secondary metabolites, comprise a significant portion of structurally

diverse and biologically active natural products. These valuable compounds, utilized as
antibiotic, anticancer, anti-tumor and anti-cholesterol drugs, are generated by three classical
types of polyketide synthases (PKSs). Although type III PKSs were thought to exist only in
plants and bacteria, the genes for these iterative homodimeric enzymes were recently
discovered in fungi. In the industrially important fungus, Aspergillus oryzae, four similar yet
distinct type III PKS genes have been identified, providing an attractive opportunity to
understand chemical diversity in fungal secondary metabolites. Here we report the 1.75 Å
apo-crystal structure of one of these enzymes, CsyB. The structure reveals a homodimeric
protein with a highly conserved Cys-His-Asn triad that catalyzes the condensation of different
saturated fatty acyl-CoAs into a variety of aromatic polyketides. The apo structure sets the
stage for investigation of complexes with substrate and product bound in the multi-functional
active site. The goal is to delineate the structural basis for the specific function and unique
products of CsyB. Future work will examine the other PKSs present in A. oryzae. The
structural characterization of these enzymes will greatly inform the design of biosynthetic
PKSs to produce novel bioactive "unnatural" natural products.
S-047

Structural Investigation of the Mechanism and Regulation of PLP Synthase.

1,2 2 2 1,2
Amber Smith , David Akey , Markos Koutmos , Janet Smith
1 2
University of Michigan, Ann Arbor, MI, United States, Life Sciences Institute, Ann Arbor, MI,
United States

PLP synthase (PLPS) from the bacterium G. stearothermophilus generates pyridoxal 5’-
phosphate (PLP), the active form of vitamin B6. PLPS is a 24-mer complex made up of two
subunits, a 32 kDa PdxS synthase subunit and a 25 kDa PdxT glutamine amidotransferase
(GAT) subunit. PdxS, possessing the ( / )8 barrel fold, forms a cylindrical dodecamer of two
1 2
hexameric rings . PdxT binds in a 1:1 ratio around the outside of the PdxS stacked rings .
PdxT functions as a Triad GAT catalyzing the hydrolysis of glutamine to glutamate and
ammonia. The ammonia is then channeled to the synthase active site of PdxS, where PLP is
formed from ammonia, ribose 5-phosphate and glyceraldehyde-3-phosphate. The mechanism
of this reaction is unknown. A flexible C-terminal tail of PdxS is essential to PLP synthesis and
3
to the cross talk between the active sites of the two subunits . Using an inactive mutant of
PdxT, we co-crystallized PLPS with substrates glutamine and ribose 5-phosphate. Initial
crystals diffracted to ~4 Å and revealed density for the C-terminal tail. Dehydration
experiments improved the diffraction quality of the crystals to 2.75 Å. The higher resolution
structure will provide more structural information in order to determine the functional
relevance of the C-terminal tail.

Supported by NIH grant: DK42303.

1 Zhu, J., Burgner, J. W., Harms, E., Belitsky, B. R. & Smith, J. L. A new arrangement
of ( / )8 barrels in the synthase subunit of PLP synthase. J Biol Chem 280, 27914-
27923, (2005).

2 Zein, F., Zhang, Y., Kang, Y., Burns, K., Begley, T., & Ealick, S. Structural insights
into the mechanism of the PLP synthase holoenzyme from Thermotoga maritima.
Biochemistry 45, 14609-14620, (2006).

3 Raschle, T., Speziga, D., Kress, W., Moccand, C., Gehrig, P., Amehein, N., Weber-
Ban, E., & Fitzpatrick, T. Intersubunit cross-talk in pyridoxal 5’-phosphate synthase,
coordinated by the C terminus of the synthase subunit. J Biol Chem 284, 7706-7718,
(2009).
S-050

Inhibition of Saccharomyces cerevisiae S-formylglutahtione Hydrolase and Activation

of the H160I variant by Peroxide

Patricia M. Legler, Charles B. Millard

Walter Reed Army Institute of Research, Silver Spring, MD, United States

S. cerevisiae S-formylglutathione hydrolase (SFGH, homologous to human esteraseD)

belongs to a class of serine hydrolases that are resistant to organophosphates but sensitive
to classical thio-enzyme inhibitors such as Hg and N-ethylmaleimide. Under oxidizing
conditions, inhibition of SFGH activity is attributable to a cysteine (Cys-60) adjacent to the
catalytic triad and approximately 8.0 Å away from the O of the active site serine. Cys-60 is
oxidized to a sulfenic acid in the structure of the diethylphosphate inhibited W197I variant
(PDB 3C6B). While this structural snap-shot captured an unstable reversibly oxidized state, it
remained unclear as to whether the oxidation occurred before, during or after the reaction
with the organophosphate inhibitor. To determine if the oxidation of Cys-60 was linked to
ester hydrolysis we used kinetic experiments and site-directed mutagenesis in combination
with X-ray crystallography. In the presence of substrate, the rate of inhibition of the WT
SFGH by peroxide increases 14-fold suggesting that oxidation is linked to ester hydrolysis.
We also found one variant, H160I, which is activated by peroxide. This variant is activated at
comparable rates in the presence and absence of substrate suggesting that the conserved
His-160 is involved in the inhibitory mechanism linking ester hydrolysis to the oxidation of
Cys-60. Mechanisms for inhibition by both peroxide and CuCl2 are proposed as well as a
mechanism for peroxide activation of the H160I variant. A Dali structural similarity search
uncovered two other enzymes (B.subtilis RsbQ, 1WOM and C. acetobutylicum lipase-
esterase, 3E0X) that contain a similar Cys adjacent to a catalytic triad. We speculate that the
regulatory motif uncovered is conserved in some D-type esterases and discuss its structural
similarities in the active site of Human Protective Protein (HPP; also known as Cathepsin A).
This work was funded by the U.S. Defense Threat Reduction Agency JSTO award
1.D0015_06_WR_C (CBM). The opinions or assertions contained herein belong to the
authors and are not necessarily the official views of the U.S. Army or the U.S.
Department of Defense
S-053

A Short, Strong Hydrogen Bond in the Active Site of Human Carbonic Anhydrase II

1 2 1 2 1
Balendu Avvaru , Chae Kim , Katherine Sippel , Sol Gruner , Mavis Agbandje-McKenna ,
1 1
David Silverman , Robert McKenna
1 2
University of Florida, Gainesville, FL, United States, CHESS, Cornell University, Ithaca, NY,
United States

The crystal structure of human carbonic anhydrase II (HCA II) obtained at 0.9 Å resolution
reveals that a water molecule, termed deep water, Dw, and bound in a hydrophobic pocket of
the active site forms a short, strong hydrogen bond with the zinc-bound solvent molecule, a
conclusion based on the observed oxygen-oxygen distance of 2.45 Å. This water structure
has similarities with hydrated hydroxide found in crystals of certain inorganic complexes. The
energy required to displace Dw contributes in significant part to the weak binding of CO2 in
the enzyme-substrate complex, a weak binding that enhances kcat for the conversion of CO2
into bicarbonate. In addition, this short, strong hydrogen bond is expected to contribute to the
low pKa of the zinc-bound water and to promote proton transfer in catalysis.
S-056

New Insights for the Role of Calcium in Human Calcium Activated Nucleotidase (CAN)
Activity and Dimerization

Stefanie Ward, Terrence Kirley, Andrew Herr

University of Cincinnati, Cincinnati, OH, United States

Blood-sucking insects secrete nucleotidases that prevent host blood clotting by hydrolyzing
ADP, a platelet agonist, to AMP in the blood. Mammals also express a homologous, soluble
calcium-activated nucleotidase (CAN), however, the mammalian ortholog prefers GDP to
ADP as a substrate and therefore is not efficient at preventing thrombosis. Calcium is
required for CAN catalytic activity of CAN and potentiates CAN’s activity by inducing its
dimerization, although the allosteric mechanism is unknown. Therefore, we have introduced
a point mutation at a key residue in the dimer interface, Ile170Lys (I170K), to produce an
obligate monomer species in order to study calcium’s role in catalysis and dimerization
through crystallographic analysis. I170K demonstrates a significant reduction of ADP
hydrolysis, which correlates with the loss of dimerization. I170K was crystallized in two
crystal forms and the structures were solved at 1.6 and 1.8 Å resolution by molecular
replacement. Additionally, the wild-type CAN was co-crystallized with the non-hydrolyzable
substrate GMPCP in the presence of calcium. Structural alignments with the CAN wild-type
apo structure 2H2N indicate significant main chain and side chain rotamer shifts for residues
within the enzymatic active site and dimer interface, suggesting a likely mechanism by which
dimerization can modulate activity. In addition, our high resolution data reveal the location of
multiple calcium ions within the active site that are likely to play important roles in substrate
binding and hydrolysis. These data demonstrate the importance of calcium-induced
dimerization for nucleotidase function, which could lay the groundwork for the development of
an engineered human CAN with improved ADP cleavage for use as a blood clot inhibitor.
S-059

A sulfotransferase and thioesterase working together to produce the terminal alkene in

curacin A

Jennifer Gehret, David Sherman, Janet Smith

University of Michigan, Ann Arbor, MI, United States

Curacin A is a small-molecule antimitotic natural product, produced by the marine

cyanobacterium Lyngbya mujuscula. Curacin is unique among polyketide natural products
because of its many interesting chemical groups, including a terminal double bond, created
during offloading of the final product from the polyketide synthase (PKS). The double bond
contrasts with the more common cyclized macrolactone or linear carboxylic acid produced by
a thioesterase (TE) domain at the end of the PKS. The curacin pathway instead contains
both sulfotransferase (ST) and TE domains, which work together to create the terminal
1
alkene . The ST sulfonates a -hydroxyl in the penultimate intermediate, after which the TE
hydrolyses, decarboxylates, and desulfates the intermediate to offload curacin A with its
terminal double bond. This scheme includes the first observation of biological sulfation for
chemical activation as well as novel decarboxylation/desulfation activity in a TE. This system
has potential in bioengineering hydrocarbon production for biofuels.

The crystal structures of the ST (1.6Å) and TE (1.7Å) were determined as individual domains
excised from the PKS module. The TE has the expected / hydrolase fold but differs from
other offloading TEs in lid structure, dimer interface position, and an open-cleft active site.
Comparison with uncharacterized sequences of putative tandem ST-TE domains with
presumably similar activity reveals dense conservation within the cleft. A model of the
predicted acyl enzyme intermediate shows a conserved Arg205 which may confer specificity
to TE for the -sulfate, a prediction that is supported by site-directed mutagenesis studies.
1
L. Gu et al., J Am Chem Soc 131, 16033 (2009).

This work was supported by NIH grant DK42303

S-062

Distal Pocket Effects on Nitrite Binding to Heme Iron

Jun Yi, George Richter-Addo

University of Oklahoma, Norman, OK, United States

The binding of the nitrite anion to metal centers occurs via a number of ways. The "nitro"
N-binding mode (metal-NO2) is quite common for synthetic heme models and is present in the
crystal structures of the nitrite adducts of cytochrome cd1 nitrite reductase (NiR), E. coli sulfite
reductase hemeprotein, and cytochrome c NiR. We reported the X-ray crystal structure of
the nitrite adduct of ferric horse heart myoglobin (hh Mb) and showed that the nitrite ligand
was bound to heme Fe in an unprecedented O-binding mode. We hypothesized that the
distal His64 residue in this Mb(ONO) complex was responsible for directing the O-binding of
the nitrite ligand. To test this hypothesis, we prepared and characterized the nitrite adducts of
the mutant H64V and the double mutant H64V/V67R. The lack of a distal pocket His64
residue in the H64V-nitrite adduct resulted in the nitrite ligand adopting the more common N-
binding mode. Reintroducing a distal pocket H-bonding side chain (i.e., in the H64V/V67R
double mutant) resulted in the restoration of the observed nitrito O-binding mode. These
results will be presented and discussed in context of the proposed (by others) nitrite
reductase activity of the Mb protein.
S-065

Structure Based Insight into the Mechanism of Type I Dehydroquinate Dehydration

1,3 1,3 1,3 2 2
Samuel Light , George Minasov , Elisabetta Sabini , Arnon Lavie , Michael Caffrey ,
1,3
Wayne Anderson
1
Department of Molecular Pharmacology and Biochemistry, Feinberg School of Medicine,
2
Chicago, IL, United States, University of Illinois at Chicago, Department of Biochemistry and
3
Molecular Genetics, Chicago, IL, United States, Center for Structural Genomics of Infectious
Diseases, Chicago, IL, United States

The shikimate pathway links metabolism of carbohydrates to biosynthesis of aromatic

compounds. In a sequence of seven metabolic steps phosphoenolpyruvate and erythrose 4-
phosphate are converted to chorismate, the precursor of the aromatic amino acids and many
secondary aromatic metabolites. The third step in the pathway, consisting of the dehydration
of dehydroquinate to dehydroshikimate, is performed by two distinct enzymes which catalyze
the same reaction through disparate mechanisms. Crystal structures of the type I
dehydroquinate dehydratase from Salmonella enterica typhimurium and Clostridum difficile
are reported here. Structures of the un-liganded, substrate, and product bound enzyme
illustrate protein movement over the course of the reaction. Upon substrate binding, a 7
residue flexible region hydrogen bonds with the substrate, closing over the active site. The
substrate bound structure shows a 1.5 Å movement of histidine-143 to interact with the
leaving hydroxyl. Following dehydration, the product bound structure shows histidine-143
returned to its original orientation. The direct interaction of histidine-143 with the leaving group
positions the residue for proton shuttling between ring and leaving hydroxyl. The mechanism
of histidine-143 proton transfer proposed here is consistent with previous observations of cis-
elimination in type I dehyroquinate dehydration (Harrison and Rose, 1963).

The Center for Structural Genomics of Infectious Diseases has been funded by the National
Institute of Allergy and Infectious Diseases under Contract No. HHSN272200700058C.
S-068

Catalysis in the nitrilase superfamily amidases; implications from active site structure
1 2 3 3 2
Serah Kimani , Brandon Weber , Andrew Nel , Don Cowan , Trevor Sewell
1
Molecular and Cell Biology Department, University of Cape Town, Western Cape, South
2
Africa, Electron Microscope Unit, University of Cape Town, Western Cape, South Africa,
3
Department of Biotechnology, University of the Western Cape, Western Cape, South Africa

The nitrilase superfamily amidases catalyze the conversion of various amides to their
corresponding acid and ammonia using a highly conserved Glu, Lys, Cys catalytic triad in an
acid-base catalysis mechanism. Some of these enzymes are potential biocatalysts in the fine
+
chemical industry, while others like the amidase domain of the NAD synthetase from
Mycobacterium tuberculosis (MTB) are attractive drug targets.

We have recently solved the crystal structure of the amidase from Geobacillus pallidus
RAPc8. The most interesting observation in this structure arises from the size and the
geometry of the active site pocket, which is arranged in such a way that the reaction
intermediate restricts access to the glutamic acid (Glu59) previously thought to be the general
base catalyst for the hydrolysis of the acyl intermediate. An alternative choice for a general
base catalyst is another glutamic acid residue (Glu142), which has not been characterized
before, and which we found to be highly conserved in other structures from the nitrilase
superfamily. We have also very recently solved the structure of another amidase from
Nesterenkonia sp. The position and coordination of the second glutamic acid residue
(Glu139) is also conserved in this amidase. We have proposed a catalytic mechanism that
postulates the involvement of this additional glutamic acid as a fourth catalytic residue in the
amidases of the nitrilase superfamily. We are presently investigating the role of this residue
using both biophysical and structural methods.

Mass spectra from the Geobacillus and Nesterenkonia sp. amidase mutants where the
proposed general base catalyst glutamic acid residue has been changed to a leucine and a
glutamine respectively, indicate that tetrahedral intermediates of various substrates are being
trapped in the active site. This confirms that this residue is indeed involved in catalysis. To
further confirm these findings, crystals of the E139Q Nesterenkonia amidase mutant reacted
with various substrates have been prepared. The progress on this work will be presented.
S-071

Structural investigation of the biotin carboxylation reaction in the BC domain

of Rhizobium etli pyruvate carboxylase

Sudhanshu Kumar, Martin St. Maurice

Marquette University, Milwaukee, WI, United States

Pyruvate carboxylase (PC) is a biotin-dependent multifunctional enzyme which plays

an important role in gluconeogenesis, lipogenesis, glucose mediated insulin secretion
and neurotransmitter biosynthesis. PC contains three functional domains on a single
polypeptide chain: an N-terminal biotin carboxylase (BC) domain, a central
carboxyltransferase (CT) domain and a C-terminal biotin carboxyl carrier protein
(BCCP) domain. BCCP-biotin “swings” between BC and CT active sites to affect
catalysis. Recent X-ray crystal structures have revealed the complete domain
architecture of PC, showing BCCP-biotin and pyruvate positioned in the CT domain
and free biotin and ADP/ATP bound in the BC domain. However, the catalytically
relevant position and orientation of BCCP-biotin in the BC domain remains unknown.
To structurally investigate this unknown conformation, we rationalized crystallizing
the kinetically incompetent mutant of Rhizobium etli PC, T882A, in the hopes of
trapping the BCCP domain in the desired conformation. It has been proposed that
the biotin carboxylation reaction is a stepwise process. First, bicarbonate is
phosphorylated to form a carboxyphosphate intermediate which subsequently leads
to carboxylation of biotin. So far, there has been no direct and conclusive evidence
for the existence of this proposed intermediate. Thus, the R.etli PC T882A mutant
was co-crystallized with the carboxyphosphate intermediate, phosphonoacetate, with
the intent to mimic the binding of the proposed carboxyphosphate intermediate. The
X-ray crystal structure at 2.6 Å resolution reveals BCCP-biotin bound in the BC
domain of PC. Furthermore, the carboxyphosphate analogue, phosphonoacetate, is
present in the active site. This structure offers new insight into the molecular basis of
catalysis in biotin-dependent carboxylase enzymes and strengthens proposals
invoking a carboxyphosphate intermediate in the catalytic mechanism for this
reaction.
S-074

Structural Studies on Mutants of HMG CoA Reductase from Pseudomonas mevalonii

Moumita Sen, C Nicklaus Steussy, Chandra Duncan, Victor Rodwell, Cynthia Stauffacher

Purdue University, West lafayette, United States

HMG-CoA reductase catalyzes the four-electron reduction of HMG-CoA to free CoA and
mevalonate. This is one of the few double oxidation/reduction reactions in intermediary
metabolism that take place in a single active site. In addition to the unusual enzymology, this
reaction is of interest because it is the committed step of the fundamental mevalonate
isoprenoid pathway. In animals this pathway produces cholesterol, the steroid hormones and
a variety of signaling molecules based on the isoprenoid building block (1). In bacteria the
pathway is equally important, and has been shown to be essential to the virulence of
Staphylococcal and Streptococcal bacteria (2). To better understand the nature of this
reaction, our laboratory has undertaken a comprehensive structural study of the mechanism
of HMG-CoA reductase in bacteria utilizing the enzyme from Pseudomonas mevalonii.

HMG-CoA reductase is an obligate dimer, with each monomer consisting of a large domain, a
small domain, and a flap domain (2, 3) that is disordered in the apoenzyme structure. The flap
domain is ordered in the crystal structure only in the presence of ligand and co-factors, where
it closes over the active site, positioned by a network of hydrogen bonds that include the
ligand and co-factor. Two residues proposed to be important in flap domain movement have
been mutated. Mutant proteins have been crystallized, soaked with various combinations of
ligands and co-factors, and their structures have been solved at 1.95-2.40Å. These
structures, reinforced with kinetic analysis of the mutants, demonstrate the essentialness of
this closure in the reaction and reveal how these residues are involved in flap domain
movement.

1. Goldstein JL, Brown MS, Nature, 343, 425-430 (1990)

2. Wilding, EI et al. J. Bacteriol., 182 5147-5152 (2000)

3. Lawrence CM et al. Science, 268, 1758-1762 (1995)

4. Tabernero L et al. Proc Natl Acad Sci USA, 96, 7167-7171 (1999)
S-077

Structural studies of thrombin-PAR1 interaction

Prafull S. Gandhi1,2, Zhiwei Chen1, Enrico Di Cera1

1
Saint Louis University Medical Center, Saint Louis, MO, United States, 2Washington
University School of Medicine, Saint Louis, MO, United States

Abundant structural information exists on how thrombin recognizes ligands at the

active site or at exosaites separate from the active site region, but remarkably little is
known on how thrombin recognizes substrates that bridge both the active site and
exosite I. Likewise, abundant data exist on how binding of ligands to exosite I alters
the activity of thrombin, but the molecular basis of this long-range allosteric effects
has remained elusive. The protease activated receptor PAR1 is particularly relevant
in view of the plethora of biological effects associated with its activation by thrombin.
Here we present the 1.8 Å resolution crystal structure of thrombin S195A in complex
with a 30-residue long, uncleaved extracellular fragment of PAR1 that documents for
the first time a productive binding mode bridging the active site and exosite I. We
also show how binding of the cleaved extracellular fragment of PAR1 to exosite I
causes a massive conformational change of thrombin from the inactive E* to the
active E form, as well the details of the long-range allosteric communication between
exosite I and the active site. These results fill a significant gap in our understanding
of the mechanisms of substrate recognition by thrombin and the molecular basis of
its allosteric function.
S-080

Structural and Functional Studies of Staphylococcus aureus Pyruvate Carboxylase

Linda Yu, Song Xiang, Liang Tong

Columbia University, New York, United States

Pyruvate carboxylase (PC) catalyzes the ATP-dependent transformation of pyruvate to

oxaloacetate, which marks the first step in gluconeogenesis. Oxaloacetate is also an
important intermediate in the tricarboxylic acid cycle. Therefore, PC is considered an essential
enzyme in intermediary metabolism. The structural architecture of PC consists of four
domains, the biotin carboxylase (BC) domain, the carboxyltransferase (CT) domain, the biotin
carboxyl carrier protein (BCCP) domain, and a novel PC tetramerization (PT) domain. It
belongs to a group of biotin dependent enzymes where the biotin is covalently bound to
BCCP, which swings between the distinct active sites on the BC and CT domains to carry out
the catalysis. The newly discovered PT domain is essential for keeping the tetramer intact
and mutations in this domain disrupt the tetramer in both human and Staphylococcus aureus
PC. In terms of regulation, it is known that PCs from vertebrate sources are highly activated
by acetyl-CoA, while PCs from prokaryotes have varying degrees of dependency on acetyl-
CoA.
We have recently reported the crystal structure of the S. aureus PC, alone and in the
presence of acetyl-CoA. The acetyl-CoA binding site can be found at the interface where the
BC dimer meets one PT domain. Upon CoA binding, the enzyme becomes more symmetrical,
which is supported by cryo-EM data. Activator binding also seems to stabilize the BC dimer,
which is hypothesized to be a possible mechanism by which acetyl-CoA enhances enzyme
activity. The latest results from our structural and biochemical studies on this important
enzyme will be presented.
S-083
+
Residue 143 is the Key Residue for the Thrombin Activation by Na
Weiling Niu, Zhiwei Chen, Leslie Bush-Pelc, Alaji Bah, Prafull Gandhi, Enrico Di Cera

Saint Louis University, Saint Louis, MO, United States

+ +
Thrombin is a Na -activated trypsin-like protease for which the binding of Na enhances
allosterically activity toward synthetic and physiological substrates. Although this effect has
been known for over three decades, no information has been available on its molecular basis.
We extended the classical Botts-Morales theory for the action of a modifier on an enzyme to
+
account for the contribution of the E*, E and E:Na forms of thrombin and established that
+
analysis of kcat unequivocally identifies allosteric transduction of Na binding into enhanced
catalytic activity. Next, we constructed and purfied to homogeneity the thrombin mutant
N143P to selectively destabilize the environment of the oxyanion hole of the enzyme. The
+ +
mutant binds Na with an affinity comparable to that of wild-type but features no Na -
+
dependent enhancement of kcat. The crystal structure of N143P in the absence of Na is in the
+ +
inactive E* form. Binding of Na converts the enzyme to the active E:Na form. The N143P
mutation abrogates the important H-bond between the backbone N atom of residue 143 and
the carbonyl O atom of E192, which in turn controls the orientation of the E192-G193 peptide
+
bond and the correct architecture of the oxyanion hole. We conclude that Na activates
thrombin by securing the correct orientation of the E192-G193 peptide bond. Conservation of
the 143-192 H-bond in trypsin-like proteases and the importance of the oxyanion hole in
+ +
proteases function suggest that this mechanism of Na activation is present in all Na -
activated trypsin-like proteases.
S-086

Role of His 265, the most conserved residue for a family of C-C bond hydrolases, in the
catalytic mechanism of BphD from Burkholderia xenovorans LB400
1 1,3 2,4 2 1
Subhangi Ghosh , Shiva Bhowmik , Geoff Horsman , Lindsay Eltis , Jeffrey Bolin
1 2
Purdue University, West lafayette, IN, United States, University of British Columbia,
3
Vancouver, BC, Canada, The Scripps Research Institute, La Jolla, CA, United States,
4
University of Wisconsin, Madison, WI, United States

BphDLB400, a C-C bond hydrolase from the biphenyl degradation pathway of Burkholderia
xenovorans LB400, is a key determinant in the degradation of biphenyl and PCBs. Homologs
play a similar role in the degradation of dioxins and other xenobiotic pollutants as well as
steroids. BphDLB400 catalyzes the cleavage of the C5-C6 bond of 2-hydroxy-6-oxo-6-
phenylhexa-2,4-diene (HOPDA). The reaction is believed to proceed via two steps and is
known to depend on residues S112 and H265. In the first step HOPDA undergoes
tautomerization to yield a keto intermediate, which facilitates the second step, hydrolysis of
the C5-C6 bond. The present study will further explore the role of H265 in the first step.

For the wild type enzyme, stopped flow spectrophotometry demonstrated the rapid formation
of an intermediate species with a spectrum red shifted (λ max=492nm) from that of the
substrate (λ max=434nm). The intermediate decays concurrently with the formation of spectral
features corresponding to the product. In the BphDLB400 S112A mutant, this intermediate
decays extremely slowly and is effectively trapped. In BphDLB400 variants carrying the
mutation H265A, the intermediate species is not observed. Crystal structures of
enzyme:HOPDA complexes revealed remarkably different conformations of HOPDA for the
S112A and S112A/H265A variants. In the S112A:HOPDA complex, the dienoate portion
HOPDA adopts a non-planar conformation with the 2-hydroxy/oxo oxygen near H265. In the
S112A/H265A:complex, HOPDA is in a planar conformation with the 2-hydroxy/oxo oxygen
distant from H265. The difference in conformation could be driven by the ability of H265 to
act as a base or its hydrogen bonding capacity of H265.

To resolve this issue, the present study investigates the interaction of HOPDA with BphDLB400
in the H265Q mutant, a variant that preserves hydrogen bonding while ablating the ability of
the residue to function as a base. Data from stopped-flow spectrophotometry and crystal
structures of mutants BphDLB400 H265Q, S112A/H265Q and their complexes with HOPDA will
be presented. Microspectrophotometry on single crystals of the complex of BphDLB400 S112A
with HOPDA, before and after X-ray diffraction data collection, are being performed to
correlate the crystal structures of enzyme:HOPDA complexes with the transient or trapped
species observed in solution.
S-089

Structural basis for substrate recognition by mouse N-acetylglucosaminyltransferase II

(GnT II)

1 1,2 1,3 4 1,2

Zhijie Li , Malathy Satkunarajah , Jenny Tan , Jayaraman Seetharaman , James Rini
1 2
Department of Biochemistry, University of Toronto, Toronto, ON, Canada, Department of
3
Molecular Genetics, University of Toronto, Toronto, ON, Canada, Division of Structural
4
Biology and Biochemistry, The Hospital for Sick Children, Toronto, ON, Canada, Biology
Department, Brookhaven National Laboratory, Upton, NY, United States

GnT II is a Golgi-resident glycosyltransferase in the N-glycan biosynthetic pathway of

multicelluar organisms. It recognizes the GnT I and mannosidase II processed N-glycan and
catalyzes an essential step in the biosynthesis of complex N-glycans. Here we report the X-
ray crystal structure of the soluble catalytic domain of mouse GnT II in both its apo form and
in complexes with its substrates/products: i) UDP-Mg2+; ii) UDP-GlcNAc-Mg2+; iii) UDP-
Mg2+ and GlcNAc-Man3-octyl and iv) GlcNAc-Man3-octyl alone. The enzyme is metal ion
dependent and as shown by the structure a member of the GT-A fold family. The
tetrasaccharide acceptor substrate, GlcNAc-Man3-octyl, is well-ordered and extensive
interactions with the β 1,2-linked GlcNAc moiety reveals the structural basis for GnT II's
requirement for the prior action of GnT I. Comparison with other GnT I dependent enzymes
shows that recognition of the β 1,2-linked GlcNAc does not stem from evolutionarily conserved
structural features. A flexible loop covering the nucleotide sugar binding site was ordered in
the ternary complex of GnT II with UDP and the acceptor sugar. Unlike some other
glycosyltransferases, this loop does not form a binding surface for the acceptor sugar.
However, a partial stacking of the donor and acceptor substrates, suggested by the
superimposition of the donor and acceptor complexes, may contribute to the observed Bi Bi
ordered sequential kinetics shown by GnT II.
S-092

Novel chemistry in the ureide catabolism pathway as revealed by structural and

functional analysis of enzymes from the Klebsiella pneumoniae Hpx gene cluster

Jarrod French, Katherine Hicks, Steven Ealick

Cornell University, Ithaca, New York, United States

The degradation of ureides into more soluble compounds occurs in many organisms as
diverse as mammals, plants and bacteria. In plants, some fungi and several bacteria the
catabolism of these molecules provide a source of nitrogen, carbon and energy. While much
of the biochemistry of this degradation has been worked out, there are still many questions to
be answered. Recent genetic studies on Klebsiella pneumoniae have uncovered a gene
cluster believed to contain all of the enzymes required for the breakdown of uric acid to
allantoin and those responsible for the further catabolism of allantoin to carbon dioxide and
ammonia. In this work, insights into the novel chemistry that occurs along this pathway are
provided by several crystal structures and supporting biochemical studies. Recent debate
over the mechanism of the decarboxylation of 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline
is discussed in the context of the 1.6 – 1.8 Å crystal structures of the enzyme, HpxQ, in
unliganded and product-bound forms. The 2.3 Å structure of HpxA, the first reported structure
of an allantoin racemase, sheds light on the mode of ligand binding and the two-base
mechanism of catalysis within the active site. Finally, the biochemical and structural
characterization of HpxJ, an enzyme that catalyzes a novel aminotransfer reaction, is
presented.
S-095

Human UDP-glucose Dehydrogenase and the Pin in Fischer’ s Lock

1 2 1 1 1
Renuka Kadirvelraj , Stephen Weitzel , Nicholas Sennett , Samuel Polizzi , Zachary Wood
1 2
University of Georgia, Athens, GA, United States, University of Oregon, Eugene, OR, United
States
+
Human UDP-glucose dehydrogenase (hUGDH) catalyzes the NAD -dependent oxidation of
UDP-glucose (UDP-Glc) to UDP-glucuronic acid, a key building block of the extra cellular
matrix. hUGDH is believed to be allosterically regulated by the feedback inhibitor UDP-xylose
(UDP-Xyl). The substrate UDP-Glc and inhibitor UDP-Xyl are isosteric except at the C5’
position; UDP-Glc has a hydroxymethyl substituent at C5’, while UDP-Xyl has a hydrogen.
We report two crystal structures of the hUGDH:UDP-Xyl complex and compare them to an
existing hUGDH:UDP-Glc structure. We show that both substrate and inhibitor UDP-sugars
bind in the hUGDH active site. When UDP-Glc binds, a buried, mobile loop in the active site
adopts a conformation that promotes three hUGDH dimers to oligomerize into a torus-shaped
+
hexamer. When UDP-Xyl binds, the buried loop repacks, blocking the NAD binding site and
altering the hUGDH oligomer interface. This results in an inactive, horseshoe-shaped
hexamer. Sedimentation velocity studies confirm that the binding of either UDP-Glc or UDP-
Xyl favors hexamer formation. Thus, the hUGDH active site is bifunctional in that it carries out
both catalysis and regulation. This is in contrast to classic, heterotropic allostery, where the
functional and regulatory sites are separate. We present evidence showing how this
bifunctional active site evolved from a non-allosteric, ancestral UGDH.
S-098

The Role of Structural Change in UGDH Regulation

Nicholas Sennett, Renuka Kadirvelraj, Zachary Wood

University of Georgia, Athens, GA, United States

+
Human UDP-glucose dehydrogenase (UGDH) catalyses the NAD -dependent oxidation of
UDP-glucose (UDP-Glc) to UDP-glucuronic acid. We show UGDH activity is linked to the
oligomeric state of the enzyme. In solution apo-UGDH favors a dimeric state but can also
oligomerize to form tetramers and hexamers. Binding of the substrate UDP-Glc causes a
buried loop to adopt a conformation that induces UGDH to form a torus-shaped hexamer. In
contrast, the feedback inhibitor, UDP-xylose (UDP-Xyl), shifts the buried loop to favor a stable
horseshoe-shaped hexamer. To see how the buried loop moves we designed a mutant in the
protein core. The crystal structure of the mutant shows that the domain containing the loop
opens with a clamshell-like motion, allowing the loop to repack. Crystal structure and
sedimentation velocity results confirm that the loop mutant forms the torus-shaped hexamer,
suggesting that domain opening and substrate exchange can occur without dissociation of the
oligomer. To determine if the hexamer state is required for activity we designed a mutation to
stabilize the dimer state. Sedimentation velocity results confirmed that the mutant is a dimer
and kinetics showed the mutant to be inactive. We propose that the loop of dimeric UGDH
freely moves between its two conformations and that substrate binding drives the dimer to the
active torus-shaped hexamer while inhibitor binding drives the dimer to the inactive
horseshoe-shaped hexamer.
S-220

Apo Form of Human FABP5 Solved at High Resolution in the Inactive Conformation

Eric Armstrong, Eric Ortlund

Emory University, Atlanta, GA, United States

The fatty acid binding protein (FABP) family includes nine known members, each ~14-15kDa
in size and found throughout the animal kingdom. Though they share relatively little sequence
homology, all form a twisted β -barrel, composed of 10 anti-parallel β -strands arranged into
two orthogonal β -sheets, with a helix-turn-helix lid near the N-terminus. Belonging to the
superfamily of intracellular lipid binding proteins (iLBP), they have traditionally been thought to
be mainly involved in the solubilization/protection of their various hydrophobic cargos,
facilitating ligand transport via passive diffusion across the various compartments of the cell.
However, research within the past decade has increasingly bestowed a newfound importance
upon the iLBPs as specific mediators of vital signaling pathways. FABP5, like its family
members, displays a rather promiscuous ligand binding profile, and has been shown to form a
complex with numerous long chain fatty acids as well as several synthetic small molecules.
Interestingly, only a subset of these have been demonstrated to serve as "activators," i.e., to
result in the protein's nuclear translocation from the cytoplasm in cellular assays. We
hypothesize that this differential response upon binding can be explained structurally via an
activator-unique conformational change in FABP5, leading to the formation of a tertiary
nuclear localization sequence. Surprisingly, incubation of the protein with non-activating
ligands facilitated the crystallization of a new apo form that has been solved at 1.67Å. To our
knowledge this is the first high resolution structure of an empty iLBP, which we believe will
provide a basis for understanding the molecular switch that triggers FABP5 nuclear import.
S-101

Structural Studies of Wolinella Succinogenes O-acetylhomoserine Sulfhydrylase

1 1 1,2 1,2
Timothy Tran , Andrew Torrelli , Kalyanaraman Krishnamoorthy , Tadhg Begley , Steve
1
Ealick
1 2
Cornell University, Ithaca, NY, United States, Texas A&M University, College Station, TX,
United States

O-acetylhomoserine sulfhydrylase (OAHS, EC 4.2.99.10) is a pyridoxal-5’-phosphate (PLP)

dependent sulfide-utilizing enzyme in L-cysteine and L-methionine biosynthetic pathways of
various enteric bacteria and fungi. As the name implies, OAHS catalyzes the conversion of O-
acetylhomoserine to homocysteine using hydrogen sulfide in a process known as direct
sulfhydration. However, the source of sulfur has not been identified and no structures of
OAHS have been reported.
Using molecular replacement, the crystal structure of Wolinella Succinogenes OAHS
(WsMetY) has been determined at 2.2 Å resolution. WsMetY crystallized in space group C2
with two monomers in the asymmetric unit. Size exclusion chromatography showed that the
biological unit exists as a tetramer. This observation is supported using crystallographic two-
fold symmetry to generate the tetramer, and confirmed by the free energy calculation from the
PISA server. The structure has been refined to a current R/Rfree of 23.0/25.7. Superposition of
homologous structures revealed that WsMetY has the same fold as cystathionine gamma
lyase and methionine gamma lyase. Their active sites, containing PLP, are also very similar
to one another. The structure of WsMetY, together with biochemical data, provide useful
insight to the mechanism of sulfur transfer.
S-104

Crystallographic studies on two nickel-alkyl species in methyl-coenzyme M reductase.

1 2 2 2 1
Peder E. Cedervall , Xianghui Li , Mishtu Dey , Stephen W. Ragsdale , Carrie M. Wilmot
1 2
University of Minnesota, Minneapolis, MN, United States, University of Michigan, Ann Arbor,
MI, United States

In methanogenic archaea, methyl-coenzyme M reductase (MCR) catalyzes the final and rate-
limiting step in methane biogenesis; the reduction of methyl-coenzyme M (methyl-SCoM) by
coenzyme B (CoBSH) to methane and a heterodisulfide (CoBS-SCoM). MCR is a 300 kDa
protein with six subunits arranged in a α 2β 2γ 2 oligomer. Crystallographic studies have shown
that the two active sites of MCR each contain a highly reduced nickel-tetrapyrrole, coenzyme
F430, that sits at the base of a 30 Å long substrate channel. No true catalytic intermediate for
MCR has ever been observed so the reaction mechanism remains illusive. Based on
mechanistic studies in solution, DFT calculations and previous X-ray crystal structures three
different mechanisms have been proposed. One of the proposed mechanisms involves a high
valent Ni(III)-alkyl intermediate. This species can artificially be produced by treating the
enzyme with either methyl iodide or bromopropanesulfonate, generating Ni(III)-methyl and
Ni(III)-propylsulfonate, respectively. Here we present the crystal structures of MCR in complex
with these two alkyl species. The resulting structures show a mixture of the expected alkyl
species and the substrate analogue HSCoM (demethylated methyl-SCoM), which co-purifies
with MCR and cannot be fully removed by extensive buffer exchange. By using multi-
wavelength X-ray diffraction studies we were able to differentiate the components via
anomalous electron density maps and allow structural analysis of the Ni-alkyl species.
S-107

Structural Insights into Thiopurine S-Methyltransferase Pharmacogenetics

1 2 1 2 2
Yi Peng , Qiping Feng , Dennis Wilk , Araba Adjei , Oreste Salavaggione , Richard
2 1
Weinshilboum , Vivien Yee
1 2
Case Western Reserve University, Cleveland, OH, United States, Mayo Clinic College of
Medicine-Mayo Clinic, Rochester, MN, United States

Thiopurine S-methyltransferase (TPMT) modulates the cytotoxic effects of thiopurine

prodrugs such as 6-mercaptopurine (6MP) by methylating them in a reaction using S-
adenosyl-L-methionine (AdoMet) as the donor. Patients with TPMT variant allozymes exhibit
diminished levels of protein and/or enzyme activity and are at risk for thiopurine drug-induced
toxicity. We have compared the catalytic activity of wild-type and two variant TPMTs, and
found that enzyme activities were decreased for both variants while expression levels were
substantially decreased for only one, compared to the wild-type enzyme. We have also
determined an ensemble of murine TPMT (mTPMT) crystals structures, as binary and ternary
complexes containing either wild-type or a variant TPMT. Comparison of the wild-type
structures reveals that an active site loop becomes ordered upon acceptor substrate binding.
The positions of the two ligands are consistent with the expected SN2 reaction mechanism.
Crystal structures and molecular dynamics (MD) simulation calculations for one variant reveal
conformational differences in the active site loop that could explain compromised acceptor
substrate binding and reduced enzyme activity. Computational modeling and MD simulation
calculations for the second variant are consistent with the observed decrease in both enzyme
expression and activity. Together, the crystallographic and computational structural studies
provide a detailed view of the molecular contributions in a classic example of
pharmacogenetics.
S-111

Structural insight into the allosteric inhibition of aspartate kinase from

Corynebacterium glutamicum

Ayako Yoshida, Takeo Tomita, Tomohisa Kuzuyama, Makoto Nishiyama

Biotechnology Research Center, The University of Tokyo, Tokyo, Japan

Aspartate kinase (AK) is the enzyme that catalyzes the first committed step of the
biosynthesis of aspartate family amino acids; lysine, threonine, and methionine. AK is known
to be regulated by the end products via feedback inhibition as seen in other enzymes at the
first step in amino acid biosynthetic pathway. AK from Corynebacterium glutamicum, a
bacterium used for industrial fermentation of amino acids including glutamate and lysine, is
inhibited by lysine and threonine in a concerted manner. AK from C. glutamicum (CgAK) also
has a characteristic 2 2-type heterooligomeric quaternary structure. The 2 2-type structure
is composed of two subunits and two subunits, which are encoded by in-frame
overlapping gene. To elucidate the unique regulatory mechanism and quaternary structure,
we determined the crystal structures of CgAK in several forms; an inhibitory T-state form
complex with both lysine and threonine, an active R-state form complex with only threonine,
and feedback inhibition-resistant mutant complex with both lysine and threonine.

We previously showed that threonine binding stabilizes an interaction between subunit and
the regulatory domain of the subunit, which is essential for catalytic regulation, by the
crystal structure determination of regulatory domain ( subunit) dimer with threonine and
some mutational experiments. In T-state, we showed the allosteric binding sites of both
inhibitors, and comparison of the crystal structures between T and R-state revealed that
lysine binding causes a conformational change to a closed inhibitory form, and the interaction
between the catalytic domain in subunit and subunit (regulatory subunit) is a key event for
stabilizing the inhibitory form. We propose that the regulatory mechanism of CgAK is
composed of two steps, i) the interaction between regulatory domain (subunit) triggered by
threonine-binding, ii) the conformational change at the C-terminus of subunit to interact
between catalytic and regulatory domain provoked by lysine-binding. This study shows not
only the first crystal structures of 2 2-type AK but also the mechanism of concerted inhibition.
Moreover, since AK is a candidate of antibacterial drug because of its absence in humans,
this study will lead to the development of novel antibacterial drugs.
S-114

Introduction of a disulfide bond leads to stabilization and crystallization of a ricin

immunogen
1 1 3 1
Jaimee R. Compton , Patricia M. Legler , Mark A. Olson , Benjamin V. Clingan , Charles B.
2
Millard
1 2
Walter Reed Army Institute of Research, Silver Spring, MD, United States, U.S. Army
3
Medical Research & Materiel Command, Frederick, MD, United States, U.S. Army Medical
Research Institute of Infectious Diseases, Frederick, MD, United States

RTA1-33/44-198 is a catalytically inactive, single-domain derivative of the ricin toxin A-chain

(RTA) engineered to serve as a stable protein scaffold for presentation of native immunogenic
epitopes. To improve the stability and solubility of RTA1-33/44-198, we have undertaken the
design challenge of introducing a disulfide (SS) bond. Nine residue-pairs for placement of the
SS-bond were selected based on molecular dynamics simulation studies of the modeled
single-domain chain. Disulfide formation at either of two positions involving a specific surface
loop (44-55) increased the protein melting temperature by ~5°C compared with RTA1-33/44-
198 and by ~13°C compared with RTA. Prolonged stabil ity studies of the R48C/T77C variant
confirmed a >40% reduction in self-aggregation compared with RTA1-33/44-198 lacking the
SS-bond. Introduction of the disulfide bonds promoted crystallization, and the X-ray
structures of the variants were solved. The structures confirm formation of a SS-bond and a
single-domain structure that is globally compacted in volume compared with RTA. Loop 44-
55 is partly disordered as predicted by simulations and is positioned to form self-self
interactions between symmetry-related molecules. We discuss the importance of loop 34-55
as a nucleus for unfolding and aggregation and draw conclusions for ongoing structure-based
minimalist design of the RTA immunogen. This work was funded by the U.S. Defense
Threat Reduction Agency JSTO award S.S.0003_06_WR_B (CBM) and National
Institutes of Health U01 A1082120-01 (CBM). The opinions or assertions contained
herein belong to the authors and are not necessarily the official views of the U.S. Army
or the U.S. Department of Defense.
S-117

Mechanism for allosteric regulation of glutamate dehydrogenase from Thermus

thermophilus

Takeo Tomita, Tomohisa Kuzuyama, Makoto Nishiyama

Biotechnology Research Center, The University of Tokyo, Tokyo, Japan

Glutamate dehydrogenase (GDH) catalyzes the reversible conversion between glutamate and
2-oxoglutarate using NAD(P)(H) as coenzymes. Due to the important role in balancing
nitrogen metabolism in cells, GDH is widely distributed among living organisms. An extremely
thermophilic bacterium, Thermus thermophilus, possesses two glutamate dehydrogenase
genes, gdhA and gdhB, in a tandem manner on its genome. To elucidate the functions of
these genes, the gene products were expressed, purified, and characterized. GdhA showed
no GDH activity, while GdhB showed GDH activity for reductive amination activity 1.3-fold
higher than for oxidative deamination. When GdhA was expressed with his-tag fused GdhB,
GdhA was co-purified with the his-tagged GdhB. The co-purified GdhA/GdhB had decreased
reductive amination activity and increasing oxidative deamination activity, resulting in 3.1-fold
preference of oxidative deamination to reductive amination. These results demonstrate that
GdhB undergoes conformational change through hetero-complex formation with GdhA.
Addition of leucine elevated the GDH activity of co-purified GdhA/GdhB hetero-complex by
974 and 245% for reductive amination and oxidative deamination, respectively, while GdhB
alone did not show such a strong activation by leucine. These results suggest that the
allosteric activation by leucine occurs through formation of hetero-complex, where GdhA and
GdhB act as a regulatory subunit and as a catalytic subunit, respectively. To elucidate the
allosteric mechanism of GdhA/GdhB, we determined the crystal structures of GdhA and GdhB
complexed with glutamate at 2.2 and 2.1 Å resolution, respectively. GdhA consists of catalytic
domain and nucleotide binding domain (NBD) and takes a homotetrameric structure with
novel subunit interfaces between its NBDs, while small type GDHs including GdhA and GdhB
are known to have homohexameric structure. GdhB takes a homohexameric structure and
glutamate are bound in the active sites and the subunit interface position far from active sites.
Glu in the active sites are recognized by several electrostatic interactions and hydrogen
bonds similar with the other GDH/Glu complexes. Glu in the subunit interface are recognized
by the residues from three distinct subunits. This observation raises the hypothesis that the
second Glu site may function allosteric effector binding site for recognition of leucine.
S-120

Protein-protein complexes of the E. coli phenylacetic acid utilization pathway

1 1 2 2 2
Andrey Grishin , Eunice Ajamian , Limei Tao , Linhua Zhang , Allan Matte , Miroslaw
1,2
Cygler
1 2
Department of Biochemistry, McGill University, Montreal QC, Canada, Biotechnology
Research Institute, National Research Council, Montreal QC, Canada

The metabolic pathway of phenylacetic acid utilization in E. coli K12 is poorly characterized
both biochemically and structurally. This pathway is important as it represents an aerobic
route for the utilization of phenylacetic acid as a coenzyme A derivative. Of the five
consecutive reactions utilized to metabolize phenylacetic acid, three are presumed to involve
enzyme complexes. The oxygenase reaction of this pathway is catalyzed by PaaA-E. We
performed a search for stable protein-protein complexes involved in the oxygenase reaction
of phenylacetic acid metabolism by co-expression of various combinations of pathway
enzymes (PaaA-B-C-D-E) followed by co-purification experiments. These studies revealed
that PaaA-B-C and PaaA-C could be purified as complexes, although no strong interaction
was found between either PaaE or PaaD with PaaA-B-C. The presence of functional protein
complexes was verified by detecting reaction products using LC-MS. Our studies show that
PaaA, PaaB, PaaC and PaaE, but not PaaD, are indispensable for activity in vitro. The crystal
structure of PaaA-C was determined as well as its complexes with coenzyme A, 3-
hydroxybutyrl-coenzyme A, benzoyl-coenzyme A and the true substrate, phenylacetyl-
coenzyme A. Despite low sequence identity, PaaA and PaaC are structurally similar to
methane monooxygenase and to other di-Fe monoxygenases. This represents the first
structure of a multi-component monoxygenase that utilizes a CoA-derivative of an aromatic
compound as substrate. The search for additional protein-protein interactions for other
components of this pathway, as well as their biochemical and structural characterization, is in
progress. Supported by CIHR.
S-123

Structural analysis of Bacillus phytase in complex with phytate and metal ions

Yi-Fang Zeng, Rey-Ting Guo, Je-Ruei Liu

Institute Of Biotechnology, National Taiwan University, Taipei, Taiwan

The hydrolysis of phosphomonoesters in biological systems is an important process.

Phytase, which hydrolyzes phytate to less phosphorylated myo-inositol derivatives and
inorganic phosphate, is widespread in nature. Bacillus phytases, which exhibit their desirable
activity profile under neutral pH, higher thermal stability, and strict substrate specificity for the
calcium–phytate complex, have considerable potential for commercial and environmental
applications. Thus, we are interested to know how the substrate binding domain of Bacillus
subtilis phytase PhyC attracts the substrate and metal ion involvement.

Here, we have determined the high resolution X-ray structures of the Bacillus subtilis phytase
PhyC in complex with phytate analog in the presence of 5 mM CaCl2. In this structure, the
phytate analog was bound the catalytic site of beta-propeller phytase. In addition, we
2+ 2+
determined the enzyme activity in both Ca and Cd loaded state from the structure, and
2+ 2+
found the Cd competed against Ca in the metal ion binding position. Thus, the phytase
2+
activity of PhyC was greatly inhibited by metal ion Cd . These findings provided the evidence
2+
for the binding interaction between the enzyme and substrate in the present of Ca and/or
2+
Cd metal ions.
S-126

The crystal structure of Thermotoga maritima CelA

Ya-San Cheng, Je-Ruei Liu, Rey-Ting Guo

Institute of Biotechnology, National Taiwan University, Taipei, Taiwan

Plant matter is the most abundant renewable biomass on earth and cellulose is the major
component of plant cell wall. Cellulose is a kind of polysaccharide consisting of glucose linked
together via  -1,4-glycosidic bond. Thermotoga maritima is an anaerobic hyperthermophilic
bacterium and it can produce some thermostable carbohydrate-degrading enzymes which
have potential industrial applications. CelA from T. maritima is a thermostable -1,4-
endoglucanase which is classified into glycoside hydrolase family 12. The three-dimensional
structure of TmCelA has not been solved yet. Therefore, we are interested in knowing the
TmCelA protein structure and the catalytic mechanism by solving the X-ray structure. We
already purified the TmCelA protein and obtained the well diffracted protein crystals. The
phase problem was solved by using some heavy atom derivatives recently and the structure
refinement is ongoing now. Once we obtained the TmCelA protein structure, we can have
more understanding of the catalytic mechanism. We may also have more idea of knowing
CelA protein properties such as hyperthermostability by analyzing TmCelA protein structure.
S-129

Structural analysis of Piromyces rhizinflata 2301 CelAcd

Chih-Wen Tseng, Rey-Ting Guo, Je-Ruei Liu

Institute of Biotechnology, National Taiwan University, Taipei, Taiwan

The enzymatic degradation of the plant cell wall is environmentally friendly routes to biomass
conversion, including the production of biofuels. Cellulase program is the most widely used
scheme in green energy developments. The Piromyces rhizinflata CelA2, a bifunctional endo-
and exoglucanase, belong to the glycosyl hydrolase family 5 and showed hydrolysis activity
toward barley β -glucan、 lichenin、 oat spelt xylan, and carboxymethyl cellulase（ CMC） . The
three dimensional structure of the catalytic domain of CelA has not been solved yet. Here we
conduct a study to determine the structure of CelA by solving its X-ray structure. So far, the
recombinant CelA was purified to homogeneity by immobilized metal ion-affinity
chromatography and DEAE-Sepharose ion exchange column. The molecular weight of the
purified CelA was estimated 49 kDa on SDS-polyacrylamide gel electrophoresis. Afterward,
the catalytic mechanism and detail binding interactions between enzyme and substrates will
be studied by solving the protein structure.
S-132
Structure of RedJ: A Thioesterase from the Prodiginine Biosynthetic Pathway
1 2 2 1
Jonathan R. Whicher , Galina Florova , Kevin A. Reynolds , Janet L. Smith
1 2
University of Michigan, Ann Arbor, MI, United States, Portland State University, Portland,
OR, United States
Microbial polyketide synthase (PKS) and non-ribosomal peptide synthase (NRPS)
pathways synthesize small-molecule natural products from acylCoA and amino acid
precursors, respectively. Modular PKS and NRPS are arranged in an assembly line fashion,
with each enzyme catalyzing a specific elongation or modification of an intermediate in the
biosynthetic pathway to the natural product. The natural products are secreted by the
microbes and have multiple biological activities, which are thought to provide the producing
organism with a competitive advantage in its environment. Most natural products are
cytotoxic, and as a result, they have been investigated as possible therapies for human
diseases. Thus the study of the enzymes involved in the PKS and NRPS systems has
become an important area of research in hopes of engineering these pathways to produce
novel therapeutics. Prodiginines are a class of tripyrrole small- molecule natural products
produced by hybrid PKS/NRPS systems. Analogues of prodiginines have
immunosuppressant and anticancer activity. As a result, the prodiginine biosynthetic pathway
in Streptomyces coelicolor has been extensively studied and most enzymes within this
pathway have been assigned functions. However, one enzyme, RedJ, has an unknown
function. Biochemical, bioinformatic and genetic evidence indicate that RedJ is a thioesterase
with a novel role in facilitating transfer of a 12-carbon intermediate from one enzyme to
another. The 2.12-Å crystal structure of RedJ presented here suggests a structural basis for
RedJ specificity for long-chain aliphatic substrates. In addition, distinct conformations of an
active-site “lid” region in different crystal forms provide insights into the mechanism by which
RedJ regulates access of substrates to its active site.

JRW is supported by an NIH Chemistry Biology Interface Training Grant.

Research supported by NIH grant DK42303 to JLS.

S-135

Crystal structure of Plasmepsin I (PM-I) from Plasmodium falciparum

1 2 2 1 3
Prasenjit Bhaumik , Yasumi Horimoto , Huogen Xiao , Alexander Wlodawer , Yoshiaki Kiso ,
1
Gustchina Alla
1
Protein Structure Section, Macromolecular Crystallography Laboratory, National Cancer
2
Institute at Frederick, Frederick, Maryland, United States, University of Guelph, Guelph,
3
Ontario, Canada, Kyoto Pharmaceutical University, Kyoto, Japan

Malaria is contributing to death of nearly two million people every year, most of them children.
Although several antimalarial drugs are available, rapid development of resistance to the
currently available treatments makes it necessary to discover and develop a new generation
of therapeutics. Plasmodium falciparum, the parasite that causes the deadliest form of
malaria, consumes large amounts of hemoglobin from the blood cells of the human host to
generate amino acids for its growth and maturation. Hemoglobin is degraded by several
aspartic proteases (plasmepsins) present in the acidic digestive food vacuole of the parasite.
Plasmepsin I (PM-I) is one of the enzymes directly involved in hemoglobin degradation, thus it
is considered a promising target for new antimalarial drugs. We have crystallized the
recombinant PM-I complexed with a potent inhibitor of several plasmepsins, KNI-10006.
Crystal structure of the complex was determined with data extending to the resolution of 3.1
Å, with R-factor and R-free of 21.1% and 29.9%, respectively. The PM-I-KNI-10006 complex
crystallized in the tetragonal space group P43 with four molecules in the asymmetric unit,
related by non-crystallographic symmetry. The structure elucidates the unique binding mode
of KNI-10006 in the PM-I active site, with the central hydroxyl group of the inhibitor positioned
between two catalytic aspartates, Asp32 and Asp215. Analysis of the PM-I-KNI-10006
complex and its comparison with the structures of other plasmepsins will help to elucidate the
inhibition mechanism of KNI-10006, and also should guide future design of specific inhibitors
that could be developed into antimalarial drugs.
S-138

An X-ray Crystallographic Investigation of Nitric Oxide Binding to Beef Liver catalase.

1 2 2 1
Namrta Purwar , Jennifer McGarry , A. Andrew Pacheco , Marius Schmidt
1 2
UW milwaukee, Department of Physics, Milwaukee, WI, United States, UW-Milwaukee,
Departmen of chemistry and Biochemistry, Milwaukee, WI, United States

The function of catalase in the elimination of H2O2 from living aerobic organisms has drawn
the interest of scientists for a long time. The enormous enzymatic activity makes this enzyme
very efficient. Catalase catalyzes the heterolytic decomposition of H2O2 into non-toxic water
and oxygen, thus avoiding the formation of highly reactive and toxic radicals from homolytic
H2O2 decomposition. Herein we report on the interaction of NO with Beef liver Catalase
(BLC). NO mimics H2O2 binding at the active site, but does not undergo further reaction to
compound I. Using X-ray crystallography on BLC crystals, we show how NO binds to the
heme iron of the catalase. X-ray data of three BLC species were collected at BioCARS 14-
BMC, Advanced Photon Source, Argonne IL. Initially, single crystals of BLC were grown in
th
the presence of trace NH4OH, and had an NH3 ligand bound at the 6 coordination site of the
heme-iron (occupancy 100%). After soaking the crystals in NH3 free buffer, a second species
th
was characterized, which had no electron density at the 6 coordination site. To investigate
NO binding, crystals were soaked in 100mM of 1-(N,N-diethylamino)diazen-1-ium-1,2-diolate
(DEANO), which releases controlled amounts of NO when dissolved in neutral or acidic
solutions. Crystals soaked with DEANO showed about 60% NO occupancy at the iron binding
site. An NO molecule fit to the electron density displays a Fe-N-O angle that is significantly
3+
bent away from the normal of the heme plane. Typically, NO bound to (ferric) Fe displays
2+
linear or nearly linear Fe-N-O angles, whereas NO bound to (ferrous) Fe shows substantial
bending. In catalase the iron is in the ferric Fe(III) state. Therefore the large bending angle is
a remarkable result. Two possible explanations are: (i) the proximal ligand of the heme in
catalase is Tyr 357, whose deprotonated hydroxyl group might donate substantial electron
density to the iron, so that it resembles more an Fe(II); (ii) by exposing the crystals to X-rays
during data collection, photo-electrons are generated that might reduce the Fe(III) to Fe(II).
S-141

Crystal Design Based on Charge-assisted Hydrogen Bonds

Yuzhou Liu, Michael Ward

New York University, New York, United States

Inclusion compounds comprise host frameworks, assembled through non-covalent

interactions, and “guest” molecules. A particularly attractive feature of inclusion compounds is
the ability to incorporate functional guests within a host framework with controllable
architecture. Whereas most crystal engineering strategies rely on structural control and
function combined in the same molecular building block, inclusion compounds allow
separation of structure and function, permitting improved control of material properties. One
challenge that frequently appears is the ability to manipulate and control the host architecture
while varying guest molecules.

We have reported inclusion compounds based on hydrogen bonded frameworks comprising

+ +
the guanidinium cation (G =C(NH2)3 ) and organosulfonate anions including
- - -
organomonosulfonate (MS=R-SO3 )and organodisulfonate (DS= O3S-R-SO3 ; R=alkyl, arene).
The three-fold symmetry of the ions enable self-assembly, through charge-assisted (N-H· · · O)
hydrogen bonds, into infinite 2-D quasi-hexagonal guanidinium sulfonate (GS) sheet
decorated with organic groups that project from the surface of the sheet. This GS sheet can
be described as an assembly of GS ribbons connected by hydrogen bonds acting as “hinges”.
This feature enables the GS network to pucker like an accordion so that its inclusion
compound can achieve dense packing. The distance between adjacent sheets is primarily
dictated by the dimension of the R groups.

Most of our work has the similarity that different sulfonate nodes in one GS sheet belongs to
different sulfonate molecules, and the inclusion cavities are formed between the organic parts
of different sulfonate molecules. Intra-connecting sulfonate nodes in the GS sheet, which can
be achieved by using multi-sulfonates with suitable sulfonate spacing for GS sheet formation,
will introduce the capsule structures. This will lead to a way to systematically construct
capsule structures from interchangeable molecular modules with predicated crystal
structures. The impulse to construct capsule structures comes from their interesting
application as molecular containers. Therefore constructing molecular capsules using GS
system will not only advance the ability in controlling crystal packing motifs three
dimensionally, but also benefit the development of functional materials which always suffers
from the poor control over the solid state structures.
S-144

The Crystal Structures of 4,4’ -(methylenediimino)bis-1,2,5-oxadiazole-3-carboxylic acid

and carboxamide
1 1 2
Mark Frisch , Jeffrey Deschamps , Rodney Willard
1
The Laboratory for the Structure of Matter, Naval Research Laboratory, Washington, DC,
2
United States, School of Polymers and High Performance Materials, University of Southern
Mississippi, Hattiesburg, MS, United States

The needs of the US defense for advanced energetics have been evolving over the past
several years. Energetic materials, compounds which under certain stimuli will release large
amounts of energy, are essential ingredients in explosives and rocket propellants. An
important property to take into account when designing organic compounds for use as
energetic materials is the density; density is directly related to performance. This program to
produce densely packed organic compounds suitable for use as energetic materials led to the
synthesis of two compounds derived from amino-1,2,5-oxadiazole. The bis-carboxamide and
bis-carboxylic acid analogues were characterized by single crystal X-ray diffraction using
MoK. Both of these compounds crystallize in a monoclinic space group however the bis-
3
carboxamide is calculated to have a higher density (1.800 vs. 1.623 Mg/m ). Presented
herein will be a comparison of the two compounds along with a detailed crystallographic
description.
S-147

Characterization of a Novel Pathway Essential For Survival In Macrophages Across

Distally Related Pathogens

Rodrigo Torres, Benson Lan, Celia Goulding

Unviversity of California Irvine, Irvine, CA, United States

The ability of pathogens to evade our immune defenses has been well documented and is the
cause of many diseases today. One subset has the ability survive in macrophages and
includes Yersinia pestis, the causative agent of plague, and Salmonella enterica, the cause of
food poisoning. These pathogens are currently treated with various antibiotics, but further
developments are needed to identify new targets for treatment, due to the advent of drug
resistant strains and the possibility of reemergence of potent pathogens such as Y. pestis, as
a possible source of bioterrorism. Of note, a novel rip (required for intracellular proliferation)
operon has been specifically shown to be involved in Y. pestis and S. enterica survival when
endocytosed into activated macrophages. This rip operon is conserved among a distally-
related subset of macrophage-residing pathogens, including Burkholderia mallei, suggesting
that this uncharacterized pathway involving the Rip proteins (RipA, RipB, RipC) is required for
their survival. Thus, we purpose building a structural understanding of these

three proteins, followed by testing a functional hypotheses about their substrates, products
and interaction partners, in order to shed light on their involvement in pathogenicity. To that
end, preliminary structures have been obtained for RipA to 1.9A and RipC to 2.6 A, with RipB
currently under crystallization trials. In addition, initial activity assays for RipA suggest CoA
transferase activity, the proposed function based on structural homology searches.
S-150

X-ray powder diffraction residues characterization to avoid water contamination in oil

fields.
1 1 1 2
Maria Lara , Samantha Rendon , Monty Rendon , Luis Rendon
1 2
marudecori consultants, cuernavaca, Mexico, Mexican Institute of Water Technology,
jiutepec, Mexico

When petrochemical equipment is subject to maintenance it is common to

obtain residues from inside natural gas containers, these residues most of the
cases are non hazardous materials, nonetheless it is always a good practice
to make a complete characterization of them.

X-rays powder diffraction characterization is a excellent method to avoid

water contamination in oil fields facilities; water is a subject of environmental
interest. The use of X-rays powder diffraction characterization as a tool to
avoid its contamination is growing continually, as an alternative to a regular
and tedious chemical analysis.

Petrochemical industry is a dormant risk due to the formation of chlorinated

organic compounds or any other kind
of hazardous compound which are
considered to be toxic or carcinogenic.
Prevention appears to be a more
convenient method in comparison with
any water treatment of industrial
residual water containing waste from
processes sources such as oil
industry, textiles, agriculture, paper
and pulp; that leave residues that are
fairly easy to remove.

We are presently experimenting a great deal of success with preventing the contamination
process and avoiding the removal of organic residues in water.
HS-004

HFE: An Iron Uptake Regulation Molecule

Justin Fu, Yuhan Chen, Bryan Dongre, Shariq Moore, Nick Nabar, Vick Nabar, Nikil Prasad,
Joshua Speagle, Sai Vangala, Louise Thompson
1 2
Brookfield Central High School, Brookfield, WI, United States, Blood Center of Wisconsin,
Milwaukee, WI, United States

Accumulation of excess iron results in a common hereditary disease, Hereditary

Haemochromatosis (HH). There are various genetic mutations that lead to different forms of
the disease. HH-I is a form of this disease in which iron accumulates in hepatocytes and
intestinal epithelial cells and is associated with a mutation in the HFE (high iron protein) gene.
The Brookfield Central SMART Team (Students Modeling A Research Topic) developed a
model of HFE using 3D printing technology. The HFE gene encodes for a non-classical MHC
class I protein. In physiological conditions, HFE is expressed and translocated to the cell
surface where it may interact with a transferrin receptor (Tfr). The binding of the 1/2 domains
of HFE to the Tfr allows for controlled release of iron bound to transferrin-transferrin receptor
complex. A mutation (845G>A) causes the replacement of a cysteine with a tyrosine (C282Y).
This replacement prevents the 3 subunit of HFE from folding properly and from interacting
with 2 microglobulin, abrogating the translocation of the HFE-microglobulin complex to the cell
membrane and promoting its rapid degradation. This defect hinders the regulatory capability
of HFE. Current treatments include phlebotomy to prevent organ damage from accumulated
iron. Further study to increase understanding of the regulatory mechanism may lead to
improved treatment design. Supported by a grant from NIH-NCRR-SEPA.
HS-003

SMART Teams: Using 3D Rapid Prototyping Technology to Model DNA Replication

Mechanisms and Engage High School Students in the Scientific Process.

1 1 1 2 2
Susan Huang , Aimee Marceau , Nicholas P. George , Muhammad Cheema , Zoe Havlena ,
2 1 2 2 2
Amy Hua , Basudeb Bhattachryya , Dylan Meacham , Jade Moon , Connie Wang , David L.
1 1
Nelson , James L. Keck
1 2
Madison West Senior High School, Madison, WI, United States, Univ. of Wisconsin,
Madison, WI, United States

DNA replication is a vital process in all organisms and understanding the fundamental
biochemical interactions that drive replication is essential. Single-stranded DNA-binding
(SSB) proteins form an important component of the replication machinery that facilitates the
transfer of RNA primers from the enzyme primase to the replicative polymerase. This activity
occurs throughout lagging-strand DNA replication. The crystal structure of the E. coli has
modeled this interaction using 3D Rapid Prototyping Technology to gain insights into the
physical interactions that drive DNA replication. The SMART team program allows students to
experience the scientific process beyond the textbook by investigating the experimental
methodology of structural biology and takes students out of the classroom and into the
laboratory. Supported by grants from the Howard Hughes Medical Institute and NIH-NCRR-
SEPA.
HS002

Lighting Up Science: Firefly Luciferase.

Elana Baltrusaitis, Allyson Bigelow, Rachel Brielmaier, Pamela Burbach, Jake Dowler, Johnny
Fuller, Caroline Hildebrand, Teagan Jessup, Molly Jordan, Josh Kramer, Jenna Lieungh, Alex
Mikhailov, Pat O’Grady, Andrew Pelto, Quin Rowen, Rachelle Schmude, Bobby Schultz, Katherine
Seubert, Alex Sherman, Parker Sniatynski, Alex Venuti, Erin Verdeyen, Molly Wetzel, Donna
LaFlamme.

St. Dominic School, Brookfield, WI, Medical College of Wisconsin, Milwaukee, WI.

Luciferase is the generic name for an enzyme responsible for bioluminescence reactions and
is commonly associated with fireflies. It is also found in many other organisms including
bacteria, fungi, anemones, and dinoflagellates. Since the gene for the North American firefly
(Photinus pyralis) luciferase was cloned in 1985, scientists have been genetically engineering
the gene into living cells. The luciferase reaction is now widely used in scientific research to
study protein production in cells, to analyze gene promoter activity, to study stem cell function
in vivo, and in cancer studies, to trace the metastasis of cancer cells in living test animals.
The scientific study of the luciferase enzymes themselves is also continuing. In recent
research, single amino acid mutations to the active site cause the emission of different
colored light in a predictable way. The uses of and improvements in bioluminescent imaging
are increasing exponentially in cell biology, molecular biology, and in medical research. The
St. Dominic SMART Team (Students Modeling A Research Topic) developed a model of
luciferase using 3D printing technology.
S-163
Isovaleryl-CoA Dehydrogenase: Dehydrogenate This!
Nick Grabon, Beth Bougie, Matt Cira, Colin Erovick, Anne Fahey, Kelsey Jeletz, Elanore Kukla, Matt
Murphhy, Tim Rohman, Alyssa Sass, Laura Tiffany, Sam Wolff, Karen Tiffany, Jung-Ja Kim.

Cedarburg High School, Cedarburg, WI, Medical College of Wisconain, Milwaukee, WI.

Although rare, isovaleric acidemia (IVA) is a potentially fatal metabolic disorder that affects
one in every 250,000 people in the US. IVA results from lack of an enzyme, isovaleryl-CoA
dehydrogenase (IVD), involved in the breakdown of leucine. Without this enzyme, leucine
catabolism stops and organic acids accumulate within the body, causing symptoms of IVA,
including vomiting, diarrhea, and fatigue. IVD belongs to a family of related enzymes called
acyl-CoA dehydrogenases. IVD catalyzes the dehydrogenation, or removal of a pair of
hydrogen atoms, of a small, branched-chain substrate, isovaleryl-CoA, during the third step of
leucine catabolism. Glutamate 254 of IVD removes one hydrogen as a proton from the
substrate, and flavin adenine dinucleotide, FAD, a cofactor of the enzyme, takes away the
other hydrogen from the substrate. The three-dimensional structure of IVD, as determined
through X-ray diffraction, illustrates how a small-branched chain substrate is able to fit into the
active site of this enzyme and enables further investigation of how mutation of the IVD gene
could affect IVD function, thus resulting in IVA. To further understand the structural impact on
substrate specificity, a physical model of IVD has been designed and built by the Cedarburg
High School SMART (Students Modeling a Research Topic) Team using 3D printing
technology. Supported by a grant from NIH-NCRR-SEPA.
S-166

Crystal Structures of Flt3 and cFMS in Complex with Inhibitors

Hu Pan, May Lin, Kari Callaway, Terence Hui, Weimei Xing, Shendong Yuan, Jeanne Baker,
John Anderson, Ying-Zi Xu, Hing Sham, Frederique Bard, Brian Wipke, Rick Artis, Nanhua
Yao

Elan Pharmaceuticals, S. San Francisco, CA, United States

FLT3 and cFMS are type III receptor tyrosine kinases and play important roles in innate
immunity, cancer, and inflammatory diseases. As part of a structure-based drug discovery
project, we have determined a number of crystal structures of the kinase domain of Flt3 and
cFMS in complex with inhibitors of different chemo types, respectively. These structures
revealed the inhibitory mechanism of the JM domain. The different sequences and
conformations of JM domains adopted by Flt3 and cFMS in crystal structures imply
conformational flexibility in this region, which could be exploited for developing more selective
inhibitors. Detailed structural analysis of these co-crystal structures provides great insights
into the binding modes and selectivity of the inhibitors among the members of type III RTK
family and guides the design of novel inhibitors targeting autoimmune diseases.
S-170

Combining 3D Structures and Sequences to Assign Substrates and Functions of 16,000

Enzymes.
1 1 1 1
Robert Huether , Jimmitti Teysir , David Dziak , Courtney McEachon , Dana
1 1,2
Hogan , William L. Duax
1
Hauptman Woodward Medical Research Inst., Buffalo, NY, United States,
2
State Univ. of New York at Buffalo, Buffalo, NY, United States

We have been able to align 16,000 short chain oxidoreductase enzymes that have the Rossman
fold recognition element TGxxxGxG and the catalytic hexad (N)SYKP(T) (acronym TGYK). The
alignment is sufficiently accurate that we can separate gram-positive from gram-negative bacteria
and isolates all members of most bacterial classes, orders, families and genus. We can correlate
variation in 2 positions that determine cofactor recognition with 5 residues that define at least 100
known or potential substrates with additional residues that determine the details of specific
oligomeric aggregation. On the basis of amino acids in three positions in the sequence, we can
separate the two largest TGYK subfamilies, the 1800 member β -keto acyl carrier protein
reductase family present in all bacteria and the acetoacetyl CoA reductase family that is present
only in α , β and γ proteobacteria. We achieve accurate alignment by locating a few residues
(primarily Gly, Pro, Ala and Arg residues, GARP) that are fully conserved in all 16,000 members
of the family and by determining precisely the location and minimum size of indels required to
align all members of family. The GARP residues are critical to the alignment because of their
stereochemical properties. Glycines, having positive phi values that were embedded early in
folded proteins, are conserved throughout the evolution of those proteins families. These results
support conclusions based upon analysis of multiple open reading frames and codon bias in
actinobacteria and proteobacteria that some species in these phylums evolved at a time when
the defined genetic code was composed of only triples that end in G and C. Support in part by: Mr
Roy Carver, Stafford Graduate Fellowship, Caerus Forum Fund and The East Hill Foundation.
S-172

Disrupting Quorum Sensing: Two Mechanisms Underlying Antagonist Function

1 1 1 1 1,2
Guozhou Chen , Lee Swem , Danielle Swem , Philip Jeffrey , Bonnie Bassler , Fred
1
Hughson
1 2
Princeton Univ, Princeton, NJ, United States, HHMI, Chevy Chase, MD, United States

Quorum sensing bacteria communicate via small molecules called autoinducers to coordinate
collective behaviors. Gram-negative bacteria employ acylated homoserine lactones (AHLs)
as autoinducers. AHLs enter cells and bind dimeric LuxR-type transcription factors, which
subsequently regulate quorum-sensing target genes. Membrane-permeable quorum-sensing
antagonists that prevent population-wide expression of virulence genes offer a potential route
to novel antibacterial therapeutics. Here, we report structure-function analyses of a LuxR-
type protein called CviR from Chromobacterium violacein. We find that two CviR antagonists
function by distinct mechanisms: one by blocking RNA polymerase engagement, the other by
preventing operator DNA binding. In the former case, RNA polymerase binding is blocked by
the relocation of only two non-hydrogen atoms in the LuxR-type receptor. In the latter case,
the bound antagonist acts by stabilizing a domain-swapped configuration in which the
receptor’s DNA-binding helices are held apart by ~60 Å, double the ~30 Å separation required
for operator binding.
S-174

Crystal Structure of RHCC Interacting with the Anti-cancer drug (Cis-platin) and its
Potential as Novel Chemotherapeutic Delivery System in cancer

1 2 1 1
Efehi Ogbomo , Suat Ozbek , Sabine Hombach-Klonisch , Thatchawan Thanasupawat ,
1 1 1
Jerry Krcek , Thomas Klonisch , Joerg Stetefeld
1 2
University of Manitoba, Winnipeg, Manitoba, Canada, Heidelberg Institute of Zoology,
Heidelberg, Germany

Right handed coiled‐ coil (RHCC) is a 24 kDa tetrameric protein that originates from the
archaebacterium Staphylothermus marinus. S. marinus is an extremophile capable of
surviving wide ranges of temperature, salt, pressure and pH. The crystal structure of RHCC
reveals a new structural motif with four large cavities inside the tetrameric channel. The
3
cavities vary in size (320 - 360 Å ) and can be loaded with metallic compounds. Based on our
new Cis‐ platin‐ RHCC crystal structure, we hypothesize that the binding properties of the
cavities make RHCC a potential storage and delivery system for one of the most efficient
anti‐ cancer drugs. Here we present the crystal structure of the chemotherapeutic drug
Cis‐ platin bound to RHCC at 3.2 Å resolutions. RHCC was crystallized in space group P3121
with unit cell dimensions of a, b=112.8 Å, c=71.6 Å and α , β =90°, γ =120°. Employing
fluorescence microscopy we show that Alexafluor labelled RHCC molecules are internalized
by the human hypopharyngeal squamous carcinoma cell line FaDu, human glioblastoma cell
line T98G, and primary glioblastoma cells from patients. RHCC may provide a novel mode for
the delivery of chemotherapeutic drugs into tumour cells and represent a unique and novel
approach in the treatment of cancer patients.
S-175

Molecular characterization of a Class I P450 electron transfer system from

Novosphingobium aromaticivorans DSM12444
1 2 3 1 1 2
Wen Yang , Stephen Bell , Hui Wang , Weihong Zhou , Mark Bartlam , Luet-Lok Wong , Zihe
1,3
Rao
1 2 3
Nankai University, Tianjin, China, University of Oxford, Oxford, United Kingdom, Tsinghua
University, Beijing, China

Cytochrome P450 enzymes of the CYP101 and CYP111 families from the oligotrophic
bacterium Novosphingobium aromaticivorans DSM12444 are heme monooxygenases that
receive electrons from NADH via ArR, a ferredoxin reductase, and Arx, a [2Fe-2S] ferredoxin.
These systems show fast NADH turnovers that are efficiently coupled to product formation.
The three-dimensional structures of ArR, Arx and CYP101D1, which form a physiological
class I P450 electron transfer chain, have been solved by X-ray crystallography. The general
structural features of these proteins are similar to their counterparts in other Class I systems
such as putidaredoxin reductase (PdR), putidaredoxin (Pdx) and CYP101A1 of the camphor
hydroxylase system from Pseudomonas putida, and adrenodoxin (Adx) of the mitochondrial
steroidogenic CYP11 and CYP24A1 systems. However significant differences in the proposed
protein-protein interaction regions of the ferredoxin reductase, ferredoxin and P450 enzyme
are found. There are regions of positive charge on the likely interaction face of ArR and
CYP101D1 and a corresponding negatively charged area on the surface of Arx. The [2Fe-2S]
cluster binding loop in Arx also has a neutral, hydrophobic patch on the surface. These
surface characteristics are more in common with those of Adx than Pdx. The observed
structural features are consistent with the ionic strength dependence of the activity.
S-177

“Structure-based identification of ceruloplasmin as a hypothetical human p53-binding

protein and its possible role in carcinogenesis”
1 2 2
Chris Pacheco Rivera , Birgit Eisenhaber , Chandra Verma
1 2
Universidad Peruana Cayetano Heredia (UPCH), Lima, Peru, Bioinformatics Institute (BII),
A-STAR, Singapore, Singapore

The p53 tumor suppressor protein is the most commonly mutated protein identified in cancer.
p53 activation promotes the upregulation of various target genes responsible for cell cycle
arrest or apoptotic cell death depending on the cellular environment, a critical role in cellular
defense against cancer. Molecular interactions between the p53 protein and azurin, a redox
Pseudomonas aeruginosa protein, were demonstrated to trigger apoptosis in human cancer
cells. Since the protein-protein interaction between azurin and p53 allows the stabilization of
the latter and cancer regression, it was of great importance to determine the domains
involved in their physical association. Models based on crystal structures of p53 and azurin
suggest that their interaction take place at the DNA-binding core domain of p53, where the
95% of cancer-associated mutations take place. Thus, p53 DNA-binding domain, might
potentially represent a new target for tumour cell death induction or growth arrest in cancer
treatment.

Here, by means of tertiary structure alignment methods, we report a hypothetical human p53
binding protein, ceruloplasmin, a multicopper oxidase protein comprised of multiples domains
each of which has the typical fold of a single domain of cupredoxin proteins which shows a
significantly tertiary structural similarity to azurin. Protein docking approaches allowed us to
identify a potential p53-ceruloplasmin binding interface by the extrapolation of residues
involved in the p53-azurin complex formation; experimental data suggest the involvement of
ceruloplasmin in cancer development.
S-179

Local caching to improve efficiency of CIF DDLm function import references in

SBEVSL

Elena Zlateva, Herbert Bernstein

Dowling College, Oakdale, NY, United States

The introduction of the DDLm import attributes into CIF [1] has allowed for the development of
specific domain dictionaries without the overhead of redundant common definitions. These
modularized dictionaries can be located anywhere on the web, and importation facilitates the
access and sharing of specialized definitions. In an attempt to increase efficiency of DDLm
dictionary expansion through importation, we have created an expansion utility, CIFGET
(available for download at http://sourceforge.net/projects/cifget/), that follows all importation
tags, much like HTML links, and fetches a local copy files tree of referenced dictionaries. The
advantages of having a local copy of the tree are similar to the benefit of running local
database queries and transactions versus querying a remote database. Local queries
provide virtually no delays. Thus, running a dictionary expansion utility on a local dictionary
tree is faster and more reliable than fetching dictionary definitions only when needed.

Local database queries are an efficient way to create look-ups of any dictionary definition or
value. The Structural Biology Extensible Visualization Scripting Language Project has
proposed an extension to the current DDLm specification standards to allow for function
definitions anywhere within a CIF data file. This would allow user-defined functions to
manipulate the data from the data file to provide scripting, data conversion, or animation
capabilities. These functions could be exported into modularized dictionaries, thereby creating
a library of functions, which would then be accessible through importation. Function calls
from a CIF data file to a SBEVSL functions dictionary, fetched on the local files tree, will be
very fast as they are analogous to querying a local database with key-value pairs of function
names and definitions.

Work supported in part by grants from IUCr, NIH and DOE.

[1] Hall, SR, Spadaccini, N., Westbrook, J., “Dictionary Definition Language DDLm”, IUCr,
2007. http://www.iucr.org/__data/assets/pdf_file/0020/16382/DDLm_spec_aug08.pdf
S-181

Host Cell Pre-Selection, Towards the Development of a Generally Applicable “Tough”

Protein Over-Expression in Escherichia coli

Jiapeng Ruan, Wayne Anderson

Department of Molecular Pharmacology and Biological Chemistry, Northwestern University

Feinberg School of Medicine, Chicago, United States

Overproduction of proteins from heterologous genes has limitations in the production of

functionally folded soluble proteins. For the Escherichia coli expression system, although
numerous efforts have published, the benefits from current strategies have been case-
specific. Establishing generally applicable soluble expression methods for protein
overexpression remains a significantly challenging task. We have determined that soluble
expression in E. coli requires the coupling of tightly controlled target gene expression and
target protein specific folding environments. To alleviate solubility problems, we chose E. coli
host cells containing vectors for target protein expression and the co-expression of
transcription factors, followed by pre-selection of host cells under protein folding and
overexpression stress. Selected host strains were then used for the induction of
overexpression. We show here that host pre-selection optimizes the overexpression of some
previously totally insoluble “tough” proteins with dramatically improved solubility. Approaches
to generate expression regulation and host pre-selection are discussed and evaluated.
S-184

In-silico docking study of Mitogen-activated Protein Kinase Kinase 4 (MAP2K4/MEK4)

with Genistein and its Analogs

Sankar Narayan Krishna, Li Xu, Rebecca Farmer, Xiaoke Huang, Antoinette Nibbs, Karl
Scheidt, Wayne Anderson, Raymond Bergan

Northwestern University, Chicago, IL, United States

Metastasis or spread of Prostate Cancer (PCa) to other parts of the body is the second
highest cause of death due to cancer among men in the United States. Our lab has shown
that 4,5,7-trihydroxyisoflavone (genistein), inhibits the initiating step of cell invasion, as well
as the downstream formation of metastasis associated with PCa, by inhibiting Mitogen-
activated protein kinase kinase 4 (MAP2K4/MEK4) activity. In particular, it has been
shown that MEK4, a 399 amino acid protein, activates the established pro-invasion protein,
p38 MAPK. MEK4 increases cell invasion and induces matrix metalloproteinase type 2 (MMP-
2). Genistein inhibits MEK4 kinase activity in vitro, and MEK4-mediated signaling in intact
PCa cells. In related studies, a series of genistein analogs have been synthesized and
tested for their ability to inhibit prostate cell invasion, using a Boyden chamber assay system.

In order to investigate the structure-activity relationship of MEK4 with genistein and

synthetic analogs, an in silico model of MEK4 was constructed. Using this model, while
blinded to the Boyden chamber assay results, activity prediction after virtual docking
(Autodock 4.0) was done. In particular, the analogs and genistein were first segregated
based on their optimal scoring binding sites on the protein target and then the Autodock
estimated Inhibition constant (Ki) was determined. In silico results were then compared to
Boyden chamber assay results. The similarities and differences between in silico and
experimental findings and thus the potential of using this in silico modeling technique for
predicting analog activity will be discussed.

As a direct extension of this study, efforts are now being directed at crystallizing MEK4
and deriving it both with and without genistein and relevant analogs. The biochemical
mechanism by which genistein and selected analogs inhibit MEK4 kinase activity will be

determined by measuring their e ect upon MEK4 enzyme kinetics in vitro. These results

combined with in silico will be crucial in guiding the synthesis of new lead compounds with
higher specificity and lower cytotoxicity.
S-187

Structural studies of topoisomerase V, a novel type IC topoisomerase

Rakhi Rajan, Alexandra Gast, Bhupesh Taneja, Alfonso Mondragon

Northwestern University, Evanston, United States

Topoisomerases are ubiquitous enzymes that regulate the topology of DNA inside the cell
and thus facilitate several fundamental cellular processes. They function by creating a
transient DNA break and passing another DNA through this break before resealing the break.
Topoisomerase V (Topo-V) is a novel type IC topoisomerase, and is unique because it
contains both topoisomerase and DNA repair activities within the same protein. The
topoisomerase domain is located at the N- terminus of the protein and this is followed by
twelve helix-hairpin-helix (HhH)2 domains. Previous studies of an N-terminal 61 kDa fragment
of Topo-V (Topo-61) revealed that the topoisomerase domain of Topo-V has a new fold
entirely different from other topoisomerases. In the present study, different fragments of Topo-
V containing either the topoisomerase domain (Topo-44) or both topoisomerase and repair
domains (Topo-78) have been constructed and analyzed by structural and biochemical
methods. Crystal structures of Topo-44 determined under three different conditions show
significant conformational changes in the (HhH)2 domain near the topoisomerase active site
compared to Topo-61 crystal structure. These conformational changes are required for
exposing the topoisomerase active site so that DNA can gain access to the active site. Five
phosphate ions bound in the topoisomerase active site helped to model a DNA molecule, and
the model gives initial information on how the protein and DNA interacts. Biochemical studies
are in progress to understand the cleavage/religation mechanism and DNA and metal binding
characteristics of Topo-V. Crystallization studies of Topo-78 gave a model for the
th
topoisomerase domain and the first seven (HhH)2 domains. The last (8 ) (HhH)2 domain,
which has the DNA repair activity, is highly disordered and hence studies are in progress with
a deletion mutant of Topo-78 to understand the structure of the repair domain. In addition,
biochemical experiments are being carried out to identify the residues involved in DNA repair
activity of Topo-78. Successful structure determination of Topo-78 will give structural details
of the topoisomerase and repair active sites for the first time and show whether these two
domains interact each other.
S-190

The Crystal Structure of CARDS Toxin from Mycoplasma pneumoniae

1 1,2 1,2 1,2
Argentina Becker , Alexander B. Taylor , Ahmad Galaleldeen , Olga N. Pakhomova ,
1,2 3 3 1,2
Stephen P. Holloway , T.R. Kannan , Joel B. Baseman , P. John Hart
1 2
Department of Biochemistry, UTHSCSA, San Antonio, Texas, United States, The X-ray
Crystallography Core Laboratory, UTHSCSA, San Antonio, Texas, United States,
3
Department of Microbiology and Immunology, UTHSCSA, San Antonio, Texas, United States

Mycoplasma pneumoniae is a bacterial pathogen that colonizes the lung and is known to
cause asthma, pneumonia and other infections in humans. M. pneumoniae produces a toxin
known as CARDS (Community-Acquired Respiratory Distress Syndrome) toxin that has been
shown to display cytotoxic effects on mammalian cells similar to those observed during M.
pneumoniae infection, suggesting that the toxin plays a key role in M. pneumoniae
pathogenesis. The 591 amino acid protein has ADP-ribosylating and vacuolization activities
and biochemical evidence suggests that it gains entry into host cells through binding to
surfactant protein A (SP-A), an abundant glycoprotein in the lung. Here, we present the
crystal structure of CARDS toxin determined to 2.6 Å resolution. The crystals grow in space
group R3 with one molecule in the asymmetric unit. The catalytic N-terminal CARDS domain
shares structural similarity with the S1 subunit of the toxin from Bordetella pertussis
(Pertussis toxin) while the receptor-binding C-terminal domain, with no detectable sequence
homology to any known proteins, folds into two topologically similar subdomains. The new
structural data provide insight into various aspects of CARDS toxin trafficking and function.
S-193

Evaluating the Bruker SMART X2S bench-top system: A means to bringing x-ray
crystallography into the undergraduate curriculum

Uzma Zakai, Ilia Guzei, Nicholas Hill

University of Wisconsin-Madison, Madison, United States

X-ray crystallography is a powerful and increasingly common tool for routine structural
characterization yet remains poorly represented at the undergraduate level. Several
strategies have been adopted to bring X-ray crystallography into the mainstream
undergraduate experience, including forming alliances between institutions to economize
equipment and establish joint teaching and research projects. Herein we report our evaluation
of Bruker SMART X2S, a single crystal X-ray diffractometer designed for institutions lacking
any crystallographic infrastructure. Bruker SMART X2S is a portable benchtop diffractometer
that requires only a 110 V outlet to operate. The instrument operation is intuitive and facile
with an automation layer governing the workflow from behind the scenes. Based on our
examination of 19 samples, the Bruker SMART X2S yields publishable quality data. Although
data quality is a function of sample quality, this instrument is a bold advance towards bringing
chemical crystallography in the undergraduate curriculum.
S-196
Assessing the stability of theophylline cocrystals in the presence of competing
coformers in the solid state

Jennifer Urban, Heba Abourahma

The College of New Jersey, Ewing, NJ, United States
Cocrystals have become an increasingly popular subject of study in the chemistry
community. It is generally known that physical properties of a compound, including solubility,
hygroscopicity, and stability, can be altered when cocrystallized with other molecules, termed
"coformers". Our goal is to assess the stability of a cocrystal in the solid state in the presence
of competing coformers and determine the possibility of displacing one coformer in a cocrystal
with another. The results could be important in the pharmaceutical industry if a cocrystal were
to make it to the formulation stage as it would have to be ground into a tablet with excipients,
binding materials and others.
We have focused our attention on theophylline, an active pharmaceutical ingredient (API) that
is used in asthma medication. Theophylline makes a good model system for this study since a
number of theophylline cocrystals with a variety of coformers have been reported to date. To
achieve our goal, two general types of experiments were conducted: selectivity and
competition experiments. In the selectivity experiment the theophylline is wet-ground with
equimolar amounts of two coformers and the relative affinity of the theophylline to the
coformers is determined. In the competition experiment, a prepared cocrystal of theophylline
is wet-ground with a competing coformer that is known to form a cocrystal with theophylline.
With each experiment, grinding time is varied between 20, 40, and 60 minutes to determine
its effect on the experimental outcome. Cocrystals of theophylline that were studied in this
project include those with 4-nitrophenol, 4-hydroxybenzoic acid, salicylic acid, 2-
hydroxynaphthoic acid, melamine and acetamide. The products from each experiment were
analyzed by powder x-ray diffraction (PXRD), differential scanning calorimetry (DSC),
thermogravimetric analysis (TGA) and infrared spectrometry (IR). This poster will present the
results from the selectivity and competition experiments of theophylline cocrystals with the
coformers mentioned above.
S-199

Cocrystals as a Means to Control Polymorphism in Pyrazinamide

Devon Cocuzza, Heba Abourahma

The College of New Jersey, Ewing, New Jersey, United States

Polymorphism is the ability of a molecule to exist in more than one possible form in
the solid state. Each polymorphic form has its own unique crystal structure which is
responsible for determining the physical properties such as stability, solubility, hygroscopicity
and dissolution rate among other things.

The objective of our research is to examine cocrystals as a method to control polymorphism.

Cocrystals are crystalline materials that are comprised of at least two different components
that are solid at room temperature and are held together by non-covalent interactions. The
target polymorphic compound in our study is pyrazinamide (PZA). PZA is an active
pharmaceutical ingredient (API) used for the treatment of tuberculosis and is known to exist in
four different forms: alpha, beta, gamma, and delta. Since amides are known to have high
affinity to carboxylic acids and amides, we considered cocrystal formers (coformers) that
contain these functionalities such as benzoic acid, anthranilic acid, 3,5-dintrosalicylic acid,
isophthalic acid, and nicotinamide. Our experiments consisted of reacting equimolar amounts
of PZA with each coformer via liquid-assisted solid state grinding and in solution. The
products were characterized using Differential Scanning Calorimetry (DSC),
Thermogravimetric Analysis (TGA), Nuclear Magnetic Resonance (NMR), Infrared
Spectroscopy (IR) and Powder X-Ray Diffraction (PXRD). We further examined the
persistence of a particular cocrystal formation in the presence of different solvents during
liquid-assisted solid state grinding including methanol, ethanol, THF, DMSO, DMF, acetone,
acetonitrile, and water. Herein we will present the synthesis and characterization of PZA
cocrystals obtained to date from the above mentioned experiments.
S-202

Why is P21/n a Standard Non-Standard Space Group?

James Haestier1, Amber L. Thompson1, David J. Watkin1, George C. Feast2, Jeremy

Robertson2, Lee W. Page3
1
Chemical Crystallography, Oxford, United Kingdom, 2Chemistry Research
Laboratory, Oxford, United Kingdom, 3GlaxoSmithKline, Harlow, United Kingdom

The structure of an unusual methylene aziridine was initially determined in the

monoclinic space group P21/n, with a cell of a = 13.8593(3) Å, b = 10.5242(2) Å, c =
14.8044(4) Å and β = 92.0014 (7)°, and two molecules in the asymmetric unit.

Prior to publication [1], the CIF was verified with checkCIF, which gave the following
alert:

PLAT128_ALERT_4_G

Non-standard setting of Space-group P21/c…P21/n

The hyperlink explains further: ‘The reported monoclinic space-group is in a non-

standard setting. Transformation to the conventional setting is indicated unless there
is a good (scientific) reason not to do so.’

The unit-cell obtained from the initial indexing was then transformed, and
reprocessed (including integration, scaling and cell final refinement) to give the data
in the space group P21/c, with a cell of a = 13.8594(2) Å, b = 10.5243(2) Å, c =
19.9230(3) Å and β = 132.0439(7)°. The atomic coordinates from the original
structure were transformed, and the structure re-refined.

It has long been known that refinements in oblique cells have increased correlation
between selected parameters, potentially making refinements less stable [2]. A
comparison of these results in P21/n and P21/c clearly displayed an increase in the
correlation between coordinates in the ac plane for the oblique cell. The increase in
the corresponding covariances makes a significant contribution to the standard
uncertainties of derived parameters, e.g. bond lengths. Thus, there are clear,
scientific advantages to reporting this (or any structure) in the “more orthogonal”
space-group setting.

1. Feast G. C. et al. (2009). Acta Cryst. C65, o635–o638.

2. Dunitz, J. D. (1979). X-ray Analysis and the Structure of Organic Molecules, p
205–206. Cornell University Press.
S-205

Structural Determination and Identification of Receptor Binding Site of Adeno

Associated Virus Serotype 6 using X-ray Crystallography

Robert Ng, Govindasamy Lakshmanan, Hyun-Joo Nam, Brittney Gurda, Jude Samulski,
Robert McKenna, Mavis Agbandje-McKenna
1 2
University of Florida, Gainesville, FL, United States, University of North Carolina, Chapel Hill,
NC, United States

Adeno-associated viruses (AAVs) are nonpathogenic single-stranded DNA viruses which

belong to the Parvoviridae family and the genus Dependovirus. Due to their non-
pathogenicity, attention has been directed towards their development as gene therapy
vectors. Representative members of the AAV antigenic clades/ clonal isolates display capsid
sequence-associated receptor attachment, tissue tropism, transduction efficiency, and
antigenic reactivity properties. To identify the structural features that dictate these different
properties, we have initiated the structure determination of these viruses. Unlike most of the
AAVs that either bind sialic acid or heparin sulfate as a primary cell surface receptor, AAV6 is
a unique serotype that binds both carbohydrate on cell surfaces. To identify the AAV6 capsid
regions involved in these interactions we aim to determine the structure of this serotype alone
and complexed with carbohydrate ligands. The structure of AAV6 determined to 9.7Å
resolution by cryo-electron microscopy and image reconstruction shows the capsid features
already described for the AAV. An X-ray diffraction data has been collected for AAV6 alone
and AAV6 complexed with heparin sulfate or sialic acid, and structure determination using our
available crystal structure of AAV1 as a phasing model is underway. These structures will be
used to identify variable surface loop regions on the AAV6 capsid that dictate its unique dual
carbohyrdrate binding phenotype. In addition, a comparison of the AAV1 and AAV6 structures
will provide information on the role of the capsid in dictating the differential liver and lung
tropism observed between these two viruses which differ by only 6/736 amino acids in their
capsid viral protein. This data will impact our understanding of the capsid determinants of
tissue tropism and transduction efficiency for the AAVs and will be applicable for the
engineering of recombinant AAV vectors for improved tissue targeting specificity.
S-208

Structural Studies of MG289, an Extracytoplasmic Thiamine Binding Lipoprotein from

the Sexually Transmitted Infection Mycoplasma genitalium

Katherine Sippel, Balasubramanian Venekatakrishnan, Susan Boehlein, Govindasamy

Lakshamanan, Yoshihisa Sakai, Jeanne Quirit, Mavis Agbandje-McKenna, Charles Rosser,
Robert McKenna

University of Florida, Gainesville, Florida, United States

Mycoplasma genitalium (Mg) is a sexually transmitted infection that causes non-gonococcal

urethritis in men and endocervicitis in women. This pathogen is resistant to tetracyclines and
azithromycin indicating the need for novel drug targets to treat this disease. Studies have
identified MG289 as a minimal gene necessary for Mg survival. It is homologous to Cypl, an
extracytoplasmic thiamine pyrophosphate (TPP) binding lipoprotein from Mycoplasma
hyorhinis. The structure of Cypl was solved previously using magic triangle providing a
molecular replacement model for structure solution. The crystal structure of MG289, has been
solved and refined to 2.1 Å resolution. The current model has an Rcryst of 18.6% and Rfree of
23.1%. MG289, like Cypl, is a mixed α /β protein with two well-defined domains, which are
separated by a deep cleft. As predicted from sequence alignments and homology modeling,
the structure shows thiamine bound to MG289, rather than the TPP seen in Cypl. Analysis of
the thiamine in the binding cleft shows numerous interactions that can be translated into an in
vitro fluorescence-based kinetic assay for drug binding. The insights derived from the
structure solution and characterizations of MG289 are being used as a starting point for
structure-based drug design of a novel antibiotic to fight mycoplasmal infection.
S-211

Structural Investigations of TopBP1 in DNA Replication Stress

Charles Leung, Mark Glover

University of Alberta, Edmonton, AB, Canada

Replication stress often compromises the replication machinery and can lead to DNA damage
at replication forks. This induces a complex repair mechanism, which is characterized by the
early accumulation of Replication protein A (RPA) onto chromatin. Studies have shown that
independent loading of RPA onto exposed ssDNA and the trimeric ring complex Rad9-Hus1-
Rad1 (9-1-1) at DNA junctions facilitates activation of the crucial signalling kinase, Ataxia
telangiectasia and Rad3 related (ATR), via the ATR activating function of Topoisomerase II
binding protein 1 (TopBP1). The ability of TopBP1 to activate ATR results from its ability to
form protein-protein interactions. We are currently investigating the structural biology of
TopBP1 involved in these specific processes.
S-214
2
Structural Investigation of trans-4-hydroxynonenal-derived 1,N -deoxyguanosine
Adduct in protein-DNA complex

Surajit Banerjee, Plamen P. Christov, Albena Kozekova, Carmelo J. Rizzo, Michael P. Stone

Vanderbilt University, Nashville, TN, United States

trans-4-Hydroxynonenal (HNE) is produced by peroxidation of -6 polyunsaturated fatty

acids. HNE exhibits a range of biological effects, from alteration in gene expression and cell
signaling to cell proliferation and apoptosis. The Michael addition of deoxyguanosine to HNE
2
yields four diastereomeric exocyclic 1,N -dG adducts. Site specific mutagenesis reveals that
these induce primarily G to T mutations. Here we present the replication bypass studies and
structures of the Sulfolobus solfataricus DNA polymerase Dpo4 with the template:primer 5'-
TCAYXGAATCCTTCCCCC-3' 5'-GGGGGAAGGATTC-3', where X is the site of adduction
with the (6S,8R,11S) HNE-dG adduct and Y is either C or T for two sequences respectively.
In-vitro bypass studies with Dpo4 shows misincorporation of dATP at the adduct site for both
2
sequences. The 1,N -HNE-dGua adduct maintains the exocyclic structure in case of -1 primer
and intercalates between the neighbouring template bases. The adduct shows a ring open
conformations when placed opposite to dCyd to expose the Watson-Crick base pairing face of
the adducted dG, as observed previously by NMR for duplex DNA.
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S-223

Crystal structure of Helicobacter pylori MinE, a cell division topological specificity

factor

Hyung-Seop Youn, Jung-Gyu Lee, Jun Yop An, Lai San Woo, Yeong-Jin Lee, Won Ju Jeong,
Soo Hyun Eom

Gwangju Institute of Science and Technology, Gwangju, Korea, Republic of

In gram negative bacteria, proper placement of the FtsZ ring, mediated by nucleoid occlusion
and the activities of the dynamic oscillating Min proteins MinC, MinD and MinE, is required for
correct positioning of the cell division septum. MinE is a topological specificity factor that
counters the activity of MinCD division inhibitor at the mid-cell division site. Its structure
consists of an anti-MinCD domain and a topology specificity domain (TSD). Previous NMR
analysis of truncated Escherichia coli MinE showed that the TSD domain contains a long α -
helix and two antiparallel β -strands, which mediate formation of a homodimeric α /β structure.
Here we report the crystal structure of full-length Helicobacter pylori MinE and redefine its
TSD based on that structure. The N-terminal region of the TSD (residues 19-26), previously
defined as part of the anti-MinCD domain, forms a β -strand (β A) and participates in TSD
folding. In addition, H. pylori MinE forms a dimer through the interaction of anti-parallel β A-
strands. Moreover, we observed serial dimer-dimer interactions within the crystal packing,
resulting in the formation of a filamentous structure. We therefore redefine the functional
domain of MinE and propose that a multimeric filamentous structure is formed through anti-
parallel β -strand interactions.
S-226

ER α -glucosidase I in the N-glycosylation pathway

1 2
Megan Barker , David Rose
1 2
University of Toronto, Toronto, Ontario, Canada, University of Waterloo, Waterloo, Ontario,
Canada

Many proteins on the eukaryotic cell surface are covalently linked to complex carbohydrates,
leading to a heterogeneously sugar-coated cell. The process of protein N-glycosylation
begins in the endoplasmic reticulum (ER) with the transfer of a standard N-glycan,
Glc3Man9GlcNAc2, to an asparagine residue of a nascent protein. The terminal glucose
residue is subsequently cleaved by the transmembrane enzyme ER α -glucosidase I (GluI),
followed by further processing in the ER and Golgi. Recent studies on the S. cerevisiae
homolog of GluI have determined several key residues within the catalytic domain, located in
the ER-lumenal C-terminal region (Faridmoayer et al, 2007). However, the structure of GluI is
presently unknown.

I have achieved secreted expression of a transmembrane-deletion construct of S. cerevisiae

GluI using the methyltropic yeast Pichia pastoris, giving purified protein at a yield of three
milligrams of protein per litre of growth culture. Optimizing crystal growth by microseeding
from an initial hit, I obtained rod-like crystals 0.7mm in length. Using a high-pressure cooling
system (Kim et al, 2005) developed at the Cornell High Energy Synchotron Source (CHESS),
and paratone oil as a cryoprotectant, 2.1Å datasets of the native protein and with an inhibitor
soak have been collected. Work towards solving the phase problem is currently in progress.
This poster will describe the ongoing efforts to determine the structure and understand the
catalytic mechanism of this key member of the N-glycosylation pathyway.
S-229

E2 Interaction and Dimerization in the Crystal Structure of TRAF6 and Structural Basis
for the Lack of E2 Interaction in the RING Domain of TRAF2
1 1 1 1 1 3
Qian Yin , Su-Chang Lin , Betty Lamothe , Miao Lu , Yu-Chih Lo , Gregory Hura , Lixin
1 3 2 5 4
Zheng , Rebecca L. Rich , Alejandro D. Campos , David G. Myszka , Michael J. Lenardo ,
2 1
Bryant G. Darnay , Hao Wu
1 2
Weill Medical College, New York, NY, United States, Univ . of Texas, Houston, TX, United
3 4
States, ALS Lawrence Berkely National Laboratory, Berkely, CA, United States, NIAID, NIH,
5
Bethesda, MD, United States, Univ. of Utah, Salt Lake City, UT, United States

Tumor necrosis factor (TNF) receptor-associated factor (TRAF) proteins are intracellular
signal transducers for a number of immune receptor superfamilies. TRAF2 interacts with
members of the TNF receptor superfamily and connects the receptors to downstream
signaling proteins; whereas TRAF6 mediates signalling emanated from both TNF receptors
and interleukin-1 receptor/Toll-like receptors. Both TRAF2 and TRAF6 are proposed to
function as E3 ubiquitin ligase, eliciting NF-κ B activation via Lys63-linked polyubiquitination.
E3 ligase activity has been mapped to their N-terminal RING and zinc finger domains. Here
we report the crystal structures of N-terminal TRAF6 and its complex with the ubiquitin-
conjugating enzyme (E2) Ubc13, and the structure of the RING and the first zinc finger
domains of TRAF2. The RING and zinc fingers of TRAF6 assume a rigid, elongated structure.
Interaction of TRAF6 with Ubc13 involves direct contacts of the RING and the preceding
residues, and the first zinc finger has a structural role. Unexpectedly, this region of TRAF6 is
dimeric both in the crystal and in solution, different from the trimeric C-terminal TRAF domain.
Structure-based mutagenesis reveals that TRAF6 dimerization is crucial for polyubiquitin
synthesis and autoubiquitination. Fluorescence resonance energy transfer analysis shows
that TRAF6 dimerization induces higher-order oligomerization of full-length TRAF6. The
mismatch of dimeric and trimeric symmetry may provide a mode of infinite oligomerization
that facilitates ligand-dependent signal transduction of many immune receptors. On the other
hand, although TRAF2 adopts similar linear arrangement of RING and zinc finger domains
and same dimeric status, its RING structure displays multifaceted differences from that of
TRAF6. These structural differences prevent TRAF2 from interacting with Ubc13 and other
related E2s due to steric clash and unfavorable interfaces. Our structural observation should
prompt a re-evaluation of the role of TRAF2 in TNFα signaling and may indicate that TRAF2-
associated proteins such as cIAPs may be the ubiquitin ligases for NF-κ B signaling.
S-232

Functional Structures: XLF and XRCC4 in Mammalian DNA Double-strand Break

Repair
1 2 2 3 1
Sara Andres , Sunetra Roy , Katheryn Meek , Mauro Modesti , Murray Junop
1 2
McMaster Univ., Hamilton, ONT, Canada, Michigan State Univ., Madison, WI, United
3
States, Aix-Marseille Univ. CNRS, Marseille, France

DNA double-strand breaks are one of the most lethal forms of DNA damage that can occur in
a mammalian cell. However, some double-strand breaks are part of programmed genomic
rearrangements, such as V(D)J recombination. Non-homologous end-joining is the
predominant repair pathway to fix these breaks and requires a core set of proteins to do so.
Two of these proteins, XLF and XRCC4, interact with one another and are essential for non-
homologous end-joining, yet have no enzymatic function. They carry out their roles strictly
through their architecture. To elucidate the mechanism by which these two proteins function
in complex, we solved the structure of human XLF (1-224) to 2.5 Å using SAD. This structure
bears similar resemblance to human XRCC4 (PDB 1FU1), except for the striking difference in
the elongated tail of XRCC4, compared to the tail of XLF, which winds back up and around
towards the head domains of the protein. Using information from both protein structures and
conserved regions, we identified amino acids that were key to the interaction of both proteins.
This interaction is necessary for repair, as XRCC4 mutants that were unable to bind to XLF
caused a decrease in frequencies of coding joint formation during V(D)J recombination. How
this physical interaction actually occurs, though, is still unknown. Therefore, we attempted
crystallization of an XLF-XRCC4 complex. Crystals were obtained for multiple truncations of
both proteins, but overall diffracted to >20 Å. Application of microseeding, however, and
extreme dehydration led to crystal diffraction at 4.6 Å. Herein we describe our work towards
solving and refining the low-resolution structure of an XLF-XRCC4 complex, and the insights
it provides on the XLF-XRCC4 complex function in DNA double-strand break repair.
S-235

Small changes on the substitution pattern of pyridines and pyridine-N-oxides: Their

influence on the molecule aggregation

Vera Vasylyeva, Klaus Merz

Ruhr-University Bochum, Bochum, Germany

One challenge in chemical engineering is the lack of correlation between crystal packing and
the molecular structure. The nature of self-organisation in the solid state is complicated and
depends on different parameters such as symmetry, secondary interactions and
supramolecular synthons. Our strategy for analysing weak dipole-dipole-interactions is to
reduce the complexity of parameters and investigate small molecules, such as fluoro- and
deutero-substituted pyridines and pyridine-N-oxides. The in situ crystallisation with an IR-laser
and a low temperature device allows a crystallisation of the compounds with a low melting
point under the direct control of the crystal growth via X-ray analysis.

Fluorine is known to influence the electronic structure of aromatic backbone and therefore the
entire molecules but the nature of the C-F…H hydrogen bond is discussed controversially. On
the other hand, fluorine forms only weak intermolecular interactions and seems to have no
influence on the crystal packing. Pauling’s definition of the hydrogen bond would imply that
fluorine, as the most electronegative atom, should be a stronger hydrogen-bond acceptor
then oxygen and nitrogen. But the C-F group, the so-called “organic fluorine”, does not form
hydrogen bonds commensurate with electronegativity considerations in contrast to the C-O
and C-N groups. Hydrogen/deuterium exchange is without doubt the smallest possible
alteration of the molecular structure. Very few examples are known that show a remarkable
influence of deuterium substitution on the aggregation of molecules. Recently
pentadeuteropyridine was found to crystallise completely different in comparison to a not-
deuterated pyridine [1]. We pose two questions: How can the influence of fluorine/deuterium
on molecule structure be useful for crystal engineering? A comparison of fluoro-substituted
pyridines shows different intermolecular interactions depending on the substitution pattern of
the fluorine atoms at the pyridine backbone [2]. Furthermore already a partial deuteration of
pyridine-N-oxide leads to great changes in the crystallisation behaviour [3]. The second
question is: Can the weak influence of secondary interactions of fluorine/deuterium
substituents on the crystal packing be amplified through the increase of the number of F/D-
atoms?

[1] R. Boese et. al, Angew. Chem. Int. Ed., 2009, 48, 755-757

[2] V. Vasylyeva, K. Merz, J. Fluor. Chem., 2010, 131(3), 446-449

[3] V. Vasylyeva, T. Kedziorski, C. Schauerte, K. Merz, Angew. Chem., 2010, submitted

S-238

Fragment-Based Lead Discovery for Urokinase-Type Plasminogen Activator Inhibitors

1 2 2 1 1
Li Qiu , Longguang Jiang , Mingdong Huang , Edward J. Meehan , Liqing Chen
1 2
University of Alabama in Huntsville, Huntsville,AL, United States, State Key Laboratory of
Structural Chemistry, Fujian Institute of Research on the Structure of Matter, Chinese
Academy of Sciences, Fuzhou,Fujian, China

The Urokinase-Type Plasminogen Activator (uPA) is a trypsin-like serine protease that activates
plasminogen to plasmin. uPA also plays crucial roles in regulating arterial remodeling, angiogenesis,
cell migration and proliferation. uPA has been widely recognized as a target against tumor metastasis in
various animal models. Upregulation of uPA expression has been shown to correlate with cellular
proliferation and invasiveness. Various strategies have been proposed to intervene with either uPA
proteolytic activity or the zymogen activation. High affinity inhibitors have been identified that
intervene the uPA proteolytic activity. However, these uPA inhibitors are typically quite basic and thus
have poor bioavailability. Fragment-based screening using high throughput X-ray crystallography is an
established method to identify low molecular weight fragments that can specifically bind to the target
active site. Those fragments may be evolved to larger lead compounds, either by linking or merging
fragments together or by growing the fragments to pick up additional interactions. In this investigation,
high quality uPA crystals were soaked with a library of 384 low molecular weight fragments and
screened by X-ray diffraction for compounds binding. Data were collected at the SER-CAT
synchrotron beamlines 22ID/22BM at the Argonne National Laboratory. Small molecule fragment
specifically binds to uPA as determined by structural analysis was biochemically tested on its
binding/inhibition profile. The results of this study will be implemented towards the development of
novel drug compounds.
S-247

Supramolecular Structural Variation in a Series of Ni(X)(NO)(PPh3)2 Complexes.

Nongnaphat Khosavithitkul, Kenneth J. Haller

Science, Muang, Nakhon Ratchasima, Thailand

Concerted weak interactions are important building blocks of extended solid state
structures. One such building block for molecular compounds is the multiple phenyl-phenyl edge-
to-face (ef) C−H∙ ∙ ∙ π attractive noncovalent interactions of the concerted sextuple phenyl embrace
(6PE). The resulting sum of interaction energy is sufficient to make it a dominant supramolecular
motif for crystals of complexes containing triphenylphosphine or similar ligands.

The supramolecular structures of a series of three four-coordinate Ni(X)(NO)(P(C6H5)3)2

− − −
complexes, where X = NCS , N3 , and Cl are examined. Although the molecular complexes are
quite closely related, their supramolecular structures are different. The primary extended
interactions in the isothiocycanato complex are zig-zag chains of the expected 6PE between
adjacent triphenylphosphine ligands. Chains are connected together into a three dimensional
network by additional weaker concerted interactions. The chloro complex also contains one-
dimensional chains of 6PE. However, it crystallizes with a benzene molecule of solvation, which
accommodates considerably more C−H∙ ∙ ∙ π interactions than a phenyl ring, thus becoming the
major link interconnecting three of the 6PE chains via ten C−H∙ ∙ ∙ π interactions centered about
the benzene of crystallization. While the azido complex does not contain 6PE, it does contain
one-dimensional chains of highly concerted noncovalent interactions involving the azido
group as well as phenyl rings. Thus, as the strongest hydrogen bond acceptor, the azido
ligand becomes perhaps the most important determiner of the supramolecular structure.
Additional phenyl-phenyl interactions are abundant and important in determining finer details
of the crystal structures.
S-250

The structure of 2-(naphthyl)-3-pyridinyl-1,3-thiazolidin-4-one

1 1 1 1
Jose Luis Pinto , Jose Antonio Henao , Vladimir Kouznetsov , Diego Fernando Amado ,
2 2
Teresa González , Alexander Briceño
1 2
Universidad Industrial de Santander, Bucaramanga, Santander, Colombia, Instituto
Venezolano de Investigaciones Científicas, Caracas, Distrito Capital, Venezuela

Heterocyclic compounds are largely studied due to the showed biological activities of most of
the heterocycles. Thiazolidinones are important heterocyclic compounds, which exhibit a
broad range of biological activities, including interesting profile as fungicidal, pesticide,
antibacterial, anticonvulsant, antihistaminic, antioxidant, anti-inflammatory and antinociceptive
agents, etc. As a consequence many different protocols allowing the synthesis of 4-
thiazolidinone skeletons have been developed

The compound 2-(naphthyl)-3-pyridinyl-1,3-thiazolidin-4-one was synthesized by means of a

multicomponent reaction with a Dean-Stark trap promoted by glacial acetic acid, in which
stoichiometric amounts were used of the reagents: α -aminopyridine, α -naphthylaldehide, and
thioglycolic acid, in anhydrous toluene. The molecular characterization was carried out by
means of the techniques IR, MNR and GC-MS.

The X-ray diffraction data were collected on an AFC7S single crystal diffractometer using
MoK radiation ( = 0.71070Å). The structural solution and refinement were made with
Shelxs-97 software package.

The 2-(naphthyl)-3-pyridinyl-1,3-thiazolidin-4-one (C18H14N2OS), M = 306 g/mol, crystallizes in

the monoclinic system, space group P21/c [No. 14] with unit cell parameters a = 11.958 (3) Å,
3
b = 9.656 (2) Å, c = 12.662 (2) Å, β = 97.010° (5), Vol = 1451.09 (2) Å and Z = 4.

Key Words: Crystal structure; thiazolidin-4-ones.

S-253

Crystal Structure and Molecular Modeling of Analogue of Chalcone C20H22NO3.

William B. Fernandes, Caridad Noda Perez, Hamilton B. Napolitano

Department of Chemistry, UnUCET, State University of Goias, Anapolis/Goias, Brazil

Chalcones are considered precursors of the biosynthesis of flavonoids and are obtained by
condensation reaction of Claisen-Schmidt between a ketone and an aromatic aldehyde in the
presence of basic catalysts. The importance of chalcones is based upon the wide variety of
chemical and biological properties presented by them. Furthermore, chalcones have been the
subject of several theoretical and experimental studies, that aimed at determining their
molecular structures, chemical reactivity, antimicrobial activity, among other applications in
[1,2]
the therapy field . In order to develop new drugs the analogue of chalcone of retinoid type
(1E,4E)-1-(4-nitrophenyl)-5-(2,6,6-trimetilciclohex-1-enyl)-penta-1,4-dien-3-one was obtained
from the equimolar coupling of the β -ionona with p-nitrobenzaldeide by classical Claisen-
Schimidt condensation using lithium hydroxide as catalyst.The single crystal growth was
obtained by indirect diffusion technique using a system of hexane and methanol. The
structure was solved by Direct Methods and refined by full matrix Least Square methods on
2 [3]
F using WINGX package . Non H atoms were refined anisotropically and all H atoms were
placed geometrically. The compound crystallizes in the P21/c monoclinic space group and the
cell dimensions are: a = 11.593(2) Å, b = 11.715(2) Å, c = 14.202(2) Å, α = γ = 90° and β =
110.147(5)°; Z = 4 and V = 1810.8(4) ų. 17572 measured reflections with 3211 unique and
2166 observed [I > 4σ (I)]. The final residual factor R1 is 0.0525 for 226 refined parameters.
...
One non-classical intra-molecular hydrogen bonds C–H O [2.856(3)Å] stabilizes the molecule
in partially planar arrangement. The nature of the observed disorder was investigated
theoretically within Density Functional Theory using pseudopotentials and planewave basis
set by Nudged Elastic Band Method. The transition state structure was obtained and the
calculated potential barrier energy is 9.22 eV. The effect of crystal packing was also
examined.

This work was financed by CAPES (process 2036-09-6) and PrP/UEG.

[1] Dominguez, J. N., Charris, J. E. (2001). European J. of Med. Chem., 36, 555 - 560.

[2] Valla, A., Cartier, D. (2006). European Journal of Medicinal Chemistry, 41, 142 - 146.

[3] Farrugia, L. J. (1999). Journal Appl. Cryst., 32, 837 - 838.

S-256

Experimental Electron Density Determinations of CB1 Receptor Agonists

Steven Fournet, Edwin D. Stevens, Mark L. Trudell

Department of Chemistry, University of New Orleans, New Orleans, LA 70148, United States

Of the two cannabinoid receptors that have been identified, CB1 and CB2, the CB1 receptor
is found primarily in the central nervous system. Some agonists of the CB1 receptor,
including the drug Rimonabant, have been shown to be effective in the treatment of obesity in
clinical trials. Recently, a new series of compounds have been synthesized that have been
shown to be agonists with a wide range of binding affinities for the CB1 receptor in rat brains.
We have undertaken the high-resolution measurement of the electron density distributions of
several of these compounds in order to correlate features of the electron structure with
-1
biological activity. Highly redundant, high-resolution (typically sin max/ 1.1 Å ) x-ray
diffraction data sets have been collected at 120 K using MoK radiation, and the data refined
using the Hansen-Coppens aspherical atom multipole model with the XD2006 program.

Analysis of the resulting electron density

distributions includes deformation density
maps and topological parameters obtained
using the Atoms in Molecules theory. Of
particular interest are plots of the
electrostatic potential calculated on the
surface of the molecular electron density
distribution. The molecular electrostatic
potential provides information on chemical
reactivity, identifying for example sites of
protonation and electrophilic attack, and is
a major component of intermolecular
interaction energies.

Funded in part by the National Institute on

S-259

Experimental Electron Density Distribution of Dimethoxygossypol, a Derivative of the

Disesquiterpene Gossypol Isolated from Cotton Plants
1 2 1
Carlos Zelaya , Michael K. Dowd , Edwin D. Stevens
1
Department of Chemistry, University of New Orleans, New Orleans, LA 70148, United
2
States, Southern Regional Research Center, USDA, New Orleans, LA 70124, United States

Gossypol is a natural product isolated from the cotton plant that is of interest because of its
wide sphere of bioactivity. We have isolated and synthesized a number of derivatives of
gossypol to explore their anticancer and antifungal activity. Crystals of the 6,6’-dimethoxy
derivative were found to be suitable for a high-resolution study of the electron density
distribution. A highly redundant set of x-ray diffraction intensity measurements was
collected to (sin /)max of 1.19 Å at 120 K. The experimental electron density distribution
-1

was obtained by least-squares refinement of the x-ray data using the Hansen-Coppens
aspherical atom multipole model.

In addition to maps of the molecular

deformation density, the topology of
the electron distribution of
dimethoxygossypol has been
analyzed using the Atoms in
Molecules approach. The locations
of the critical points of the electron
distribution, and the values of the
2
density, (rb), Laplacian (rb), and
bond ellipticity, , at the bond critical
points have been determined for the
wide variety of different covalent
bonds and hydrogen bonds present
in the structure.
S-262

N-alkyl-DABCOnium trihalozincates: Privileged scaffolds for the preparation of

noncentrosymmetric solids

Aaron Finke, Danielle Gray, Jeffrey Moore

University of Illinois, Urbana-Champaign, Urbana, IL, United States

A major requirement for the development of materials with nonlinear optical susceptibilities is
the absence of inversion centers in the solid state, i.e. crystallization in noncentrosymmetric
(NCS) space groups. Nonetheless, a priori determination of a compound's preference for
acentricity remains a challenge. Few achiral, organic moieties are known to act as privileged
NCS scaffolds, due in part to the difficulty of systematic investigations of such moieties on
solid-state morphology.

We present studies of a new, achiral, metal-organic scaffold, which exhibits an unusual

preference for polar, NCS space groups. Compounds containing the scaffold are easily
prepared from DABCO (1,4-diazabicyclo[2.2.1]octane), ZnBr2, and an alkyl bromide; all
compounds are highly crystalline solids, which are stable under ambient conditions. A library
of compounds containing the scaffold was analyzed by single-crystal X-ray diffraction.
Systematic modification of the alkyl functionality was crucial to understanding the role of the
scaffold toward preferential crystallization in NCS space groups.
S-264

Rapid temperature switching for time resolved measurements

1 1 1 2
Kevin Beyer , Peter Chupas , Karena Chapman , Mark Newton
1 2
Argonne National Laboratory, Argonne, Illinois, United States, ESRF, Grenoble, France

The ability to probe materials and reactions in real time under real operating conditions is
pivotal to understanding of their structure and functional behavior. Towards this goal it is
important to develop appropriate sample environments generating non-ambient operating
conditions. In particular, probing the kinetics and mechanism for a reaction rely on the ability
to initiate the process on a time scale that is fast relative to the reaction itself. Here we
present apparatus that enables the rapid switching of temperature or reactive gas streams to
initiate and characterize solid state reactions.
S-265

Structural Analisys of the Phenyl Sulfonylamide Acetophenone as an intermediary of a

Sulfonamide Chalcone
1 1 2 2
Lorraine Malaspina , Carlito Lariucci , William Fernandes , Caridad Perez
1 2
Federal University of Goias, Goiania, Goias, Brazil, State University of Goias, Anapolis,
Goias, Brazil

Chemically, the chalcones are flavonoids with open-chain, in which the two aromatic rings are
connected by a system of three carbons, forming ketones α , and β unsaturated, where both the carbonyl
and the olefinic portion are linked to aromatic groups. They are found in nature, in undergrowth plants,
in different plants organs, especially at the flowers. They constitute a class of antifungal and anticancer
agents that, according to some authors, has shown promising therapeutic efficacy against a wide variety
of tumor cells both in vivo and in vitro, especially in the treatment of stomach cancer.

The importance of chalcones is due to the wide variety of chemical and biological properties that they
present. For this reason, chalcones have been the subject of several theoretical and experimental
studies, mainly aimed at determining their molecular structures, their chemical reactivity, its
antimicrobial activity, its capacity of inhibition and enzyme induction, among other applications in the
therapy field.

The compound object of this work was obtained by Prof. Caridad Noda Perez from the State University
of Goiás and her student William Borges Fernandes.

Crystal Data: Data collection in a KappaCCD diffractometer, MoKα radiation. The solution,
anisotropic refinement, geometrical calculations, molecular packing and drawings were done with the
program package WINGX. Molecular formula: C14H13NO3S. Structure: a = 12.5179(6) Å, b =
8.3615(4) Å, c = 13.0007(5) Å, α = γ = 90º, β = 98.118(3)º, monoclinic, space group P21/c, Z = 4, V =
1347.13(6) ų. 9797 measured reflections with 3009 unique and 6061 observed. Final indices R1 =
0.0456 for 177 refined parameters.

There is an intra-molecular hydrogen bond with N−H…O angle equal to 137.58º and distance equal to
2.182 Å, and symmetry [ x, y+1, z].

Acknowledgements: This work was partially financed by CNPq, CAPES and FUNAPE/UFG. The
Data Collection were done by Prof. Carlos Alberto Simone at the Institute of Physics of USP-São
Carlos (IFSC).
S-268

Common Recognition Motifs used by Prokaryotic LysR-type Transcriptional

Regulators are Evident from the Structures of BenM DNA Binding Domain with its
Operator-promoter DNA
Amer Alanazi, Ellen Neidle, Cory Momany

University of Georgia, Athens, GA, United States

The LysR-type transcriptional regulator (LTTR) BenM is involved in controlling benzoate

degradation in the soil bacterium Acinetobacter baylyi strain ADP1. The 1.8 Å resolution
crystal structure of the unbound BenM DNA binding domain subunit (BenM DBD) confirmed
that the BenM DBD forms a compact globular domain composed of three helices with a
winged helix-turn-helix motif (a2-a3) and longer linker-helix resembling that of the DBD of
winged helix proteins. BenM DBD was crystallized with its cognate benA and catB DNA
promoter sites in two different crystal-packing arrangements. In these nucleic acid complexes,
BenM DBD dimers span a large region of bent DNA where the DNA recognition helices (a2)
of one dimer bind into two consecutive DNA major grooves in a sequence-dependent manner.
The specific DNA major groove interactions that define the LTTR conserved recognition motif
(T-N11-A) include van der Waals interaction of two proline residues at the N-terminal end of
the recognition helices with the methyl group of the thymine base of the recognition motif.
Also involved in sequence specific interactions are the side chain of Gln 29 with the imino and
amino groups of the recognition motif adenine base respectively (5'-ATAC-3') and the side
chain of Arg 34 with the carbonyl oxygen of guanine (5` -GTAT-3` ) in the complementary
strand. The wing of the winged HTH motif interacts mainly with the phosphate backbone of
the DNA minor groove and assists in the proper positioning of the N-terminal end of the
recognition helix.
S-271

Understanding blue-to-red conversion in monomeric fluorescent timers and hydrolytic

degradation of their chromophores
1,2 3 2 2
Sergei Pletnev , Fedor Subach , Zbigniew Dauter , Alexander Wlodawer , Vladislav
3
Verkhusha
1 2
SAIC-Frederick, Frederick, IL, United States, National Cancer Institute, Frederick, IL, United
3
States, Albert Einstein College of Medicine, New York, NY, United States

Crystal structures of a fast fluorescent timer (Fast-FT) and its precursor with blocked blue-to-
red conversion (Blue102) have been determined at the resolution of 1.15 Å and 1.81 Å,
respectively. Structural data suggest that blue-to-red conversion, taking place in Fast-FT and
in related fluorescent timers (FTs) of the same family, is associated with the oxidation of Cα 2-
Cβ 2 bond of the chromophore. Site directed mutagenesis revealed a crucial role of Arg70 and
Tyr83 in the delayed oxidation of Cα 2-Cβ 2 bond, introducing the timing factor in maturation of
the fluorescent timer. Substitutions Ser217Ala and Ser217Cys in Fast-FT substantially slow
down formation of an intermediate blue chromophore but do not affect much blue-to-red
conversion, whereas mutation Arg70Lys, having little effect on the blue chromophore
formation rate, markedly accelerates formation of the red chromophore. The chromophore of
FTs adopts a cis-conformation stabilized by a hydrogen bond between the phenolate oxygen
of the chromophore and the side chain hydroxyl of Ser146. In case of Blue102, a bulky side
chain of Ile146 precludes the chromophore from adopting a “cis-like” conformation, blocking
its blue-to-red conversion. Both Fast-FT and Blue102 structures revealed hydrolytic
degradation of the chromophores. In Fast-FT, chromophore-forming Met66 residue is
eliminated from the polypeptide chain, whereas Leu66 in Blue102 is cleaved out from the
chromophore, and although decarboxylated, remains attached to the preceding Phe65.
Hydrolysis of the chromophore competes with chromophore maturation starting from ether the
keto or enolate intermediates and is driven by the same residues that participate in
chromophore maturation.
S-274

Structural and functional analysis of LR11 Vps10p domain

Zenzaburo Nakata, Masamichi Nagae, Norihisa Yasui, Terukazu Nogi, Junichi Takagi

Institute for Protein Research, Suita, Osaka, Japan

LDLR relative with 11 binding repeats (LR11) is a 250-kDa type-1 membrane protein highly
expressed in cortex and cerebellum. This protein contains a domain that is structurally similar
to the Vacuolar protein sorting 10 protein (Vps10p), a sorting protein in yeast. LR11 is known
as a major risk factor of Alzheimer disease and is hypothesized to be involved in the
intracellular trafficking of the amyloid presursor protein, regulating the production of
amyloidgenic- peptide. Here we have analyzed the structure and function of LR11 Vps10p
domain to gain insights into the mechanism of intracellular protein sorting mediated by this
domain.

The binding affinity of LR11 Vps10p domain to a ligand, its own propeptide, was
measured by fluorescence polarization assay at various pH. The binding was markedly
reduced at acidic pH. This suggests that LR11 Vps10p domain may be involved in the
intracellular sorting in a pH dependent manner. The crystal structure of LR11 Vps10p domain
under the acidic condition was solved at 2.3 Å resolution. Vps10p domain assumes a ten-
bladed -propeller fold followed by two small domains, designated 10CC-a and 10CC-b
domains. Superposition of Vps10p domain of LR11 with that of sortilin reveals that the
putative ligand-binding site is masked by a loop connecting blade 6 and 7 of LR11. This result
suggests that the ligand recognition property of LR11 Vps10p domain is completely different
from that of sortilin.
S-277

Characterizing heme uptake and iron storage from pathogenic and non-pathogenic
Mycobacteria.
1 2 1 1 1
Lisa Marie McMath , Michael Tullius , Lana Cong , Nicholas Chim , Cedric Owens , Christine
1 2 1
Harmston , Marcus Horwitz , Celia Goulding
1 2
University of California- Irvine, Irvine, CA, United States, University of California- Los
Angeles, Los Angeles, CA, United States

Iron is essential for virtually all forms of life. Similar to most pathogens,
Mycobacterium tuberculosis (Mtb) must import iron from its host. We aim to understand novel
mechanisms of mycobacterial iron metabolism to potentially provide new avenues for anti-Mtb
therapeutic development.

We have shown that Mtb has a newly discovered heme uptake system, and have
identified the genomic region responsible. Found encoded within this genomic region is a
secreted protein that binds heme tightly, which we propose to be a hemophore. We have
solved its structure, and are currently attempting to solve its structure in complex with heme.
Additionally, we observed that non-pathogenic Mycobacterium bovis BCG (BCG) has an
attenuated heme uptake system in comparison with Mtb. By sequence alignment, the
homologous hemophore in BCG is identical to that of the Mtb hemophore, except for lysine 87
substituted with threonine (K87T). We hypothesize this substitution may contribute to the
attenuation seen in the BCG pathway. To address structural consequences, we have
crystallized the BCG apo-hemophore, and are currently attempting to crystallize it in complex
with heme. Furthermore, preliminary heme transfer experiments suggest an inefficient
transfer of heme from the BCG hemophore to one of the potential heme transporters.

Bacteria usually have cytosolic iron storage proteins. The mycobacterial ferritin (BfrB),
a structurally conserved iron detoxification and storage protein, has been identified and
crystallized. However, our preliminary crystallographic data show that the C-terminus of each
subunit in the 24-mer complex is disordered. By sequence alignment, we observed that the
Mtb BfrB polypeptide is longer than most ferritins, and its secondary structure prediction is
coiled. Thus, we engineered a truncated BfrB without the last 15 residues at the C-terminus.
This truncated BfrB still assembles into a 24-subunit oligomer, readily crystallizes, and we
hope to solve its structure by X-ray crystallography.
S-280

NorthEastern CAT Beam Lines at the Advanced Photon Source

Kay Perry, Steven Ealick, Malcolm Capel, Anthony Lynch, Frank Murphy, Igor Kourinov,
David Neau, Kanagalaghatta Rajashankar, Cynthia Salbego, Jonathan Schuermann,
Narayanasami Sukumar, James Withrow

Cornell University, Argonne, IL, United States

The NorthEastern Collaborative Access Team (NE-CAT) focuses on the design, construction,
and operational support of synchrotron X-ray beamlines for the solution of technically
challenging structural biology problems and provides an important resource for the
international research community. Currently there are two operational undulator beamlines:
24ID-C - fully tunable in the energy range from 6 to 22keV and 24ID-E - fixed energy at
~12.66keV. These operational beamlines are currently open to institutional members and
general APS users. Both beamlines are equipped with MD2 microdiffractometers, Q315
detectors and robotic sample automounters. NE-CAT provides stable, well-collimated beam
from 5 to 100 microns in diameter, tools for on-axis visualization of micron-sized crystals,
focused and de-focused beam, detector two-theta rotation, a mini-kappa goniometer for
optimal alignment of crystals, automatic data collection strategy prediction and automatic data
processing. NE-CAT maintains a website at http://necat.chem.cornell.edu/.

Funding for NE-CAT is provided through a P41 grant from the National Center for Research
Resources and from the NE-CAT member institutions.
S-281

Structural studies of the interaction between MD-1 and lipid IVa.

Sung-il Yoon, Minsun Hong, Gye-Won Han, Ian Wilson

The Scripps Research Institute, La Jolla, CA, United States

MD-1 is a secretory protein that can form a stable complex with RP105 on the cell surface.
The MD-1/RP105 complex can regulate the biological function of its evolutionarily related
complex, MD-2/TLR4, which recognizes bacterial lipopolysaccharide (LPS) and initiates
innate immune responses. Here, we report structural and biophysical data to demonstrate a
previously unidentified LPS binding activity for MD-1. The crystal structure of chicken MD-1
(cMD-1) was determined at 2.0 Å resolution by SIRAS method. MD-1 exhibits a β -cup like
fold containing a large hydrophobic cavity between two β -sheets, similar to that seen in MD-2.
Based on the structural similarities between MD-1 and MD-2, we hypothesized that MD-1
directly interacts with LPS. Indeed, LPS was identified as an MD-1 ligand by electrophoresis
and gel filtration analyses. Moreover, the interaction was supported by the 2.4 Å resolution
crystal structure of cMD-1 complexed with an LPS precursor, lipid IVa. The complex structure
reveals that the MD-1 cavity embeds one lipid IVa molecule in a mode that differs from that
used by MD-2. These results imply an important biological role for soluble MD-1 as a
regulator of the host LPS response.
S-283

The SER-CAT / UGA Crystal Shipping Kit for Data Collection

John P. Rose, James Tucker Swindell II, John Chrzas, John Gonczy, Bi-Cheng Wang

SER-CAT, Department of Biochemistry & Molecular Biology University of Georgia, Athens,

GA, United States

s›? ¡‹ ·‒¡? ⁄\ ? ? «¡« ¡‒ ? ⁄\ ¡? ⁄¡? ›› ? ‹¡¦¡ \‒„? ›? ¦\‒‒„›· ? ¡¢¢ ¦ ¡‹ ? \ \? ¦› ¡¦ ›‹? \‹ ? ¦‒„ \
¦‒¡¡‹ ‹£? ›‹? ? ¡\« ‹¡ K? ⁄¡? r›· ⁄¡\ ? q¡£ ›‹\ ? b› \ ›‒\ ¡? `¦¦¡ ? s¡\«? GrdqLb`sH? ⁄\
¡ ¡ ›fi¡ ? ?›•‹?¦‒„ \ ? ⁄ fifi ‹£? „ ¡«? \ ¡ ?›‹? ⁄¡?a¡‒¤¡ ¡„N`kr?fi·¦¤M??s⁄¡?rdqLb`s?N?tf`
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¦›‹ \ ‹ ? \ ? ⁄¡? ¡fl· fi«¡‹ ? ‹¡¦¡ \‒„? ›? ›\ K? ⁄ fi? \‹ ? ‒¡¦› ¡‒? US? ¦‒„ \ ? ›? ⁄¡? ¡\« ‹¡? · ‹£? \
\‹ \‒ ?s\„ ›‒?v⁄\‒ ›‹?bwqLPOO? ‒„? ⁄ fifi¡‒?G‹› ? ‹¦ · ¡ HM???r⁄ fifi ‹£?¤ ?¦›«fi›‹¡‹ ? ‹¦ · ¡Y
S?`kr? „ ¡?fi·¦¤ P?e›\«?c¡•\‒

P?o·¦¤? ⁄ fifi ‹£?¦‒\ ¡?GS?fi·¦¤ H P?a¡‹ ?⁄¡«› \

P?l\£‹¡ ¦?fi·¦¤?⁄› ¡‒ P?a¡‹ ?¦‒„› \ ?⁄› ¡‒

P?o·¦¤? ¡fi\‒\ ›‒ P?g\«fi ›‹?¦‒„ \ ?•\‹

P?h‹ ‒·¦ ›‹\ ?bc

rdqLb`s?⁄\ ?\ ›? ¡ ¡ ›fi¡ ?\?‹› ¡ ? ¡ ¦¡?¢›‒? ›\ ‹£?\‹ ?·‹ ›\ ‹£?fi·¦¤ ?¢‒›«? \«fi ¡ ?¦›‹ \ ‹¡
‹?¦‒„› \ K? ⁄¡?rdqLb`s?o·¦¤?k›\ ¡‒M The Puck Loader is a simple "hands-free" tool, which
eliminates common errors associated with manually loading (and unloading) ALS style pucks
from cryovials. The Puck Loader has no moving parts and allows the experimenter to load a
single puck within a fraction the time required for traditional manual loading / unloading
methods. Importantly the Puck Loader allows the user to load pins directly into the puck base
while still in the cryovial thus preventing loss or damage to crystal due to operator error. The
Puck Loader also allows the experimenter to safely recover pins from the puck directly into
the cryovial for storage.
Stop by the SER-CAT booth to see the SER-CAT Shipping Kit and Puck Loader and to get a
price list and order form.
Work supported by the SER-CAT Member Institutions, University of Georgia Research
Foundation and the Georgia Research AllianceM
S-284

Regulation of the essential M. tuberculosis peptidoglycan-precursor flippase

1,4 3 1 1
Christine Gee , Kadamba Papavinasasundaram , Sloane Blair , Christina Baer , Arnold
2 2 3 1
Falick , David King , Christopher Sassetti , Tom Alber
1
Department of Molecular and Cell Biology, QB3 Institute, University of California, Berkeley,
2
Berkeley, Ca, United States, Howard Hughes Medical Institute, University of California,
3
Berkeley, Berkeley, Ca, United States, Department of Molecular Genetics and Microbiology,
4
University of Massachusetts Medical School, Worcester, MA, United States, Australian
Synchrotron, Clayton, Vic, Australia

Receptor Ser/Thr kinases control broad aspects of physiology in bacteria, but little is known
about how the kinases regulate cellular pathways. Among the Mycobacterium tuberculosis
(Mtb) proteins essential in an animal model is the flippase that delivers the peptidoglycan
(PG) precursor, lipid II, to the cell surface for incorporation into the cell wall. In mycobacteria
and several other actinomycetes, the flippase has accessory domains, which presumably add
functionality or regulate the activity. We found that the Ser/Thr protein kinase, PknB,
efficiently phosphorylates the intracellular pseudokinase domain of the Mtb flippase, and this
single modification creates a binding site for the forkhead associated (FHA) domain protein
FhaA in vitro and in vivo. To define the mechanisms of recognition, we determined the crystal
structures of the flippase extracellular domain and the intracellular pseudokinase alone and in
combination with FhaA. The extracellular accessory domain is homologous to a galactose
binding domain, while the intracellular domain has a highly diverged, inactive protein-kinase
fold. These results suggest support a model in which extracellular PG regulates PG synthesis
by controlling assembly of a protein complex containing the lipid II flippase.
S-288

GfcC shows similarities to Wza and is important for group 4 capsule polysaccharide
expression.
1 2 1
Karthik Sathiyamoorthy , Ilan Rosenshine , Mark Saper
1 2
University of Michigan, Ann Arbor, MI, United States, The Hebrew University of Jerusalem,
Jerusalem, Israel

Many bacteria produce a polysaccharide capsule necessary for resisting host defenses and
biofilm formation. The group 4 capsule operon (gfc) in enteropathogenic E. coli contains
seven genes (gfcABCDE, etp and etk), each important for polysaccharide synthesis and
export [1]. Homologs of gfcE, etp and etk are present in many other capsule systems
including a group 1 capsule system (wza, wzb and wzc respectively) also present in E. coli.
The four other gfc genes encode secreted proteins of unknown function. GfcB and GfcD are
putative outer membrane lipoproteins, while GfcC is a periplasmic protein. We have
determined the crystal structure of GfcC at 1.8-Å resolution by the single wavelength
anomalous diffraction method. GfcC has two β -grasp domains similar to domains 2 and 3 of
the periplasmic region of Wza, but with little sequence identity. The Wza structure has a C-
terminal amphipathic helix that forms a novel transmembrane helical pore (~17Å) in the
observed octamer [2]. This was proposed to be the exit hole for the growing polysaccharide
chain [2]. GfcC also has a C-terminal amphipathic helix, but in contrast to Wza, it packs
against the β -sheet of one β -grasp domain and is partially occluded by a helical hairpin insert
from the other β -grasp domain, a structure that is clearly absent in Wza. As a result, GfcC
behaves as a soluble monomer in vitro. Although the Wza homolog GfcE (95% identity) is
also encoded in the gfc operon, the unique presence of gfcABCD suggests that the
mechanism for polysaccharide translocation may be more complex. Structural predictions of
the GfcD sequence suggest that it forms a transmembrane β -barrel structure, a structure
known to be important in other polysaccharide export complexes. Interestingly, homologs of
gfcC and gfcD are fused in some Burkholderia genomes suggesting that GfcC may make
important interactions with GfcD. Experiments to understand the function of GfcC in
polysaccharide export are in progress.

[1] Peleg, A., et al. (2005). Identification of an Escherichia coli operon required for formation
of the O-antigen capsule. J Bacteriol. 187(15): 5259–5266.

[2] Collins, R.F., Beis, et al. (2007). The 3D structure of periplasm-spanning platform required
for assembly of group 1 capsular polysaccharides in Escherichia coli. Proc Natl Acad Sci USA
104(7): 2360–2365.
S-290

Structural basis of phosphatidylinositol 4-phosphate recognition by Fapp1 PH domain

1 2 3 3 1
Ju He , Robert Stahelin , Joanna Gajewiak , Glenn Prestwich , Tatiana Kutateladze
1 2
University of Colorado Denver School of Medicine, Aurora, CO, United States, Indiana
3
University School of Medicine-South Bend, South Bend, IN, United States, University of
Utah, Salt Lake City, UT, United States

The four-phosphate-adapter protein (Fapp1) regulates formation and fission of post-Golgi

vesicles and directs the endocytic transport from the trans-Golgi network (TGN) to the plasma
membrane. Upon activation, cytosolic Fapp1 is recruited to the TGN membranes through the
interaction of its N-terminal Pleckstrin Homology (PH) domain with phosphatidylinositol 4-
phosphate [PtdIns(4)P] and the small GTPase ADP-ribosylation factor 1 (ARF1). Despite the
important role of Fapp1 in secretory membrane transport, the structural and biochemical
mechanisms by which Fapp1 exerts its functions have not been established. We applied high
field Nuclear Magnetic Resonance (NMR), Surface Plasma Surface (SPR), X-ray
crystallography and Mutagenesis experiments to investigate the dual recognition of Arf1 and
PtdIns(4)P by Fapp1 PH domain. Our data reveal that Fapp1 interacts with two anchoring
components using distinct binding interfaces. Mutations in these interfaces can specifically
block the Fapp1 binding to each component. Based on the structure model, we have
developed a metabolically-stabilized PtdIns(4)P analogue that selectively targets Fapp1 PH.
Our work shows great promise for the future development of therapeutics for Fapp1 related
disorders such as Legionnaires' disease.
S-292

Structural Interactions of Pilocarpine with Human Cytochrome P450 Enzymes

Aaron Bart, Natasha DeVore, Emily Scott

University of Kansas, Lawrence, KS, United States

Cytochrome P450 (P450) enzymes are a large family of heme thiolate proteins involved in the
metabolism of both endogenous compounds and xenobiotic compounds, including drugs.
Xenobiotic-metabolizing P450 enzymes can each bind and metabolize a diverse set of
substrates and often produce a variety of metabolites. Structures of the P450 enzyme family
reveal a highly canonical global protein fold, but with large variations in the active site size,
topology, and conformational flexibility. Though in vivo and in vitro metabolism data
demonstrate both overlapping substrate selectivity and substrate specificity, the structural
basis for this is often difficult to surmise.

The goal of the current work is to determine how a related set of human cytochrome P450
enzymes bind and interact with the common inhibitor and clinical muscarinic receptor agonist
pilocarpine. Pilocarpine inhibition of CYP2A6, CYP2A13, and CYP2E1-mediated metabolism
of the substrate p-nitrophenol revealed significant differential inhibition, with pilocarpine
inhibiting CYP2A13 much more efficiently than CYP2E1. In order to elucidate key amino
acids that are involved in pilocarpine binding, a 2.8 Å X-ray structure of CYP2A13 has been
determined with pilocarpine in the active site. Data collected at SSRL indicated a P1 space
group with 12 molecules in the asymmetric unit. The CYP2A13/pilocarpine co-crystal
structure was solved by molecular replacement. Several previously determined CYP2A13
structures were used as search models to locate molecules in the asymmetric unit that have
varying conformations. Pilocarpine binds in the CYP2A13 active site, forming a coordinate
covalent bond to the heme iron.

Comparison of this structure with other structures currently being generated of pilocarpine
bound to CYP2A6 and CYP2E1 is providing an understanding of how these closely related
enzymes each interact with the inhibitor pilocarpine.
S-293

Alternating access in E. Coli maltose transporter mediated by rigid-body rotations

Michael Oldham, Shanshuang Chen, Cedric Orelle, Amy Davidson, Jue Chen

Purdue University, West Lafayette, IN, United States

ATP binding cassette (ABC) transporters couple ATP binding and hydrolysis to the
translocation of substrates across the membrane bilayer. ABC transporters are composed of
two transmembrane domains (TMDs) coupled to two cytoplasmic nucleotide-binding domains
(NBDs). Bacterial importers, including the well-studied E. coli maltose transporter, also
employ a periplasmic substrate-binding protein for delivery of the substrate to the transporter.
We have previously reported structures of the maltose transporter in two independent
conformations: an inward-facing, nucleotide-free, resting state in which the transmembrane
translocation cavity is closed to the periplasm while the cytoplasmic NBDs are held open; and
an outward-facing conformation in which the TMDs outline a substrate-binding pocket open
toward the periplasm while ATP is poised for hydrolysis along the closed dimer interface of
the NBDs. We report here the structure of an intervening, nucleotide-bound, substrate pre-
translocation state in which a closed substrate-loaded binding protein is docked atop the
closed periplasmic gate of the inward-facing TMDs. Comparison of all three structures reveals
that alternating access of the substrate involves rigid-body rotations of the TMDs that are
coupled to the closure of the NBDs around the ATP to be hydrolyzed. Prior to docking of the
binding protein, key residues that position the ATP gamma-phosphate for hydrolysis are
sequestered away from the nucleotide-binding pocket. Docking of the binding protein brings
the coupled NBDs closer together to sense the nucleotides bound at their pre-formed dimer
interface.
S-294

Comparison of the SH3-guanylate kinase (GUK) module of the tight junction protein
ZO-1 with various MAGUK core modules reveals interdomain flexibility between the
SH3 and GUK domains

1 2 1 2 1
Ming Lye , Alan Fanning , Ying Su , James Anderson , Arnon Lavie
1 2
University of Illinois at Chicago, Chicago, Illinois, United States, University of North Carolina
at Chapel Hill, Chapel Hill, North Carolina, United States

Zonula Occludens-1 (ZO-1) is a membrane-associated guanylate kinase-like

(MAGUK) protein critical for the formation of tight junctions. This property of ZO-1 and its
ability to localize to the membrane has been attributed to the core module, which consists of
the two consecutive protein-protein interaction domains Src Homology 3 and guanylate
kinase, and an insert in the SH3 domain called the U5 region (SH3-U5-GUK).

We have solved the structure of the ZO-1 core module to 2.6 Å and observed the
conservation of interdomain beta ( strand interactions as predicted by the previously solved
core module structure of the homologous MAGUK protein post-synaptic density-95 (PSD-95).
This is mediated by main chain interactions between the two beta ( strands flanking either
side of the GUK domain to form a unit that completes the SH3 fold. Interestingly however,
comparison of the core module of ZO-1 with that of PSD-95 and ZO-3 revealed significant
differences in the conformations of the core module of all three MAGUKs due to interdomain-
angle differences between the SH3 and GUK domains. The ZO-1 core module adopts a more
open and more closed conformation compared to PSD-95 and ZO-3, respectively. These
conformational differences between the different MAGUKs reflect general variations within
MAGUK core modules that could have functional and regulatory implications.

We have also made the novel discovery that the unique 6 (U6) region of ZO-1,
composed of a stretch of acidic residues immediately C-terminal to the GUK domain, binds to
the core module in a bivalent-cation dependent manner through electrostatic interactions.
Using pull-down assays, we have shown that the U6 region and the calcium sensor protein
calmodulin both compete for similar binding sites on the core module. This direct binding
interaction between the U6 region and the core module may be one general mechanism by
which U6 regulates core module function.
S-297

Crystal Structure of the MyD88:IRAK4:IRAK2 Complex Reveals A Hierarchical Helical

Oligomer in TLR/IL1-R Signaling.

Su-Chang Lin, Yu-Chih Lo, Hao Wu

Weill Cornel Med College, New York NY, United States

MyD88, IRAK4 and IRAK2 are critical signaling mediators of the TLR/IL1-R superfamily. Here
we report the crystal structure of the MyD88: IRAK4: IRAK2 death domain (DD) complex,
which surprisingly reveals a left-handed helical oligomer that consists of 6 MyD88, 4 IRAK4
and 4 IRAK2 DDs. The assembly of this helical signaling tower is hierarchical, in which
MyD88 recruits IRAK4 and the MyD88: IRAK4 complex recruits the IRAK4 substrates IRAK2
or the related IRAK1. Formation of these complexes brings the kinase domains of IRAKs into
proximity for phosphorylation and activation. Composite binding sites are required for
recruitment of each of the individual DDs in the complex, which are confirmed by mutagenesis
and previously identified signaling mutations. Specificities in the MyD88: IRAK4 interaction
and in the recruitment of IRAK2 are dictated by both detailed molecular complementarity and
correspondence of surface electrostatics. The MyD88: IRAK4: IRAK2 complex provides a
template for Toll signaling in Drosophila and an elegant mechanism for versatile assembly
and regulation of DD complexes in signal transduction.
S-298

SrRietveld: A Next Generation Rietveld Refinement Program

1 1,3 1 1 1,3 1
Jiwu Liu , Peng Tian , Wenduo Zhou , Yingrui Shang , Chris Farrow , Pavol Juhas , Simon
1,2
Billinge
1 2
Columbia University, New York, NY, United States, Brookhaven National Laboratory,
3
Brookhaven, NY, United States, Michigan State University, East Lansing, MI, United States

SrRietveld is a highly automated software tool kit for Rietveld refinements. Compared to
traditional refinement programs, it is more efficient and easier to use. It is designed for
modern high throughput diffractometers and is capable of processing large volume of data.
SrRietveld currently makes use of conventional Rietveld refinement engines, such as GSAS
and FullProf. It is built to automate and extend the functions of those engines in a flexible and
uniform way so that new refinement engines can be incorporated easily as they become
available. SrRietveld is an open source software.
S-300

Effect of ILE274 Mutation on the Structure and Function of Catalase HPII of Escherichia coli

Vikash Jha, Peter Loewen

University of Manitoba, Winnipeg, Canada

Catalase or hydroperoxidase HPII of Escherichia coli is the largest known catalase, a class of
enzyme that degrades hydrogen peroxide (H2O2) with a high turnover rate, attributed to the
presence of more than one channel leading from the molecular surface to the active site
heme. The channel approaching the active site perpendicular to the plane of heme has been
considered as the main channel for the ingress of substrate H2O2 and has been the focus of
several studies. The second channel, which approaches the heme laterally, has not been
investigated in as much detail. Ile274 is located at the entrance to the lateral channel in close
proximity to the vinyl group of ring I of heme. This study investigates the effect of Ile
mutations on the structure and activity of catalase HPII. Site directed mutagenesis of the katE
gene of E.coli was used to change Ile274 to Gly, Ala, Val, Phe, Ser, and Cys. The Ile274Gly
and Ile274Ala variants exhibit 80% and 60% reduction in activity, respectively, whereas the
Ile274Val variant retained 70% of wild type activity. The Ile274Phe mutation and most
surprisingly the Ile274Ser mutation interfered with the folding of the protein such that no
variant protein accumulated. The results indicated that the size and the hydrophobicity of the
residue at this location are important determinants of enzyme activity and protein folding. The
Ile274Cys variant folded correctly but retained only 40% activity as compared to wild type.
The heme of the Ile274Cys variant could not be extracted by acetone-HCl suggesting
covalent cross-linking to the heme and this was confirmed by mass spectrometry and its
unreactivity with the thiol reactive reagent DTNB. Crystal of the four variants including
Ile274Gly, Ile274Ala, Ile274Val, and Ile274Cys were obtained by hanging drop vapour
diffusion method and crystal structures have been determined at ~1.6Å using X-ray
technique. Two significant changes in the structures of the variants compared to the native
enzyme include the heme being present in two orientations and the presence of an oxoferryl
species that was sensitive to X-irradiation.
S-301

Fine Tuning the Activity of Liver Receptor Homologue -1, an Orphan Nuclear Receptor

Manish Pathak, Eric Ortlund

Emory University School of Medicine, Atlanta, Ga, United States

Liver Receptor Homologue-1 (LRH-1), an orphan nuclear receptor, center to the breast and
colon cancer development has also been implicated in Diabetes, Obesity, Bileacid
homeostasis and Steroidogenesis. Being at the center of multiple pathways raises the
pharmaceutical importance of the ligand on the consequent impact. Unlike human Lrh-1
Ligand Binding Domain (hLrh-1), mouse LRH-1 (mLrh-1) and Drosophila ortholog Ftz-F1 use
distinct strategy to abolish ligand binding to stay constitutive. While, Ftz-f1 redirects helix H6
into its own pocket, mLRH-1 closes the pocket mouth. Borrowing six residues from mLrh-1
mouth into hLrh-1 converted it into an unliganded & unrecruited form akin to mLRH-1 shown
by x-ray crystal structure, which retained the ability to recruit coactivator in vitro. The change
induces a series of conformational changes in the Lrh-1 mouth that accommodates the
phosphate head group.

Here we show the ligand modulated coactivator recruitment by Lrh-1. Apparently constitutive,
phospholipid stripped hLRH-1 recruits co-activator 13.4 times weaker whereas choline
enhances the recruitment suggesting a hierarchy of ligands involved. Reduced activity with
liver extract prompts us to envisage a theory of fine tuning of the ligand mediated coactivator
recruitment instead of binary active-inactive forms. The putative hypothesis refers that lower
activity is a good activity. Identification of few ligands using mass-spectroscopy will also be
explained. Moreover, change in the secondary structure content of apo LRH-1 seen by
Circular Dichroism adds into the fine-tune theory from molecular dynamics point of view
signifying controlled execution highly imperative. A diverse comparative study with other
nuclear receptors highlight the versatility associated with LRH-1 and its multifaceted role in
cellular system at different stages of development.
S-302

Toward multi-sample data collection for macromolecular crystals: frozen crystal non-
isomorphism

Rita Giordano, Sean McSweeney, Alexander Popov

ESRF, Grenoble, France

The amount of diffraction data that can be obtained from a single protein crystal, is

limited by the radiation damage to the sample and this effect cannot be avoided. In those
cases where several crystals of the same type are available, the result of a structural study
may, potentially, be improved by using all crystals. This experimental method is especially
applicable to a set of micro crystals. In general less data can be obtained from a small crystal,
before significant radiation damage occurs because the diffracting volume is lower. In order to
gain from the use of multiple-crystal data collection strategies, the experiment has to be
properly constructed. There are no well developed protocols for organizing this kind of
measurement. The challenging task is the development of a new crystal ranking method that
will be based on the determination of isomorphism between crystals. The experiments have
been carried out on the beam line ID 23 EH-1 at ESRF, where 46 data set of cubic (space
group I213) Zn-free bovine pancreatic insulin were collected to 1.5 Å. The data sets were
processed using XDS (Version 30 January 2009) and the diffraction intensities were put on a
common scale using the program XSCALE (Version 30 January 2009). One arbitrary data set
was used as a reference for XDS; this reference data set has a completeness 99.4%, R factor
3.1% and I/sigmaI 41.75 (value calculated using XDS). Using the statistical computing
program R (http://www.r-project.org/) a multivariate statistical analysis was executed. A
hierarchical cluster analysis of the matrix of the correlation coefficients of the scaled
intensities was performed in order to select the best data sets collected. This analysis
revealed two principal clusters of insulin datasets. Additionally, the principal component
analysis (PCA) was used to identify

patterns in the data, to highlight similarity and differences between data sets. Other statistical
methods were applied, for example, distance matrix analysis to differentiate

the two clusters of insulin datasets.

S-304

X-ray-induced deterioration of disulfide bridges at atomic resolution

1,2 1 3 1
Tatiana Petrova , Stephan L. Ginell , Andre Mitschler , Youngchang Kim , Vladimir Y.
2 1 3 3
Lunin , Grazyna Joachimiak , Alexandra Cousido-Siah , Isabelle Hazemann , Alberto
3 1 1
Podjarny , Krzysztof Lazarski , Andrzej Joachimiak
1 2
Argonne National Laboratory, Argonne, IL, United States, Institute of Mathematical
Problems of Biology, Russian Academy of Sciences, Pushchino, Russian Federation,
3
Département de Biologie Structurale et Génomique, IGBMC, CNRS, ULP,INSERM, Illkirch,
France

Overall and site-specific X-ray-induced damage to porcine pancreatic elastase was studied at
atomic resolution at temperatures 100K and 15K. The experiments confirmed that the
irradiation causes the small movement of protein domains and bound water molecules in
protein crystals. These structural changes occur not only at 100K but also at as low as 15K.
An investigation of the deterioration of disulfide bridges demonstrated that: (i) a decrease in
γ
the occupancy of S atoms and the appearance of new cysteine rotamers occur
γ
simultaneously; (ii) the occupancy decrease is observed for all S atoms, while new rotamers
arise for some of cysteine residues; the appearance of new conformations correlates with the
accessibility to solvent (iii) the sum of the occupancies of the initial and new conformations of
a cysteine residue is approximately equal to the occupancy of the second cysteine residue in
7
the bridge; (iv) the most pronounced changes occur at absorbed doses below 1.4*10 Gy;
with only small changes occurring at higher doses. The comparison of the radiation-induced
changes in an elastase crystal at 100 and 15K suggested that the dose needed to induce the
deterioration of disulfide bonds and atomic displacements at 15K as those seen at 100K is
two times higher.
S-306

Structural Study on Metal Aluminium Amides M[Al(NH2)4]x (M = Li, Na, K, Mg, Ca; x = 1,
2)
1 2 1 1,2 1,2
Masami Tsubota , Taisuke Ono , Keiji Shimoda , Takayuki Ichikawa , Yoshitsugu Kojima
1 2
IAMR, Hiroshima University, Higashi-Hiroshima, Japan, ADSM, Hiroshima University,
Higashi-Hiroshima, Japan

The search for alternative fuel is an urgent problem to be solved. One of the forerunners is
hydrogen. In these days, much interest has been focused on the hydrogen storage materials
composed of light elements, so-called chemical hydride. A composite technique is quite
powerful to improve gas desorption properties for chemical hydrides. Recently, Janot et al.
focused on lithium aluminium amide LiAl(NH2)4 and reported that the composite of LiH and
LiAl(NH2)4 released more than 5 mass% H2 below 130 °C. Thermal decomposition pathway of
the composite was also proposed. However, the composite become amorphous during
decomposition and the detailed reaction products are still unclear. Therefore, it is needed to
clarify the NH3 desorption mechanism of pristine LiAl(NH2)4 for better understanding the
complex thermal reaction of the composite from the structural point of view.

In this study, we have investigated the detailed structural properties of the decomposition
products by using in situ synchrotron X-ray diffraction and X-ray total scattering techniques as
well as the thermal gas desorption properties by thermogravimetry-mass spectroscopy.
Especially, the thermal decomposition pathway of M = Li had been re-examined.

M[Al(NH2)4]x was synthesized by milling the raw materials in liquid NH3. From the results of
synchrotron radiation X-ray diffraction, it was found that LiAl(NH2)4, NaAl(NH2)4, KAl(NH2)4,
Mg[Al(NH2)4]2, and Ca[Al(NH2)4]2 could be indexed with single phases with monoclinic (a =
9.50 Å, b = 7.37 Å, c = 7.42 Å, = 90.1 ), monoclinic (a = 13.24 Å, b = 6.05 Å, c = 7.34 Å, and
= 94.0 ), orthorhombic (a = 11.36 Å, b = 8.85 Å, c = 6.15 Å), hexagonal (a = 12.10 Å, c =
7.95 Å), and orthorhombic (a = 12.29 Å, b = 6.45 Å, c = 6.44 Å) unit cells, respectively. For M
= Li, the results of high temperature in situ X-ray diffraction measurements showed that
LiAl(NH2)4 became amorphous phase with NH3 desorption above 135 °C and the results of
PDF showed that a tetrahedral AlN4 unit was kept during decomposition. The decomposition
mechanism will be described.

Acknowledgement

This work was partially supported by NEDO under “Advanced Fundamental Research Project
on Hydrogen Storage Materials”.
S-308

Structural Determination of Synthetic Intermediates of Disubstituted Amino Acids –

Critical Building Blocks in the Design of Inhibitors of Amyloid Beta Aggregation

Gregory McCandless, Frank Fronczek

Department of Chemistry, Louisiana State University, Baton Rouge, LA, United States

Peptide aggregation is a common theme among major human disorders, such as Alzheimer’s
disease and Type II diabetes. The aromatic amino acids (phenylalanine and tyrosine) within
the peptide sequence of amyloid beta (linked to Alzheimer’s disease) and islet amyloid
polypeptide (associated with Type II diabetes) are believed to be the key aggregating
initiators via π -stacking. This aggregation process can be inhibited by using short peptide
mimics that will bind via self-recognition and block subsequent bonding using sterically bulky
side chains. Peptide inhibitors containing the disubtituted amino acid, dibenzyl glycine, have
been very effective (even in sub-stoichiometric amounts) in previously published in vitro
results involving aggregation studies with amyloid beta. However, the low yielding
dibenzylation step with benzyl bromide in the synthesis of this unnatural amino acid has been
disheartening – it is the first step of a series of four reaction steps. Nonetheless, a new
disubstitution strategy was established with moderate increases in product yield using
halogen exchange (similar to the Finkelstein Reaction) and new analogues were developed
using para electron donating substituents to help stabilize a positive charge on the carbon
undergoing nucleophilic attack. Also, a different synthetic mechanism has been discovered
that undergoes a radical disubtitution reaction using poor halide leaving groups and para
electron withdrawing substituents.

Single crystal X-ray diffraction results confirmed the desired synthetic intermediates for the
low yielding dibenzylation step as well as the competing substitution reaction pathways (SN2
or SN1 vs. SRN1) and types of benzylation (C-benzylation vs. O- benzylation).
S-310
Structural Biology of Rift Valley Fever Virus Nucleoprotein

Donald Raymond, Mary Piper, Sonja Gerrard, Janet Smith

University of Michigan, Ann Arbor, MI, United States
Rift Valley fever virus (genus Phlebovirus, family Bunyaviridae) is an arthropod-borne
pathogen endemic to sub-Saharan Africa that has spread to North Africa and the Arabian
peninsula. The virus infects livestock and humans and is primarily transmitted by infected
mosquitoes. The enveloped virus has a segmented negative-sense RNA genome that is
completely encapsidated by a nucleoprotein (N).

We developed a procedure to separate the RNA from the recombinant N (recN) by

denaturation, resulting in predominantly monomeric refolded N with ~10% existing as a dimer.
The approximate number of RNA nucleotides in the nucleocapsid complex was determined by
extracting the RNA from the natively purified N-RNA complex. Binding affinity and
stoichiometry were determined using a reconstituted recombinant N-RNA complex. Native
crystals of refolded N diffracted to 1.9 Å and the structure was solved using a 2.4-Å two-
wavelength MAD dataset from a crystal of SeMet N. N crystallized as a symmetric dimer in
which the N-terminus and an internal loop-helix are engaged in the subunit interface. The
compact subunit consists of two helical lobes, each with a novel fold. Potential RNA-binding
sites on opposite sides of the dimer were identified on surfaces that are uniformly
electropositive among the highly conserved phlebovirus N. The fold, dimeric organization,
lack of an electropositive cleft and EM micrographs of viral and reconstituted RNPs differ
substantially from N of other negative-sense RNA virus families, indicative of a novel RNA
encapsidation strategy.

Supported by NIH grant P01-AI055672 to JLS and an NIH Biophysics Training Grant
to DDR.
S-312

Syntheses and Crystal Structures of Some Solid-State Uranium Compounds

George N. Oh

Northwestern Univ., Evanston, IL, United States

In our continuing efforts to discover new solid-state uranium compounds and characterize
their physical properties we have recently examined compounds of the type A/U/M/Q, where
A is an alkali metal, M is Pd or Pt, and Q is S or Se. No previous work appears to have been
done in this area. From high-temperature reactions we have obtained two distinct
compositions, namely A2UM3Q6 and A2U6M4Q17, as deduced from single-crystal X-ray
diffraction determinations. A single reaction can yield both compositions.. The A2UM3Q6
compounds crystallize in the NaBa2Cu3O6 structure type. In this structure the M atoms are
coordinated in a square-planar manner by four Q atoms and these MQ4 units edge share to
form hexagons. The A2U6M4Q17 compounds crystallize in a new structure type that consists
of a network of MQ4 square-planar units, UQ7 and UQ8 units, as well as ordered AQ9 units.
Rb2U6Pt4Se17, as opposed to Rb2U6Pd4Se17, differs structurally in that the Rb atom is
disordered over two sites.
S-314

Growth of large Scandium substituted Barium hexaferrite single crystals BaScxFe12-

xO19 with small structure defects

1 2 1 2
Juergen Kraeusslich , Carsten Dubs , Ortrud Wehrhan , Peter Goernert
1 2
University, Jena, Germany, INNOVENT, Jena, Germany

Due to its outstanding magnetic properties, M-type hexagonal ferrites such as Barium
hexaferrite single crystals (BaFe12O19) or with still improved characteristics Scandium
substituted Barium hexaferrite (BaScxFe12-xO19) are a very suitable basic material for high-
frequency filter components used in the 40 to 100 GHz range of modern microwave
measurement techniques.

The growth of large Scandium substituted Barium hexaferrite single crystals up to 2 cm

edge length had been succeeded using the Top Seeded Solution Growth (TSSG) technique
(Fig. 1). Away from the seed growth sectors emerge and form larger regions

with nearly perfect crystal structure. The high

y resolution X-ray transmission topography (Fig. 2),
carried out at the synchrotron facility in Grenoble
(ESRF), had only revealed a few dislocations and
c growth striations due to the marginal fluctuation of
stoichiometric composition.

Fig. 1: Large Ba(ScFe)12O19 single crystal grown

a1 by Top Seeded Solution Growth (TSSG) technique
10 mm

Fig. 2: High resolution X-ray transmission topograph of a c-

cut Sc substituted Ba hexaferrite single crystal slice. The
shown growth sector is almost perfectly, only few dislocations
growth sector
and growth striations are seen. g - X-ray diffraction vector ┴
(1-10 0)

2 mm g
S-316

Structure-Properties Relationship along the (Co1-xNix)Al2O4 Spinel Series: An

experimental and Theoretical study
1,2 2 1 2
Delphine Gout , Gregory MacDougall , Thomas Brueckel , David Mandrus
1 2
JCNS, Oak Ridge, TN, United States, ORNL, Oak Ridge, TN, United States

The Spinel structure is one of the most common structural arrangements and mineral phases
on earth. We have recently reinvestigated the (Co1-xNix)Al2O4 Spinel series using both neutron
and X-ray powder diffraction. We have refined the crystal structures, atomic distributions of
five different Co1-xNixAl2O4 spinel compounds for x=0, 0.25, 0.5, 0.75 and 1 and the
refinements indicate specific site distributions between Ni and Co in the octahedral and
tetrahedral sites. Our goal was to understand the chemical influence on the magnetic and
optical properties. Theoretical calculations as well as optical measurements have been
performed to understand the structure-properties relationship in the spinel series.
S-318

“Unpublishable” Data: Does My R-factor Look Big in This?

Amber L. Thompson, David J. Watkin

Chemical Crystallography, Oxford, United Kingdom

One of the most common questions asked by synthetic chemists is to crystallographers is, “Is
the R-factor less than 5%?” Although often a good indicator of the quality of the data and
refinement, a bad structure may have a low R-factor and a good structure may have a high R-
factor. Thus this (and other indicators) can condemn structures to languish unpublished,
while adding conviction to incorrect results (see 70 publications in Acta Crystallographica
Section E by Zhong and Liu from Jinggangshan University, China for examples). There is no
question that in an ideal world every structure would be perfect, but we don’ t all work with
perfect crystals all of the time. Recollecting data to improve these statistics consumes
instrument time, samples and manpower as well as potentially delaying publication of results.
In cases where the structure determination is part of a package of analytical techniques, a
cost-benefit analysis may show that this is a poor use of resources if it makes no difference to
the conclusions drawn.

So, what governs whether a structure is correct and publishable? We believe that this
depends on the context, i.e. the required chemical information coupled with the
crystallographic “problems”. For example, if the starting materials are known, a chemically
sensible structure that agrees with *ALL* the available data (including bulk analysis
techniques like NMR) should be reported in the literature, even if there are crystallographic
difficulties. Based on a clearly stated "fitness for purpose", very few structures become
“unpublishable”; indeed, even where there is an unidentified problem giving a poor
refinement, a partial solution may be useful corroborative evidence when taken with other
results.

A selection of examples will be presented including good structures with high R-indices; bad
structures with low R-indices; interesting structures published with an unknown spacegroup;
published structures with exceptionally poor data, and structures with other, sometimes
unidentified difficulties.
S-320

The X-ray Beam and Biological Crystal Visualization for Macromolecular

Crystallography.

K. J. Gofron, M. Molitsky, R. W. Alkire, N. E. C. Duke, A. Joachimiak

Argonne National Laboratory, Argonne, IL, United States

The SBC on-axis visualization system allows viewing of X-ray beam and biological crystal
from X-ray beam direction, and without parallax distortion. The system was constructed using
non-dispersive optics: a long working distance Maksutov–Cassagrain reflective microscope,
and right angle (45º) mirror. This on-axis geometry allows crystal visualization during
diffraction data collection with full Kappa geometry.

An x-ray beam and biological crystal imaging system during data collection has been
developed. The direct X-ray beam uses X-ray excited ultraviolet (UV) fluorescence. The high-
energy radiation such as X-ray and Middle UV (MUV) radiation excite “visible” light
luminescence from biological materials, which can be imaged with CCD cameras. The
fluorescence from biological crystals is primarily emitted as near UV (NUV) wavelengths
between 300-360 nm depending on a biological material and surrounding environment. We
demonstrate detection of biological crystal location using X-ray excited UV fluorescence. We
discuss techniques for biological crystal location using intrinsic X-ray excited and MUV
excited, UV fluorescence from biological crystals.

The X-ray beam can be characterized using a scintillator (phosphor or a single crystal) that
converts X-ray photons into visible light photons, which can be imaged using SBC on-axis
optics. The X-ray penetration is dependent on the composition of the scintillator (especially
effective Z) and X-ray energy. Several scintillators have been used to visualize X-ray beams.
Here we compare CdWO4, PbWO4, Bi4Ge3O12, Y3Al5O12:Ce (YAG), and Gd2O2S:Tb
(phosphor). The synchrotron X-ray beam profile studies were done using on-axis and off on-
axis imaging. We determined that scintillators made of CdWO4 and similar high-Z single
crystal materials are best suited for the energy range (7-20 keV) and are most suitable for
beam visualization for macromolecular crystallography applications. These scintillators show
excellent absorption, optical, and mechanical properties.

This work was supported by the U.S. Department of Energy, Office of Biological and
Environmental Research, under contract DE-AC02-06CH11357.
S-322

THE STRUCTURE OF THE MUSCLE GROWTH INHIBITOR MYOSTATIN BOUND TO

FOLLISTATIN 288: INSIGHTS INTO RECEPTOR UTILIZATION AND HEPARIN BINDING
1 2 3 2
JENNIFER CASH , CARLIS REJON , ALEXANDRA MCPHERRON , DANIEL BERNARD ,
1
THOMAS THOMPSON
1 2
UNIVERSITY OF CINCINNATI, CINCINNATI, OH, UNITED STATES, MCGILL
3
UNIVERSITY, MONTREAL, QC, CANADA, NATIONAL INSTITUTES OF HEALTH,
BETHESDA, MD, UNITED STATES

Myostatin is a member of the transforming growth factor-

(TGF-
) family and a strong
negative regulator of muscle growth. Here, we present the crystal structure of myostatin in
complex with the antagonist follistatin 288 (Fst288). We find that the prehelix region of
myostatin very closely resembles that of TGF-
class members and that this region alone can
confer signaling through the non-canonical type I receptor Alk5. Furthermore, the N-terminal
domain of Fst288 undergoes conformational rearrangements to bind myostatin. Additionally, a
unique continuous electropositive surface is created when myostatin binds Fst288, which
significantly increases the affinity for heparin. This translates into stronger interactions with
the cell surface and enhanced myostatin degradation in the presence of either Fst288 or
Fst315. Overall, we have identified several characteristics unique to myostatin that will be
paramount to the rational design of myostatin inhibitors that could be used in the treatment of
muscle-wasting disorders.
S-324

Structure of a leucine-rich repeat domain in the NLR family.

Minsun Hong, Sung-Il Yoon, Ian Wilson

The scripps research institutue, La Jolla, CA 92037, United States

Host pattern recognition molecules (PRMs) activate the innate immune system by
recognizing conserved microbial associated molecular patterns (MAMPs) and danger-
associated molecular patterns (DAMPs). TLRs (Toll-like receptors) represent a class of
membrane-spanning PRMs that have been extensively studied in the past decade. NOD-like
receptors (NLRs, Nucleotide-binging domain, Leucine-Rich repeat containing protein) have
recently emerged as a second family of PRMs that are located intracellularly and detect
various MAMPs and DAMPs inside the host cells. Approximately 20 NLR proteins have been
found in the mammalian genome and all are characterized by three distinct domains: an N-
terminal protein-protein interaction domain, central NOD domain and C-terminal LRR.
Mutations in NLR genes have been associated with complex chronic inflammatory barrier
diseases (e.g. Crohn's disease, bronchial asthma).

However, understanding of NLRs in a molecular level has been limited due to lack of
biophysical and structural knowledge about NLR proteins and their ligands since the various
expression systems have failed to provide enough materials of recombinant proteins for
biochemical characterization. To answer questions regarding how NLRs interact with their
ligands and initiate signaling, we have been pursuing x-ray crystallographic studies on NLR
members. We have successfully expressed and determined the crystal structure of an NLR-
LRR protein which encodes a signature LRR domain in NLRs. The three-dimensional
structure of the NLR-LRR and its novel insights into other NLR, as well as other LRR protein,
families will be discussed.
S-326

Recombinant techniques for the production of protein-protein complexes for structural

characterization

Gyorgy Babnigg, Robert Jedrzejczak, Boguslav Nocek, Adam Stein, William Eschenfeld,
Lance Bigelow, Changsoo Chang, Gekleng Chhor, Marianne Cuff, Yao Fan, Grazyna
Joachimiak, Youngchang Kim, Hui Li, Jurek Osipiuk, Ella Rakowski, Kemin Tan, Christine
Tesar, Alicia Weger, Ruiying Wu, Andrzej Joachimiak

MCSG, Argonne National Laboratory, Argonne, IL, United States

X-ray crystallography provides an unparalleled insight into the functions of proteins and
became a method of choice for the understanding biology at the atomic level. While protein-
protein complexes perform most functions in cells, far fewer structures are available of them
than those of monomers or homooligomers. Since a large fraction of cellular heterooligomeric
complexes are stable, they can be directly purified from their native host for structure
determination. However, this approach is feasible for abundant protein-protein complexes.
Less abundant complexes can be expressed by recombinant techniques and reconstituting
complexes, or by co-expressing the interacting partners using a bicistronic vector. These
techniques however are neither compatible with high-throughput (HTP) operations, nor are
they economical. We have recently developed a technique amenable to HTP operation using a
standard Ligation Independent Protocol (LIC). Target selection strategies are based on
existing interaction data generated by experimental methods and on bioinformatics analyses.
A bioinformatics pipeline was developed to identify a set of 384 protein-protein complexes from
the MCSG reagent genomes utilizing the IrefIndex protein interaction database and the
MicrobesOnline resource. We employed two HTP cloning strategies: the co-expression of the
interacting partners as an operon (‘Operon-strategy’), and as a salvage pathway, as a
bicistronic cassette (‘Eps-RBS-fusion strategy’). We are able to co-express proteins from up to
three genes using this approach. We have processed more than 50 complexes in large-scale
purification and obtained crystals for more than 20 of them. Structures of 3-oxoadipate coA-
transferase and molybdopterin converting factor from Helicobacter pylori have been
determined. We demonstrate that our target selection strategy combined with the experimental
methods can lead to the successful HTP and low cost structure determination of protein-
protein complexes.
This work was supported by National Institutes of Health Grant GM074942 and by the U.S.
Department of Energy, Office of Biological and Environmental Research, under contract DE-
AC02-06CH11357
S-328

Determining the 2.2 Å Structure of Human Notch NRR1 Bound to the Fab Fragment of
an Antagonistic Antibody
Gladys de Leon, Chris Seibel, Sarah Hymowitz

Genentech Inc, South San Francisco, United States

The Notch signaling pathway is a key component in mammalian cell fate and growth.
Aberrant signaling through each receptor has been linked to numerous diseases, particularly
cancer, making the Notch family a compelling target for new drugs. To better elucidate the
discrete functions of the individual Notch receptors, phage display technology was used to
generate anti-Notch1 and anti-Notch2 inhibitory antibodies. To understand the molecular
basis of the specificity and inhibitory mechanism of these antibodies, we determined the 2.2 Å
crystal structure of the Fab fragment of the anti-Notch1 antibody bound to the Notch1
Negative Regulatory Repeat (hereafter referred to as NRR1). Initial non-single blade-like
crystals were readily obtained with commercial screens. Using synchrotron radiation sources
we collected a data set with a nominal resolution of 3.5 Å, but with significant anisotropy and
high Rsym values. Successive rounds of microseeding led to crystals with improved 3-
dimensionality and more uniform composition. A 2.2 Å data set was collected using
sychrotron radiation. Using Phaser, we found a solution with two complexes forming the
asymmetric unit with parallel orientation of the two complexes. The refined structure revealed
that NRR1 forms a compact structure very similar to that of human Notch2 or NRR1 with
2+
three Ca ion-binding LNR (Lin-Notch Repeat) modules wrapped around the core HD
domain. The structure reveals that the apparent effect of Fab binding is to stabilize the LNR-
HD interactions such that a critical cleavage site is not accessible, thus keeping the Notch1
signaling cascade quiescent. This hypothesis is supported by the observation that the Fab
does not directly occlude the processing site, but instead binds at the interface between the
HD module, LNR1 and LNR2.
S-330

Structural Basis for Recognition of Diubiquitins by NEMO

1 1 1 2 2
Yu-Chih Lo , Su-Chang Lin , Carla C. Rospigliosi , Dietrich B. Conze , Chuan-Jin Wu ,
2 1 1
Jonathan D. Ashwell , David Eliezer , Hao Wu
1 2
Weil Cornell Medical College, New York, NY, United States, National Cancer Inst. NIH,
Bethesda, MD, United States

NEMO is the regulatory subunit of the IkappaB kinase (IKK) in NF-kappaB activation, and its
CC2-LZ region interacts with Lys63 (K63)-linked polyubiquitin to recruit IKK to receptor
signaling complexes. In vitro, CC2-LZ also interacts with tandem diubiquitin. Here we report
the crystal structure of CC2-LZ with two dimeric coiled coils representing CC2 and LZ,
respectively. Surprisingly, mutagenesis and nuclear magnetic resonance experiments reveal
that the binding sites for diubiquitins at LZ are composites of both chains and that each
ubiquitin in diubiquitins interacts with symmetrical NEMO asymmetrically. For tandem
diubiquitin, the first ubiquitin uses the conserved hydrophobic patch and the C-terminal tail,
while the second ubiquitin uses an adjacent surface patch. For K63-linked diubiquitin, the
proximal ubiquitin uses its conserved hydrophobic patch, while the distal ubiquitin mostly
employs the C-terminal arm including the K63 linkage residue. These studies uncover the
energetics and geometry for mutual recognition of NEMO and diubiquitins.
S-332

X-ray Structural Studies of Four Symmetrical and Unsymmetrical Nonlinear Optical

Organic Dyes

Paul Tongwa, Andrii Gerasov, Artem Masunov, Olga Przhonska, Eric Van Stryland, Tatiana
Timofeeva

New Mexico Highlands University, Las vegas, NM, United States

We discuss the structural characterization of four linear and nonlinear dyes, including the
effects of conjugation length and terminal groups. Understanding the relations between
molecular structure and nonlinear optical (NLO) properties in organic materials has been of
interest for many years. We report the single crystal X-ray diffraction studies of some anionic
symmetrical A - π -A, (I, II, IV), and neutral asymmetrical D - π - A, (II) cyanine dyes. The
extended π -electron delocalizations/excitations that occur in these and other highly
conjugated and polycyclic organic molecules is the basic origin of their observed spectral
properties. Bond length alternation values confirm π - conjugations in these materials.
Spectral studies leading to large two-photon absorption (2PA) and excited state absorption
(ESA) cross-sections are also reported.
SP.02

Uranium and Neptunium Solid-State Compounds: Adventures in Syntheses,

Crystallography, and Characterization.

James Ibers

Northwestern Unvi., Evanston, IL, United States

The structural chemistry of the lighter actinides, though to a reasonable extent accessible,
has been neglected. This talk with discuss some aspects of our recent work on solid-state
uranium and neptunium chalcogenides and pnictides. Safety issues, problems with
syntheses, crystallographic problems, and uncertainties in complete characterization will be
illustrated for systems ranging from the cubic A5Cu12U2S15 compounds to the AAn2Q6
compounds, where A = alkali metal; An = U or Np; Q = S or Se. Some structural relationships
will be illustrated for systems ranging from the AnCuOP compounds to neptunium
thiophosphates.

*Supported by U. S. Department of Energy, Basic Energy Sciences, Chemical Sciences,

Biosciences, and Geosciences Division and Division of Materials Sciences and Engineering
Grant ER-15522.
TR.01.1

The USC-BNL Collaboration: 25 Years of Metal Hydride Structures

Thomas F. Koetzle

Brookhaven National Laboratory, Upton, New York, United States

Beginning in 1973, and continuing for 25 years, a collaboration spearheaded by Robert Bau
resulted in a series of pioneering neutron diffraction studies of transition-metal hydrides. In
work carried out at the Brookhaven High Flux Beam Reactor, the USC-BNL collaboration
produced in total more than 30 hydride structures, featuring among them terminal, edge- and
face-bridging, and interstitial hydride ligands. A wealth of information was obtained on the
bonding of hydrogen to metals. Important trends emerged including, for example, estimates
of the variation of M-H distance with H coordination number. This talk will present highlights
from the 25 years of metal hydride structures. A detailed review is included in Bau, R.;
Drabnis, M. H. Inorg. Chim. Acta 1997, 259, 27–50.

Work at BNL was supported by the United States Department of Energy Office of Basic
Energy Sciences.
TR.01.2

Commissioning of the Time-Resolved Laue Single-Crystal Neutron Diffractometer

(TOPAZ) at the Spallation Neutron Source

Xiaoping Wang, Christina M. Hoffmann, Matthew J. Frost

Oak Ridge National Laboratory, Oak Ridge, United States

The TOPAZ single-crystal neutron diffractometer entered the commissioning phase in

September 2009. It uses multiple area detectors (48 when all installed) that are highly efficient
and capable of resolving time-of-flight pulsed neutrons in microsecond time scale. This
efficient use of neutron beam flux allows data collection times on the order of minutes rather
than hours for many samples. After successful completion of the commissioning phase, we
expect that TOPAZ will be well positioned to collect data on sub-millimeter size single crystal
samples. The choices of sample environment now range from a three-circle goniometer with
fixed chi-axis for data collection at ambient temperature, a cryogenic goniometer for low
temperature experiments, to an option to mount a 5 Tesla cryogenic magnet for magnetic
structure measurements. The instrument team is working with the SNS Sample Environment
Group to expand the sample handling capabilities to accommodate the requests from the
science community. During commissioning TOPAZ will be continuously tested and improved
to accept friendly user proposals in Fall 2010.

This research is supported by UT Battelle, LLC under Contract No. DE-AC05-00OR22725 for
the U.S. Department of Energy, Office of Science.
TR.01.3

Big Metals, Small Ligands: Characterization of the 15-Coordinate Complex Thorium

Aminodiboranate [Th(H3BNMe2BH3)4] by Single Crystal Neutron Diffraction
1 2 1 3 4
Paula Piccoli , Scott Daly , Arthur Schultz , Tanya Todorova , Laura Gagliardi , Gregory
2
Girolami
1 2
Argonne National Laboratory, Argonne, IL 60439, United States, University of Illinois at
3
Urbana-Champaign, Urbana, IL 61801, United States, University of Geneva, 1211 Geneva 4,
4
Switzerland, University of Minnesota, Minneapolis, MN 55455, United States
-
The anionic borohydride ligand, [BH4] , is known to have monodentate, bidentate or tridentate
binding modes, making it a useful ligand for the achievement of high coordination numbers in
inorganic and organometallic complexes. Employing a metal center with a large atomic
radius, such as an actinide, with smaller chelating ligands like borohydrides, increases the
chance of obtaining complexes with coordination numbers higher than 12. While 14-
coordinate actinide complexes such as [U(BH4)4(thf)2] have been previously reported, no
complex with higher coordination numbers has been able to be unambiguously structurally
characterized. Due to the low sensitivity of X-rays for the detection of hydrogen atoms and
the domination of the heavy metal in the scattered X-ray data, neutron diffraction data is
invaluable for accurately characterizing such complexes.

Reaction of ThCl4 with Na(H3BNMe2BH3) in tetrahydrofuran produces the monomeric complex

[Th(H3BNMe2BH3)4] (1), a molecule for which DFT calculations predict a theoretical maximum
1
coordination number of 16 in the gas phase. Neutron diffraction data collected at the IPNS at
low temperature reveal that all but one of the hydrogen atoms on the chelating
aminodiboranate ligands coordinate to the thorium center, bringing its Werner coordination
number to 15 in the solid state, the highest for any structurally characterized complex to date.
Upon heating, complex 1 undergoes a thermal reaction to produce the sterically less crowded
14-coordinate [Th(H3BNMe2BH3)2(BH4)2].

Herein we describe the synthesis and structural characterization of complex 1, its

characterization with complementary spectroscopic methods, DFT calculations and
comparisons to analogous high coordination actinide complexes containing hydride or
borohydride ligands.

1
S. R. Daly et al, Angew. Chem.. Int. Ed., DOI: 10.1002/anie.200905797
TR.01.4

Making the Molecular Movie: First Frames

1,2
R. J. Dwayne Miller
1 2
University of Toronto, Toronto, Ontario, Canada, Center for Free Electron Laser Science
and University of Hamburg, Hamburg, Germany

Femtosecond Electron Diffraction has enabled atomic resolution to structural changes as they
occur, essentially watching atoms move in real time -- directly observe transition states. This
experiment has been referred to as "making the molecular movie" and has been previously
discussed in the context of a gedanken experiment. With the recent development of
femtosecond electron pulses with sufficient number density to execute single shot structure
determinations, this experiment has been finally realized. A new concept in electron pulse
generation was developed based on a solution to the N-body electron propagation problem
involving up to 10,000 interacting electrons that has led to a new generation of extremely
bright electron pulsed sources that minimizes space charge broadening effects. Previously
thought intractable problems of determining t=0 and fully characterizing electron pulses on the
femtosecond time scale have now been solved through the use of the laser pondermotive
potential to provide a time dependent scattering source. Synchronization of electron probe
and laser excitation pulses is now possible with an accuracy of 10 femtoseconds to follow
even the fastest nuclear motions. The camera for the “molecular movie” is now in hand.
Atomic level views of the simplest possible structural transition, melting, have been obtained
for a number of systems involving both thermal and purely electronically driven atomic
displacements. Optical manipulation of charge distributions and effects on interatomic
forces/bonding can be directly observed. New phenomena involving cooperative phase
transitions in strongly correlated electron systems have also been observed. The primitive
origins of molecular cooperativity has also been discovered. These new developments will be
discussed in the context of developing the necessary technology to directly observe the
structure-function correlation in biomolecules -- the fundamental molecular basis of biological
systems.
TR.01.5

Neutron Protein Crystallography. --------Hydrogen- and Hydration- Sensitive

Structural Biology.

Nobuo Niimura

Ibaraki University, Tokai-mura,Ibaraki-ken, Japan

The three dimensional structure determinations of biological macromolecules such as

proteins and nucleic acids by X-ray crystallography have improved our understanding
of many important life processes. In many cases, these results have clearly
suggested that hydrogen atoms and water molecules around proteins and nucleic
acids could play a crucial role in many physiological functions. However, since it is
very hard to determine the positions of hydrogen atoms in protein molecules using X-
rays alone, a detailed discussion of protonation and hydration sites often involves
some guesswork. In contrast, it is well known that neutron diffraction provides an
experimental method of directly locating hydrogen atoms, but unfortunately, to date,
there are relatively few examples of biological systems studied by single-crystal
neutron crystallography since the collection of a sufficient number of Bragg
reflections is a time-consuming process. And, perhaps more importantly, the
requirement of large single crystals (with volumes in the range of 1 - 10 mm3) has
been a serious limitation. Breakthrough technical events in the neutron
macromolecular field have been the development of the neutron imaging plate (NIP),
the adoption of Laue methods at reactor sources, and most recently the LANSCE
time-of-flight electronic detector for neutron protein crystallography. Thus these three
technical developments have allowed exploration of the main frontiers of the
capability of neutron protein crystallography. The current development of "next
generation" spallation neutron sources, such as the J-PARC (Japanese Proton
Accelerator Research Complex) in Japan and the SNS (Spallation Neutron Source)
in the USA, will enable several more powerful protein crystallographic instruments to
be installed. In these new spallation sources, a gain in neutron intensity of almost
two orders of magnitude is expected. At that point, the use of neutron diffraction is
expected to greatly expand the field of structural biology, that is, hydrogen- and
hydration-sensitive structural biology which are beyond the folding structure. The
recent results of our group will be introduced in the Symposium.
TR.01.6

Polarised-neutron Laue diffraction on a crystal containing spin-polarised hydrogen

1 2 3 3 3
Maths Karlsson , Florian Piegsa , Ben van den Brandt , Patrick Hautle , Ton Konter , Colin
1 4 2 2
Carlile , Ted Forgan , Oliver Zimmer , Garry McIntyre
1 2
European Spallation Source, Lund University, Lund, Sweden, Institut Laue-Langevin,
3 4
Grenoble, France, Paul Scherrer Institute, Villigen, Switzerland, School of Physics &
Astronomy, University of Birmingham, Birmingham, United Kingdom

Neutron diffraction on samples with large hydrogen content, e.g. on organometallic and
protein samples, generally suffers from a strong featureless background due to strong
incoherent scattering from the protons. This is particularly evident in the two-dimensional
projection of Laue diffraction, a technique which is otherwise undergoing a renaissance
thanks to the development of large-solid-angle image-plate detectors, as illustrated by
numerous recent studies from VIVALDI [1] at the ILL by Bob Bau and colleagues [2]. Despite
the strong incoherent background, the larger coherent neutron scattering length of hydrogen
relative to other elements compared with the situation in X-ray diffraction more readily yields
answers to specific questions on, e.g. protonation states, hydrogen positions, and dynamic
disorder of hydrogen. Deuteration can be used to reduce the incoherent background, but
sample growth may be difficult or even impossible, and there may be an isotopic difference in
the structure. An intriguing alternative is parallel polarisation of the incident neutron beam
and the hydrogen spins [3].

We have performed a proof-of-principle polarised-neutron Laue diffraction experiment on a

spin-polarised single crystal of Nd-doped lanthanum magnesium nitrate hydrate (LMN),
La2Mg3(NO3)12.24H2O. The experiment was carried out on the FUNSPIN beam line [4] at the
continuous spallation neutron source SINQ (PSI) with the proton spins aligned by dynamic
nuclear polarisation. It demonstrates that not only is the incoherent background indeed
reduced but also that the intensity of the Laue reflections can be enhanced or diminished
significantly to give a form of contrast variation. In the longer term, we foresee that the
technique can be employed to improve substantially the poor signal-to-noise ratio in neutron
Laue diffraction experiments on biological crystals.

[1] G.J. McIntyre et al., Physica B 385-386 (2006) 1055-1058.

[2] R. Bau et al., Inorg. Chem. 43 (2004) 555-558; T. Stewart et al., Inorg. Chim. Acta 363
(2010) 562-566; and five papers in between.

[3] J.B. Hayter, G.T. Jenkin & J.W. White, Phys. Rev. Lett. 33 (1974) 696-699.

[4] J. Zejma et al., Nucl. Instr. and Meth. A 539 (2005) 622-639.
TR.01.7

Hydrogen, Neutrons and Energy

Ian Anderson

Oak Ridge National Laboratory, Oak Ridge, TN, United States

Hydrogen, the first element, plays a major role in a sustainable energy economy through
multiple mechanisms. Neutron scattering is a well established and ideal probe for determining
the structure and dynamics of hydrogen in materials and therefore remains a critical tool in
support of basic energy research. This presentation will provide an overview of the range of
studies being carried out using neutron scattering from hydrogen, with an emphasis on the
applications to energy technology.
TR.01.8

Structure and properties of (hydroxy)alkylammonium salts of flurbiprofen

1 1 1 1 2
Carl Schwalbe , Miren Ramirez , Barbara Conway , Chris Bache , Simon Coles , Peter
3
Timmins
1 2
Aston University, Birmingham, United Kingdom, University of Southampton, Southampton,
3
United Kingdom, Bristol-Myers Squibb, Moreton, Cheshire, United Kingdom

Starting with the t-butylammonium salt of the anti-inflammatory drug flurbiprofen (FTbut), we
have systematically increased the hydrophilicity of the cation by successively changing methyl
to hydroxymethyl groups. Hydrogen bonding by the added OH groups is expected to
influence the solubility and compactability of these salts.

Me + CH 3 + CH 2 OH
H3N CH 3 to H3N CH 2 OH
COO - CH 3 CH 2 OH
F

+ - 3
In FTbut donation of hydrogen bonds from NH3 to COO forms ladders built out of R4 (10)
rings. Substitution with one OH group to make FAmp does not change this pattern: the OH
group lacks a credible hydrogen bond acceptor and is disordered. However, when a second
OH group is introduced (FAmp2), the pattern changes fundamentally. Cations deploy one NH
and one OH hydrogen atom in hydrogen bonds to one anion. The other OH group links
-
intermolecularly to the first one while NH finds OCO . The third amino H atom pairs with OH
2 2 2
as acceptor to form a centrosymmetric dimer. Thereby R2 (9), R3 (9), and R2 (10) rings are
formed. The Tris salt (FTris) exists in two polymorphs. Crystals of form II are well ordered
with similar hydrogen bonds to those in FAmp2. Crystals of FTris form I are triclinic with Z’ =
2, the independent ions being related by pseudosymmetry and also showing disorder. Each
carboxylate O atom accepts only one hydrogen bond. Whereas the twist angle between rings
in the biphenyl moiety of F is 44-46° in the other structures, it is 55° and 61° here. These
factors should imply increased energy for this form, yet FTrisI melts at a higher temperature
than FTrisII. Despite the presence of a reasonable slip plane, FtrisI displays poor mechanical
properties, producing weak compacts with troublesome elastic recovery. FTrisII has a slightly
wider slip plane, and indeed strong tablets with shiny faces and excellent mechanical
properties are formed.

We are grateful for use of the Diamond synchrotron to collect data on FAmp and FTrisI.
TR.01.9

Structural Coordination Chemistry beyond Routine Diffraction: Single Crystal

Transformations, Shape Changes and Ambiguities, and Neutron Scattering

Larry R. Falvello

University of Zaragoza - C.S.I.C., Department of Inorganic Chemistry and I.C.M.A., Zaragoza,

Spain

Diffraction analyses outside the realm of routine structure determination have been used to
explore chemical and physical phenomena that would be difficult to observe either in the
synthetic laboratory or in rapid, albeit high quality, x-ray structural studies. Chemical
reactions within single crystals have been observed to yield products that would not be
expected at the benchtop. The 1-D coordination polymer in {Cs2[Co7(citr)4(H2O)13.5]}2∙ 15H2O --
(4-)
[citr = citrate, C6H4O7 ] in the crystalline state undergoes a concerted reaction to give a 2-D
polymer in Cs2[Co(H2O)6]{[Co6.5(citr)4(H2O)9]}2∙ 3H2O, cross-linked by an unusual Co(II) center
surrounded by seven O-coordinated ligands. The experimentation needed to discover and
characterize this process is described, along with potential efficiencies for future studies of
this type. A different Co(II) citrate complex which forms a 2-D net in molecular crystals
(4n-)
(formula { -[Co(C2H6O2)(H2O)2]2[Co4(C6H4O7]4}n ), is seen to have magnetic properties that
are tunable through small structural changes outside the magnetic mesh, and which would
benefit from studies by "higher-throughput" neutron diffraction. Seemingly more routine
structural phenomena such as the planar aqua ligand in transition metal complexes are
subject to new insights through the application of high-throughput neutron diffraction and
inelastic neutron scattering, as an example system shows. A final study involves trans-
-
[Ni(cyan)2(NH3)4] (cyan = cyanurate, C3H2N3O3 ), a simple paramagnet that forms molecular
crystals with a second-order phase transformation that leads to a continuous molecular shape
change as either pressure or temperature is varied. This system was studied by neutron
diffraction to give a cogent conclusion regarding the cause of the transformation.
TR.01.10
2
The rotational dynamics of the dihydrogen ligands in RuH 2(η -H2)2(PCyp3) as seen by
neutron diffraction analysis.
1 2 2
Alberto Albinati , Silvia Capelli , Sax Mason
1 2
University of Milan, Milan, Italy, Institut Laue-Langevin, Grenoble, France

The study of the interaction of an intact H2 molecule with a transition metal center has been a
very active area of research since the discovery of this type of complex by G. Kubas in 1984.
The nature, the stability, and the fluxionality of many metal-dihydrogen complexes have been
studied by diffraction methods, NMR, inelastic neutron scattering (INS) and theoretical
calculations. Single crystal neutron diffraction has been of paramount importance in providing
accurate structural parameters; however information on the dynamics of the dihydrogen
ligands have been obtained mainly by INS and NMR. The analysis of the temperature
dependence of the Anisotropic Displacement Parameters allows dynamic information to be
extracted from diffraction data, that are useful to distinguish between molecular flexibility and
disorder or between kinematic and dynamic interpretation of structural features.

We have collected very accurate single crystal neutron diffraction data on the complex
2
RuH2(η -H2)2(PCyp3) (1) at 20, 60 and 100K on the ILL D19 diffractometer and analyzed the
data sets using a physical model (2) that explicitly accounts for the effects of temperature and
describes the structure of the complex (at the three temperatures) with a unique set of
parameters: the normal mode frequencies and associated wave vectors that describe the
rigid-body librations and translations of the complex core, and two additional rotations of the
dihydrogen moieties. This analysis yielded values for the rotational frequencies of the H2
-1 -1
groups of 104(17) cm and 170(40) cm , in good agreement with previously determined INS
values and theoretical calculations.

(1) M. Grellier, L. Vendier, B. Chaudret, A. Albinati, S. Rizzato, S. Mason, S. Sabo-Etienne J.

Am. Chem. Soc. (2005) 127, 17592.

(2) H.B. Bürgi, S. C. Capelli Acta Cryst. (2000) A56, 403.

TR.01.11

Neutron Diffraction Studies of Metal-Hydrides

Muhammed Yousufuddin

University of Texas at Arlington, Center for Nanostructured Materials, Arlington, TX, United
States

Neutron diffraction is the method of choice when locating chemically interesting hydrogen
1
atoms, particularly in systems containing a heavy metal. In this talk, two fascinating
complexes containing hydrogen atoms that were unambiguously located with neutron
diffraction will be highlighted. The first complex, OsH3Cl(PPh3)3, is a Kubas-type complex
2
containing a rare elongated dihydrogen ligand. Results from the neutron diffraction study
showed that the dihydrogen ligand demonstrated an H…H distance of 1.48(2) Å. In the
second study, a four-coordinate hydrogen atom was located and measured for the first time
3
using neutron diffraction. Results from this study showed that the hydrogen atom occupied
the interstitial space of a tetrahedral Yttrium (Y4) cluster.

This work was performed in the laboratory of Professor Robert Bau at the University of
Southern California for completion of a doctoral dissertation and will be presented at the
transaction symposium being held in Prof. Bau’ s memory and honor.

1. M. Yousufuddin and R. Bau “Neutron Diffraction” in Applications of Physical Methods to

Inorganic and Bioinorganic Chemistry, edited by Robert A. Scott and Charles M. Lukehart,
Chichester, UK; John Wiley & Sons Ltd. ISBN 978-0470-032176, 2007, 271.

2. M. Yousufuddin, T. Wen, S. A. Mason, G. J. McIntyre, G. Jia, R. Bau Angew. Chem. Int.

Ed. 2005, 44, 7227.

3. M. Yousufuddin, M. Gutmann, J. Baldamus, O. Tardif, Z. Hou, S. A. Mason, G. J. McIntytre,

R. Bau J. Amer. Chem. Soc., 2008, 130, 3888.
TR.01.12

Molecule-based Magnets:

New Materials, Chemistry, Physics, and Crystals for this Millennium

Joel Miller

University of Utah, Salt Lake City/UT, United States

Molecule-based materials exhibiting the technologically important property of bulk magnetism

have been prepared and studied in collaboration with many research groups worldwide
frequently exhibit supramolecular extended 3-D structures. These magnets are prepared via
conventional organic synthetic chemistry methodologies, but unlike classical inorganic-based
magnets do not require high-temperature metallurgical processing. Furthermore, these
magnets are frequently soluble in conventional solvents (e. g., toluene, dichloromethane,
acetonitrile, THF) and have saturation magnetizations more than twice that of iron metal on a
mole basis, as well as in some cases coercive fields exceeding that of all commercial
magnets (e.g., Co5Sm). Also several magnets with critical temperatures (Tc) exceeding room
temperature have been prepared. In addition to an overview of magnetic behavior, numerous
examples of structurally characterized magnets made from molecules will be presented. Our
groups has discovered 7 families of molecule-based magnets, mostly organic-based, and
have significantly contributed to an eight family based upon the Prussian blue structure. Four
o
examples magnetically order above room temperature and as high at 127 C. These will
III III
include [M (C5Me5)2][A], [Mn (porphyrin)][A] (A = cyanocarbon etc. electron acceptors) as
well as M[TCNE]x, which for M = V is a room temperature magnet that can be fabricated as a
thin film magnet via Chemical Vapor Deposition (CVD) techniques. A newer class of magnets
of [Ru2(O2CR)4]3[M(CN)6] (M = Cr, Fe; R = Me, t-Bu) composition will also discussed. For R =
Me an interpenetrating, cubic (3-D) lattice forms and the magnet exhibits anomalous
hysteresis, saturation magnetization, out-of-phase, "(T), AC susceptibility, and zero field
cooled-field cooled temperature-dependent magnetization data. This is in contrast to R = t-
Bu, which forms a layered (2-D) lattice. Additionally, new magnets possessing the nominal
Prussian blue composition, M'[M(CN)6]x and (Cation)yM'[M(CN)6], but not the common fcc
cubic Prussian blue structure will be described. Also discussed will be Prussian blue- like
magnets that have a different composition, namely, (Cation)2Mn3(CN)8 and (Cation)Mn3(CN)7
compositions, whose structures and magnetic behaviors will be reported.
TR.01.13

High Throughput Neutron Diffraction – New Opportunities in Hydrogen Location in

Molecular and Materials Structure
1 3 1 1 2
Chick Wilson , Paul Henry , Marc Schmidtmann , Lynne Thomas , Valeska Ting , Mark
2
Weller
1 2
University of Glasgow, Glasgow, United Kingdom, University of Southampton,
3
Southampton, United Kingdom, Helmholtz Centre Berlin (HZB), Berlin, Germany

Many of the areas recently advanced in structural studies of small molecule materials rely
heavily on the ability to study structures on a shorter timescale, either to examine a series of
samples or to study a single sample under a range of conditions. This “high throughput”
approach has only recently become feasible for neutron diffraction studies of molecular
materials as a result of a substantial revolution in the provision of instrumentation with
massive detector arrays for single crystal diffraction, and with very high count rates for
powder diffraction. This has allowed neutron chemical crystallography to respond in a very
successful fashion to modern trends in structural molecular science, extending the
applications of neutron diffraction in the area of chemical crystallography and molecular
materials; many of these exploit the power of neutron diffraction in determining accurately the
hyrogen atom parameters in materials.

This high throughput capability allows the powerful information available from neutron
diffraction to be harnessed, with complementary X-ray and computational input, to tackle
more of the problems at which neutron diffraction excels, particularly those involving hydrogen
atom location. These include prominent examples in hydrogen bonding including both strong
and weaker hydrogen bonding interactions, the location of hydrogen in inorganic systems, in
complementing charge density studies and in studying materials under conditions of variable
temperature and variable pressure.

The potential of this new capability will be illustrated by a range of recent studies on proton
migration, hydrogen atom disorder and transfer, polymorphism in molecular complexes, water
location in materials, thermochromic materials, hydrogen-bonding interactions and in the
location and full description of hydrogen in inorganic materials. The powerful complementary
use of both X-ray and computational methods with neutron powder and single crystal
diffraction in these studies will also be highlighted.
TR.01.14

Hydrogen and Hydration: The 15K neutron structure of W3Y single mutant rubredoxin
from Pyrococcus furiosus

1 2 2 3
Dean Myles , Robert Bau , I. Tsyba , Matthew Blakeley
1 2
Oak Ridge National Laboratory, Oak Ridge, TN, United States, University of Southern
3
California, Los Angeles, CA, United States, Institute Laue-Langevin, Grenoble, France

Neutron crystal structures provide insights into hydrogen bonding interactions, protonation
states and details of hydration states in protein and nucleic acid crystal structures that are not
available from x-ray analysis alone. The iron-sulfur redox protein rubredoxin from Pyrococcus
furiosus (PfRd) is stable for days in boiling water, whereas most other bacterial rubredoxins
are readily denatured within minutes at 373K. Using the LADI instrument at the Insititute Laue
Langevin, we collected high-resolution (1.7 Å) neutron diffraction data at 15K on a single
mutant form of PfRd (W3Y-PfRd) in order to probe its temperature stability. By collecting the
3
data at 15K we illustrated the feasibility of cryo-cooling large (>1 mm ) protein crystals and
hence collecting high-resolution neutron diffraction data at cryo-temperatures. Comparison of
the single mutant solvent structure at 15K against the wild-type and triple mutant forms
indicates that by lowering the data collection temperature we have observed a more complete
picture of the hydration shells of the protein. Detailed analysis of the hydrogen bonding
interactions may help explain why the triple and single mutant forms of PfRd are found to be
less stable at low pH than the wild-type form.
TR.01.15

The New Microporous materials: MOFs, COFs and ZIFs

1 2
Michael O'Keeffe , Omar Yaghi
1 2
Arizona State University, Tempe, Arizona, United States, University of california, Los
Angeles, California, United States

In the last decade there has been explosive growth in the synthesis and characterization of
new microporous materials particularly those known as metal-organic frameworks (MOFs),
covalent organic frameworks (COFs) and zeolitic imidazolate frameworks (ZIFs). MOFs are
typically neutral frameworks in which inorganic clusters (secondary building units - SBUs) are
joined by organic linkers into periodic frameworks. Ideally the inorganic part has a structure
which can be abstracted as a simple polygon or polyhedron. The organic component may be
ditopic in which event the structure topology is that of the SBU shapes joined by links. The
organic component may be polytopic and again abstracted as a polygon or polyhedron shape
and the MOF topology then corresponds to two shapes joined by linkers. COFs are similar
except for replacement of the inorganic component by an organic SBU. Recognition of the
fact that there are just a small number of known default topologies (ideally those in which
there is just one kind of link) leads to the possibility of synthesis of materials with targeted
structure, including pore size and functionality - so-called reticular chemistry. ZIFs are rather
different. They are metal imidazolates that generally adopt the structures of simple zeolites
and other zeolite-like frameworks. Here the fuctionalization of the basic imidazole ring (e.g. as
methyl- or nitro-imdazole) determines the framework topology. The new materials show
unprecedented capacity for adsorption of gases such as dihydrogen, methane and carbon
dioxide and clearly have great promise in emerging clean-energy technologies.
TR.01.16

Beyond Single Crystal Structure Determination – Interpretation of 3D Disorder Diffuse

Scattering
1,2 2 3
Hans Beat Buergi , Michal Chodkiewicz , Vickie Lynch
1 2 3
Univ. of Zurich, Zurich, Switzerland, Univ. of Bern, Bern, Switzerland, Oak Ridge National
Laboratory, Oak Ridge, TN, United States

Single crystal structure determination from Bragg diffraction has become a largely routine
operation: the process is well understood theoretically, experiments and data interpretation
are automated to a large extent, as is the validation of results. The information obtained is
limited, however: it is the content of the crystallographic unit cell averaged over the time of the
experiment and the volume of the crystal.

If the type and distribution of atoms differ between unit cells, i.e. if a crystal structure shows
occupational and displacive disorder, some of the scattered intensity is lost from the Bragg
peaks and distributed throughout reciprocal space as diffuse scattering. Some examples from
material science will illustrate some prototypical diffuse scattering: 1D streaks, 2D planes and
3D continuous diffuse signals.

The interpretation of such scattering is far from routine. Sometimes the important information
can be obtained from qualitative, ad hoc arguments and some simple modeling calculations,
sometimes significant computational resources are required to perform elaborate Monte Carlo
modeling. Both types of interpretation will be illustrated with examples. The collaborative effort
between groups at Oak Ridge National Laboratory and the University of Zürich to construct a
more general tool for the interpretation of diffuse scattering will be sketched.
07.07.1

The Use of in Situ GISAXS and GIXAS Techniques at the Design of New Classes of
Bond-Selective Catalytic Materials in the Sub-Nanometer and Nanometer Size Regime
1,2 1 1 1 1 1,6
Stefan Vajda , Sungsik Lee , Byeongdu Lee , Soenke Seifert , Randall Winans , Yu Lei ,
1 1 1 1 5
Faisal Mehmood , Larry Curtiss , Jeffrey Greeley , Michael Pellin , Luis Molina , Alessandro
4 3 3 3
Fortunelli , Kristian Sell , Viola von Oeynhausen , Karl-Heinz Meiwes-Broer , Maria Flytzani-
7 2 2 8 8
Stephanopoulos , Lisa Pfefferle , Gary Haller , Sonja Wyrzgol , Johannes Lercher
1 2
Argonne National Laboratory, Argonne, Illinois, United States, Yale University, New Haven,
3 4
Connecticut, United States, Universitaet Rostock, Rostock, Germany, IPCF-CNR, Pisa,
5 6
Italy, Universidad Valladolid, Valladolid, Spain, University of Illinois, Chicago, Illinois, United
7 8
States, Tufts University, Medford, Massachusetts, United States, Technische Universitaet
Muenchen, Muenchen, Germany

The elucidation of the size/composition/shape/structure and function correlation, the effect of

support along with the determination of the nature of the catalytic particles under realistic
reaction conditions are instrumental on the way to the design of new classes of catalytic
materials. Uniform particles on technologically relevant supports are prerequisites for such
studies, making size-selected clusters of few atoms to several nm in size as ideal model
systems.

The experimental studies are based on 1) size-selected cluster deposition on technologically

relevant support, 2) electron microscopy of nanoclusters, and 3) in situ synchrotron X-ray
characterization of clusters under working conditions, combined with mass spectroscopy
analysis of reaction products. Density functional theory calculations are used to understand
the activity of clusters and the underlying reaction mechanisms.

Using metal and metal-oxide based catalytic systems, in this presentation examples will be
given on bridging the size gap between the sub-nm and nm size regime as well as on bridging
studies of model and “real” catalysts. Select reactions will include e.g. C-H, C=C bond
activation, where new highly selective and active catalyst systems performing at low
temperatures were identified for dehydrogenation, epoxidation and other reactions.

The applied in situ X-ray techniques proved to be instrumental at gaining fundamental insights
about the properties of (sub)nanoscale matter.
07.07.2

Atomic-scale X-ray studies of redox-induced cation dynamics for oxide supported monolayer
catalysts

Michael Bedzyk1, Zhenxing Feng1, Chang-Yong Kim2, Jeffrey Elam3, Qing Ma1, Zhan Zhang3, Martin
McBriarty1, Donald Ellis1, Peter Stair1
1
Northwestern University, Evanston, IL, United States, 2Canadian Light Source, Saskatoon, SK,
Canada, 3Argonne National Laboratory, Argonne, IL, United States

Metal oxides anchored to oxide supports often exhibit greater catalytic activity as monolayers
than as thicker films. Understanding this phenomenon requires a chemically sensitive, atomic-
scale view of the interfacial processes. We use in situ X-ray standing wave (XSW) 3D atomic
imaging combined with ex situ X-ray photoelectron spectroscopy (XPS) and X-ray absorption
fine structure measurements to follow the redox-induced surface site exchange of cations on
a single crystal oxide support as well as the concurrent changes in the oxidation states of the
supported cations. This is then compared to density functional theory. As an example, we
follow the reversible changes during the redox cycle of a /3 ML WOX / -Fe2O3 (0001)
1

interface grown by atomic layer deposition. The XSW measured W atomic maps and XP
spectra show dramatic changes for the as-deposited, oxidized and reduced interfaces, which
are explained by models that account for W incorporation at Fe sites with various coordination
schemes.[1] The 3D W atomic map for each condition is measured by the summation of the
XSW measured hkl Fourier components for the XRF selected W distribution.[2] This strategy
was then also applied to redox-induced structural and chemical changes for the sub-ML and 2
ML VOX / -TiO2 (110) interfaces.

[1] Z. Feng, C.-Y. Kim, J.W. Elam, Q. Ma, Z. Zhang, M.J. Bedzyk, J. Am. Chem. Soc. 131, 18200
(2009).

[2] L. Cheng, P. Fenter, M. J. Bedzyk, N. C. Sturchio, Phys. Rev. Lett. 90, 255503 (2003).
07.07.3

Ordering and kinetics of two-dimensional self-assembly of nanoparticles at surfaces –

a study by in situ and time-resolved grazing-incidence small-angle x-ray scattering
1 1 1 1 1
Zhang Jiang , Xiao-min Lin , Michael Sprung , Suresh Narayanan , Jin Wang , Carlee
2 2 2
Ashley , Darren Dunphy , Jeffrey Brinker
1 2
Argonne National Laboratory, Argonne, IL, United States, University of New Mexico,
Albuquerque, NM, United States

The characterization of ordering and kinetics at nanostructured surfaces and interfaces has
become increasingly critical to understand and optimize the synthesis of ordered
nanomaterials on nanometer length scales. This requires high-resolution in situ and time-
resolved surface probes that can also be applied to various experimental environments.
Grazing-incidence x-ray scattering is an ideal tool for the studies. At the Advanced Photon
Source, using high-resolution small-angle x-ray scattering in grazing-incidence geometry
(GISAXS), we successfully captured the growth kinetics of two-dimensional metal nanocrystal
superlattices at the liquid/air interface and identified their ordering, phases and phase
transitions in a quantitative manner. GISAXS was also applied to study the convective
transport and the effects of non-volatile solvent during the convective assembly of two-
dimensional superlattices of virus-like nanoparticles.
07.07.4

Role of Solvent Content, Thickness, and Composition on Surface Morphology in

Diblock Copolymer Thin Films
1,2 1 2 2 2 1
Yan Sun , Kevin Henderson , Zhang Jiang , Joseph Strzalka , Jin Wang , Kenneth Shull
1 2
Northwestern University, Evanston, IL, United States, Argonne National Laboratory,
Argonne, IL, United States

The phase behavior of diblock copolymer melts and solutions offers a novel means for
templating nanoscale patterns and particles for a variety of applications. In a solution
containing a block-selective solvent, enthalpic interactions between the non-soluble block and
the solvent govern the phase behavior. In the case of poly(methyl methacrylate) (PMMA), this
interaction is temperature dependent, with the disordered state attainable around 70-80C for
solvent fractions > 0.7. However, in solventless conditions, phase behavior is entirely
governed by the XN interaction between copolymer blocks. While the phase behavior of bulk
polymer solutions and melts have been well characterized both experimentally and with self-
consistent mean field simulations, the role of confinement and surface effects in thin film
equivalents have not been as extensively studied. Though recent work has focused on the
effects of film thickness, controlling the solvent content in these films has received much less
attention. Here, we employ a poly(tert-butyl methacrylate)-poly(methyl methacrylate) (PtBMA-
PMMA) diblock to study the role of solvent content, film thickness, and block copolymer
composition. In bulk solutions, this diblock exhibits thermoreversible micellation in alcohols,
so we have chosen butanol as our solvent of interest. Using grazing incidence small-angle x-
ray scattering (GISAXS) and atomic force microscopy (AFM), we have investigated the phase
behavior in three different diblocks under various solvent conditions up to 160C. Comparisons
to bulk phase behavior and self-consistent mean field calculations are discussed.
07.07.5

Complex Epitope Engineering: A Rational Approach to Improve Protein Crystallization

2 2 2 2,1
Victor Naumov , Nicholson W. Price , Samuel K. Handelman , John F. Hunt
1 2
Northeast Structural Genomics Consortium, New York, New York, United States, Columbia
University, New York, New York, United States

In previously published work, we have shown that the prevalence of well-ordered

surface epitopes is a dominant factor controlling protein crystallization behavior (Price et al.,
2008, Nature Biotech. 27:51-57). However, straightforward efforts to exploit this insight to
improve protein crystallization have been frustrated by the fact that most single residue
mutations lowering surface entropy compromise protein solubility. In contrast, many naturally
occurring proteins have excellent solubility characteristics and nonetheless yield high quality
crystals. We infer that these proteins possess more complex surface epitopes that maintain
high solubility in dilute aqueous buffers while having a high propensity to mediate stable
interprotein packing interactions under the low-water-activity conditions used for protein
crystallization. In order to identify such epitopes, we have developed a set of new
computational methods that have been applied to all crystal structures in the Protein Data
Bank (PDB). The resulting analyses demonstrate that, on average, ~50% of surface residues
participate in packing interactions, explaining why surface mutations generally alter
crystallization behavior. They also demonstrate that protein crystals are inherently
polymorphic, with the details of intermolecular packing typically varying significantly even in
crystal structures with equivalent lattice symmetry and unit cell parameters. Most importantly,
these analyses demonstrate that most packing epitopes are small and local in the polypeptide
chain and that some sub-epitopes are highly overrepresented in crystal packing contacts
compared to their frequency of occurrence on protein surfaces in the PDB. These
overrepresented sub-epitopes generally interact with a wide variety of partner epitopes,
suggesting that they are promising candidates to use in mutational engineering of proteins to
improve their crystallization behavior. We have written software that takes a multiple
sequence alignment and determines all ways in which these overrepresented sub-epitopes
can be introduced into a protein in a manner consistent with the sequence profile of the
corresponding protein family. These computational results and tools form the foundation of a
novel approach to rational engineering of improved protein crystallization.
07.08.1

The Canadian Macromolecular Crystallography Facility at the Canadian Light Source

1 1 1 1 1 1
James Gorin , Pawel Grochulski , Michel Fodje , Shaun Labiuk , Russ Berg , Peter Thorpe ,
1 2 3
Shawn Carriere , Natalie Strynadka , Mirek Cygler
1 2
Canadian Light Source Inc., Saskatoon, SK, Canada, Department of Biochemistry and
Molecular Biology, Faculty of Medicine, University of British Columbia, Vancouver, BC,
3
Canada, National Research Council Canada Biotechnology Research Institute, Montreal,
QC, Canada

The Canadian Macromolecular Crystallography Facility (CMCF), which serves more than
60 protein crystallographers located across Canada, currently consists of two beamlines. The
first, an insertion device beamline (08ID-1) illuminated by a small-gap in-vacuum hybrid
undulator (SGU) located in the upstream half of the straight section, is capable of satisfying
the requirements of the most challenging and diverse crystallographic experiments (i.e.,
physically small crystals with large unit cell dimensions). It has been in operation since 2006
and is producing a growing number of pdb deposits and scientific publications. More than a
year ago, Mail-in Crystallography was introduced at the CMCF. It is run by a dedicated
member of our team and has proven to be very successful. Up to 25% of available beamtime
is reserved for commercial users. We typically observe increased activity of commercial users
while the US synchrotrons are closed for maintenance. The second, a bending magnet
beamline (08B1-1), has recently begun accepting users and is designed for high-throughput
data collection with the capability of being remotely accessed. Together, the complementary
beamlines will constitute a single facility, allowing for the screening and collection of data from
a variety of projects. To date, there are in excess of 40 publications and 70 pdb depositions
from 30 laboratories from Canada, the United States and the United Kingdom using CMCF
beamlines.

To facilitate remote access, SSRL Stanford-type automated mounting (SAM) robots have
been installed for both beamlines and are undergoing final commissioning. The robot is used
in combination with Uni-Pucks or cassettes which provides sufficient capacity for a shift of
screening and data collection.

Data collection software has been developed in-house and has similar functionality as Blu-
Ice. Features of the final software will include the automatic alignment and configuration of the
beamline hardware, automatic crystal mounting and centering of crystals in the X-ray beam,
automatic measurement of fluorescence spectra for MAD experiments, automatic screening
and analysis of crystals in order to assess crystal quality and determine optimum parameters
and strategies for data collection, automatic data collection and data processing.
07.08.2

Technological advances in synchrotron data collection from the perspective of a

young crystallographer.

Gina Ranieri, Richard Walter

Shamrock Structures LLC, Woodridge, IL, United States

Synchrotron X-ray data collection is in the midst of a technological revolution. From new
software to detectors to protocols there is an unprecedented ability to collect more, higher
quality data in a shorter amount of time than there ever has been in the history of the science.
From hardware improvements like fast, reliable sample automounters, new detector
technologies, all-in-one “microdiffractometers”, and auto-optimized focusing optics to software
breakthroughs like user-friendly GUI controls, diffraction centering, and automated spiral data
collection all elements are coming into place to apparently transform the discipline into a fully
automated, remotely operated, push button service. The primary drivers behind this
generation of technology are cost and capacity. On the plus side, the capacity added by
these new capabilities has increased the access of these methods to more researchers. On
the downside, we wonder what might get lost, potentially in data quality, probably in basic
learning and understanding, and whether some of these new efficiencies are truly efficient.
As a young professional who has traveled throughout the world for the last two years
“learning on the job” how to quickly and efficiently collect high-quality X-ray data, I will discuss
some of the technologies and protocols that have been developed at various facilities to
attempt to deliver faster, more efficient, higher quality X-ray data. I will cite statistics from my
data collection experiences at various facilities and discuss what I feel works and what does
not work in the name of efficiency and quality.
07.08.3

On the front lines: Using high throughput synchrotron data collection to increase
productivity downstream

Virginia L. Rath

Reciprocal Space Consulting. L.L.C., Oakland, CA, United States

Reciprocal Space Consulting, LLC, located in Oakland, California is a contract research

organization providing expertise in all aspects of structure based drug design from protein
purification through crystallization experiments to data collection and structure solution.
Utilizing both the Advanced Light Source in Berkeley and the Stanford Synchrotron
Laboratory in Menlo Park, we logged over 2000 hours of synchrotron beam time in 2009
providing synchrotron support for academic institutions as well as Biotech and Fortune 500
pharmaceutical companies. Both ALS and SSRL continue to develop robotic and software
systems to support efficient screening and data collection and these advancements have
helped drive crystallography to a new level. Protein structure information is now an essential
component in drug discovery from lead identification through lead optimization. With our
support, customers can focus on high value downstream areas such as structural analysis of
drug-protein complexes and compound design.
07.08.4

Automated Macromolecular Crystallography Facilities for Proprietary Research

Elspeth Gordon

European Synchrotron radiation facility, Grenoble, France

The ESRF Structural biology group runs a six macromolecular crystallography (MX)
beamlines and a facility for bioSAXs. This portfolio of beamlines includes three of fixed
wavelength, one of which has microfocus capability. The three variable wavelength beamlines
include two facilities with a relatively small focal spot size (50 x 30 m2) where long
wavelength ( ~2.3 A) data collections can be performed. This suite of beamlines meets the
needs of the even the most demanding experiments.

To optimise use of the beamlines and to simplify support issues we have embarked upon a
process of automation and standardisation. All beamlines are equipped with ESRF/EMBL
developed sample changer robots and are controlled via a common, intuitive graphical user
interface, mxCuBE. Within mxCuBE options including: remote access, quick realignment of
the beamline; crystal visualisation and centring; sample characterisation and experimental
strategy calculations; data collection; energy scans and the collection and analysis of X-ray
Fluorescence (XRF) spectra. A beamline database (ISpyB) allows for real-time at-a-distance
monitoring of experiments via a web interface.

Up to 30% of the beamtime on the ESRF's MX beamlines is available for those users wishing
to carry out proprietary research. Industrial users can either come to the ESRF and carry out
their experiments themselves or, once they are familiar with the technology, control their
experiment remotely from their home laboratory. The ESRF also offers a data collection
service where frozen samples are shipped to the ESRF and inhouse staff carry out the data
collection.

The increased number of samples brought to the beamlines and the high demands on
throughput and efficiency of our industrial clients have been a major driving force for technical
developments on the ESRF's MX resources. This presentation will outline key features of the
developments put in place at the ESRF and describe our plans for the future.
07.08.5

High throughput fragment screening- new reasons and new software

1 1 1 2 3
Thomas Peat , Janet Newman , Vincent Fazio , Tom Caradoc-Davies , Kim Branson
1 2 3
CSIRO, Parkville, VIC, Australia, Australian Synchrotron, Clayton, VIC, Australia, Stanford
University, Palo Alto, CA, United States

To provide an experimental basis for a comprehensive molecular modeling evaluation study,

500 fragments from the Maybridge fragment library were soaked into crystals of bovine
pancreatic trypsin and the structures determined by X-ray crystallography. The soaking
experiments were performed in both single and pooled aliquots to determine if the
combination of fragments is an appropriate strategy. X-ray diffraction data were collected on
over 1400 crystals at the Australian Synchrotron. These data were subsequently processed,
and the preliminary analysis was performed with a custom software application (Jigsaw),
which combines available software packages for structure solution and analysis.

The in silico modeling community lacks a public set of high-quality data against which
predictive rather than retrospective studies can be performed. Previous attempts to perform
such evaluations suffered from a lack of truly “blind” data. Recently, OpenEye Scientific
Software has initiated the SAMPL (Statistical Assessment of the Modeling of Proteins and
Ligands) meeting, which provides a forum for the prospective testing of

concepts, algorithms, and approaches in computational chemistry and protein modeling. One
core goal of modeling is rapid and reliable in silico screening, in which a compound library is
evaluated against a protein target of interest to select a subset of compounds, enriched for
potential activity against the target. The resulting subset can subsequently be assayed for
activity using biochemical or biophysical methods. Recently, it has been shown that this
approach may be feasible for enriching fragment libraries of low molecular weight (~200 Da)
in addition to libraries of larger “lead-like” compounds (300-500 Da).

I will discuss the strategies used to obtain this high throughput data set, the reason for
developing Jigsaw and why it was important to develop a specific process for this project.
07.08.6

Integrating Single-Crystal Spectroscopy and X-ray Diffraction at Beamline X26-C of the

National Synchrotron Light Source (NSLS)
1 1 1 1 1
John Skinner , Deborah Stoner-Ma , Annie Heroux , Dieter Schneider , Robert Sweet ,
2 1
Michael Skinner , Allen Orville
1
Biology Department, Brookhaven National Laboratory, Upton, NY 11973, United States,
2
High School Reasearch Program, Brookhaven National Laboratory, Upton, NY 11973, United
States

Correlated optical absorption spectroscopy at beamline X26-C of the NSLS is routinely

collected before, during, and after x-ray diffraction data used for crystal structure
determination. A microspectrophotometer is mounted at the beamline such that it probes
~25µm diameter region of the crystal that intersects the x-ray beam. We have integrated all of
the spectrophotometer controls with the x-ray diffraction data collection and beamline control
software, CBASS. The Ocean Optics USB4000 spectrophotometer is controlled through an
EPICS IOC developed at the Canadian Light Source, which CBASS communicates with over
EPICS Channel Access. Users typically start by performing an automated spectroscopic
screen consisting of 72 crystal images and absorption spectra collected at 5-degree
increments throughout 360 degrees of rotation. A program developed at the European
Synchrotron Radiation Facility for automatic crystal centering, C3D, is called from a CBASS
macro. The macro determines the “optimal” orientation, i.e. the flat-face of the crystal/loop, for
optical absorption spectroscopy. A Java Webstart application also allows the user to control a
"movie" of the screening run in order to evaluate quickly the relationship between crystal
orientation and the anisotropic spectroscopic data. The optimal phi/omega angle, or a user-
provided best orientation, is then revisited and an optical spectrum is collected during the
readout of the x-ray detector after each frame of the diffraction data set. Each optical
absorption spectrum is overlaid on a plot to provide instant feedback about spectroscopic
perturbations induced by x-ray dose. The entire family of spectra is documented and available
to the user in several formats through the beamline’s experiment tracking database system,
PXDB. The Protein Data Bank will link the resulting atomic coordinates and the family of
spectra upon publication and release of the structure. We are also integrating Raman
spectroscopy with two excitation lasers (785nm and 532nm) into the beamline controls. Our
diffraction and spectroscopy facility is available full-time to the general user community at the
NSLS.

Supported by the NIH National Center for Research Resources and the DOE Office of
Biological and Environmental Research.
07.08.7

SER-CAT Automation: Providing Users with A “Virtual Home Synchrotron”

John Chrzas, Jim Fait, Zheng-Qing Fu, John Gonczy, Andy Howard, Zhongmin Jin, Gerd
Rosenbaum, John Rose, B.C. Wang

University of Georgia, Athens, Georgia, United States

From its inception, SER-CAT has been working towards the concept of providing remote
users with “Light When YOU Need It!” A web-based “virtual home synchrotron beamline” was
envisioned that could be integrated into the user's research program, much like another piece
of major research equipment in the X-ray lab down the hall.
SER-CAT began exploring automation of its beamlines shortly after the signing of MOU with
APS in March 1999. Working with Oceaneering Space Systems, a conceptual design for an
automated data collection robot (ASTRO) was developed in 2000. In 2003, using funds from
the Georgia Research Alliance, automation of the SER-CAT beamlines began with the
installation of a highly modified Berkeley/ALS automounter on SER-CAT's 22BM.
Key to the SER-CAT virtual synchrotron beamline (or remote access) is the integration of
hardware with software. The SERGUI beam line control interface allows the remote user full
control of the beamline from their home lab including beamline/goniometer optimization,
wavelength selection, fluorescence scans, automated crystal screening and MAD/SAD data
collection. Today over 80% of SER-CAT members routinely collect data remotely.
Significantly, SER-CAT's robotics, system integration and added user support have allowed it
to implement 12-hour shifts with 16-hour/day on-site user support.
SER-CAT also provides its members access to automated data processing and structure
solution pipelines. Command line scripts CMDDENZO, CMDDTREK and CMDXDS are
available for automated data processing using HKL2000, d*TREK and XDS, respectively. A
cluster-based version of the SGXPro Crystallographers Workbench for automated structure
determination is available, which allows members to produce a fitted map from their SER-
CAT data in a matter of minutes.
An overview of the SER-CAT’ s automation will be described and discussed.
Work supported by the SER-CAT Member Institutions (www.ser-cat.org), University of
Georgia Research Foundation and the Georgia Research Alliance.
07.08.8

Towards serial micro crystallography of acoustically mounted crystals

Alexei Soares, Allen Orville, Marc Allaire, Matthew Engel, Ruchi Parekh, Annie Heroux,
Robert Sweet, John Skinner, Joseph Olechno, Richard Ellson

Brookhaven Naiotnal Laboratory, Upton, NY, United States

Acoustic droplet ejection (ADE) was used to transfer 2.5µL droplets of crystal slurries from
396 well plates onto MiteGen© micro mesh pins. Multiple homogenous slurries were tested
to verify that acoustic specimen preparation was effective and non-damaging for R3 insulin
and P43212 lysozyme crystals of different sizes (5µ±2µm, 10µm±4µm, 20µm±8µm), different
concentrations, and different overall purity and cleanliness. All tested conditions yielded
accurately transferred droplets of precise volumes. All transferred droplets yielded
undamaged and well diffracting crystals. Both the diffraction patterns and the resulting
structures of the specimens prepared with ADE were comparable to crystals that were hand
mounted from the same slurries prior to acoustic ejection. We conclude that ADE specimen
preparation is a strong candidate to mount micro crystals automatically from the growth plates
onto the data collection media. We discuss some difficulties unique to micro crystals;
including surface tension induced clustering, preferential orientation, and inaccessible regions
of reciprocal space.
07.09.1

Insights into Amyloid Structure from Short Assembling Peptides

1 1 2 2 3
Kyle Morris , Karen Marshall , Joost Schymkowitz , Frederic Rousseau , Pawel Sikorski ,
1
Louise Serpell
1 2
University of Sussex, Brighton, East Sussex, United Kingdom, Vrije Universiteit Brussel,
3
Brussels, Belgium, Norwegian University of Science and Technology, Trondheim, Norway

A broad range of peptides and proteins are able to spontaneously self-assemble into fibrillar
structures known as amyloid. This phenomenon is associated with a number of
neurodegenerative diseases and remarkably also with functional materials in nature. The
structural features of this unique conformation have historically been determined by X-ray
fibre diffraction (XRFD) and may be generally represented by the cross-beta architecture
where beta-strands run perpendicular to the fibre axis and are stabilised by hydrogen bonding
parallel to the fibre axis. More recently advances in X-ray crystallography and ssNMR have
consolidated features of the cross-beta architecture.
By using a multi-technique approach, developing XRFD techniques and with increased
access to new amyloid-like model systems new studies promise to reveal greater detail about
the amyloid fold. Taken together our observations define whether overall sequence or position
specific characteristics are key to amyloid formation and give insights into what stabilising
interactions are common to these fibril-forming pathological peptides.
07.09.2

A first molecular view of amyloid-like fibrils bound to small molecules revealed by X-

ray microcrystallography

Meytal Landau, Michael R. Sawaya, Kym F. Faull, Arthur Laganowsky, Jie Liu, Jorge Barrio,
David Eisenberg

University of California Los Angeles, Los Angeles,CA, United States

Amyloid protein fibrils are associated with a group of devastating human diseases. Currently
there is no approved therapeutic agent that regulates the formation of amyloid deposition and
alleviates the symptoms. Several dozen small-molecule ligands have been suggested to
affect fibrillation. In order to understand the nature of fibril-ligand interactions we sought to
determine the crystal structures of their complexes by X-ray microcrystallography. Structure
determination was impeded by the miniscule crystals, about one micrometer in width. The
difficulties include fast decay, crystal polymorphism, indexing small unit cells and merging
integrated intensities from several crystals. Co-crystallization of the fibril-segments with
ligands was particularly challenging due to low affinity and specificity of binding. Frequently
the ligands appear disordered in one dimension because their lengths span multiple unit cells
of the fibril; that is, the dimensions of the small molecule and the fibril unit cell were
incommensurate.

Previous structures of fibril-forming segments from our lab have presented the first fully
objective atomic model of the common β -spine structure of amyloids. Here we present a first
molecular insight into the interactions of such segments with ligands. The structures indicate
binding of the ligands along the fibril axis. Interestingly, in some cases the ligands induced a
unique polymorph of the fibrillar form. These structures advance our understanding of fibril-
ligand interaction and offer a starting point for the design of highly specific, potent and non-
toxic compounds that will inhibit fibrillation or will be used for diagnosis of fibrils.
OVMOXMR

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07.09.4

Structure of Point Mutants of β -Amyloid Associated with Familial Alzheimer’s Disease

2 1 1 3 1
Robert Tycko , Kimberly Sciarretta , Adam Cloe , Joseph Orgel , Stephen Meredith
1 2
The University of Chicago, Chicago, IL, United States, National Institutes of Health,
3
Bethesda, MD, United States, Illinois Institute of Technology, Chicago, IL, United States

We examined two mutant Aβ 40 peptides associated with familial Alzheimer's Disease, D23N-
Aβ and Δ E22-Aβ (Iowa and Japanese, respectively). D23N-Aβ is also associated with
cerebral amyloid angiopathy. We synthesized D23N-Aβ 40 and Δ E22-Aβ 39; both form fibrils
very rapidly and with no lag phase. EM of D23N-Aβ 40 fibrils shows multiple morphologies
with mean diameter = 6.90 nm, compared to 10.2 nm for wide-type Aβ 40. X-ray diffraction of
D23N-Aβ 40 fibrils shows cross-β pattern, with reflections at 0.47 and 0.94 nm. In contrast,
wild type Aβ 40 fibrils show reflections at 0.47 and 1.04 nm. For Δ E22-Aβ 39, fibril diameter
13 13 15
and x-ray diffraction are similar to those of wildtype Aβ 40. Solid state NMR ( C- C and N-
13
C dipole-dipole coupling) of D23N-Aβ 40 indicated molecular polymorphism of fibrils, with
only a minority containing in-register, parallel β -sheets. The majority of fibrils had antiparallel
β -sheets with 17+k ↔ 21-k registry. An intriguing possibility is that the aberrant structure of
D23N-Aβ 40 fibrils is related to the unusual vasculotropic clinical picture in these patients.
Δ E22-Aβ 39 formed fibrils instantaneously under many conditions, but without thioflavin (ThT)
fluorescence. Direct ThT binding assay (by HPLC) indicates loss of one of four putative ThT
binding sites in Δ E22-Aβ 39. Nevertheless, Δ E22-Aβ 39 fibrils have a β -sheet character. CD
of wild-type Aβ 40 indicates “random coil”, developing β -sheet character after ~ 3 days. In
contrast, Δ E22-Aβ 39 shows a β -sheet signature by CD immediately when put into aqueous
media. Soluble oligomers of Δ E22-Aβ 39 are present at extremely low concentration and are
highly transient species, which usually are not seen by size exclusion chromatographs.
Critical concentration of Δ E22-Aβ 39 is ~ one-third that of the wild-type Aβ 40. Solid-state
NMR studies of Δ E22-Aβ 39 fibrils are in progress, but preliminary studies indicate that these
fibrils, like D23N-Aβ 40 fibrils, do not have the in-register, parallel β -sheet structure that is
typical of wild-type Aβ 40 fibrils.
07.09.5

Structural Insights into Fungal Prion HET-s by X-Ray Fiber Diffraction

1 1 2 1 1
William Wan , Wen Bian , Holger Wille , Michele McDonald , Gerald Stubbs
1 2
Vanderbilt University, Nashville, TN, United States, University of California, San Francisco,
San Francisco, CA, United States

Prions are infectious proteins thought to propagate through polymerization of a misfolded

protein nucleus. The nucleus may act as a template to convert natively folded isoforms into
the misfolded state, resulting in the formation of long unbranched amyloid fibrils. The
nucleation event, and subsequent templating ability, is dependent on the environmental
conditions such as pH, salt concentration and agitation. In the filamentous fungus Podospora
anserina, the prion form of the HET-s protein is used in a self non-self recognition process
known as heterokaryon incompatibility. In this study we have obtained fiber diffraction data
from amyloids formed from the prion domain of the HET-s protein at pH 2.5 and pH 7. It has
been shown in the literature that the pH 7 fibrils are infectious while the pH 2.5 fibrils are not.
However, fiber diffraction has revealed that the two fibrils are very similar, despite infectivity
and morphological differences. Our findings imply that the structural differences that cause
infectivity are actually very subtle.

Support for this research was provided by the National Institutes of Health, grants P01
AG002132 and T32 GM008320-21.
07.09.6

Structural studies of prions and other amyloids by X-ray diffraction

1 1 1 1 1 1
Gerald Stubbs , Wen Bian , Michele McDonald , William Wan , Amy Kendall , Hayden Box ,
1 2
Adrianne Eyman , Holger Wille
1 2
Vanderbilt University, Nashville, Tennessee, United States, University of California, San
Francisco, California, United States

Amyloids are misfolded proteins forming unbranched filamentous assemblies (fibrils) that
produce a characteristic apple-green birefringence when stained with Congo red. In fiber
diffraction experiments, they exhibit characteristic meridional diffracted intensity at about 4.75
Å resolution, coming from cross- secondary structure (-strands running approximately at
right angles to the filament axis). For many years, all amyloids were assumed to have a
common structure consisting of -sheets stacked together, with the sheets approximately
parallel to the fibril axis and the strands at right-angles to the axis. In recent years, however,
work from a number of laboratories has shown that amyloid structure is much more complex
and diverse than this, although the cross- structure is always present. We have examined
amyloids formed from short peptides, the 40-residue A peptide, the 37-residue IAPP peptide,
the the prion domain of the HET-s fungal prion protein, and the mammalian prion protein PrP.
Work with A, IAPP, and HET-s makes use of information from solid state NMR, while studies
of PrP use data from electron microscopy. Fiber diffraction studies comparing brain-derived
PrP amyloid with recombinant PrP amyloid clearly indicate significant differences in structure.
Fiber diffraction data support the diversity of -structures in amyloids in general, distinguish
among competing models, and provide insight into their protofilament packing.

Supported by NIH grants P01 AG002132 and P01 AG010770.

07.10.1

Area Detector Calibration

Hakon Hope

University of California, Davis, Davis, CA, United States

Apparently, most users of commercial, CCD-based area detectors assume, without asking too
many questions, that all geometric correction factors will be automatically applied to their
data. Let us do a check to see if this implicit trust is deserved.
Here are a couple of lines copied from an otherwise innocuous shelx lst file:
Resolution(A) .68 .72 .75 .78 .83 .87 .95 1.04 1.18 1.50 inf
K 1.046 1.043 1.023 .992 .968 .965 .949 .959 .998 1.024
The scale factor K is not constant, but shows a systematic variation.
These effects are not just local phenomena. Similar observations can be readily made with
data deposited with Acta Cryst. G. Wu, B. L. Rodrigues and P. Coppens* have published a
paper “The correction of reflection intensities for incomplete absorption of high-energy X-rays
in the CCD phosphor,” J. Appl. Cryst. (2002). 35, 356-359. This paper may not have received
sufficient attention. Some time later, a feature was quietly added to sadabs to take such
effects into consideration, but to date, this author is not aware of a corresponding feature to
be accessible in the Bruker Apex user interface.
In order to simplify the assessment of detector response as a function of position on the
detector surface, the following procedure is proposed: Perform a number of scans that cover
the exact same range in crystal positions, but with the detector in different positions. For
example, perform runs with detector settings at -30°, -25° … +30° with ω covering a 60°
range. This will result in many identical reflections observed at different positions on the
detector surface. The main result of several such experiments is that identical reflections
show significant differences inmeasured intensities, varying systematically with angle of
incidence on the detector face. Variations easily exceed 5%. Corrections for these effects
lead to lower R index and smoother difference maps.
07.10.2

CrystalPlan, an experiment planning tool for crystallography

Janik Zikovsky, Christina Hoffmann

Oak Ridge National Lab., Oak Ridge, TN, United States

Instrument time at large x-ray and neutron scattering facilities such as the Spallation Neutron
Source at ORNL is always at a premium. To make the most of a user's short visit to one of
these facilities, developing a plan for an efficient experimental run would be advantageous.
The CrystalPlan program is a tool designed to take the guesswork out of acquiring large data
sets. Written in Python, CrystalPlan can run on multiple platforms. The program features an
attractive graphical user interface (GUI) including a 3D viewer.

Using the instrument's detector positions and the sample orientation, a 3D map of the
reciprocal space coverage is generated and displayed. Statistics such as the fraction of
reciprocal space measured are calculated. As users build up a list of desired sample
orientations, the reciprocal space coverage increases; the program also displays and
calculates the redundancy – how many times a specific volume of q-space was measured.
The limitations of the sample orientation goniometer, if any, are displayed to the user.

Users of the software can enter or load the sample crystal’s lattice parameters and UB matrix;
these are then used to calculate, for each H,K,L peak, whether or not it was measured, by
which detector(s), at which sample orientations and what wavelengths. For particularly
interesting reflections, users can ask the program to find the sample orientation that places
the peak at an optimal position on a detector. Once an acceptable sequence of sample
orientations has been found, data is acquired in automatic mode.

We will show a comparison between the predicted peak positions and the actual data for a
crystal measured at the TOPAZ beamline at SNS.

This research is supported by UT Battelle, LLC under Contract No. DE-AC05-00OR22725 for
the U.S. Department of Energy, Office of Science.
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07.10.4

Collecting two-wavelength MAD data without changing energy!

Kanagalaghatta Rajashankar, Igor Kourinov

NE-CAT, Cornell University, 9700 S. Cass Av., Argonne, IL-60439, United States

MAD (multiple anomalous dispersion) method utilizes the anomalous scattering nature of
specific heavy atoms at characteristic X-ray energies as well as the variation of atomic
scattering factor as a function of energy near atomic absorption edges. To maximize the
signature of anomalous scattering center, typically, diffraction data are collected at three
wavelengths, namely, peak, inflection and high-energy remote. As result of evolution of
synchrotron beamlines to deliver increasingly accurate intensity data and the development of
more robust, and powerful software for macromolecular crystal structure analysis, has
influenced SAD (single anomalous dispersion) method to take over MAD method. However,
MAD remains in the main-stream as a method of choice for challenging projects. Several
variants of MAD have been proposed that make best of both anomalous and dispersive
differences. Even, a method for simultaneous collections of multiple MAD collections has
been proposed, though no practical case been demonstrated till now. Here we propose an
easy method for collection of two wavelength MAD data, but without changing energy. This
method makes use of the inherent property of most of synchrotron beamlines, namely
presence of multiple harmonic energies. A test case of this method will be presented and
improvement over SAD phasing will be demonstrated.

NE-CAT is supported by award RR-15301 from the NCRR. APS is supported by the U.S.
Department of Energy
07.10.5

A Comprehensive Strategy - Is It Possible?

Lee Daniels, Mark Pressprich

Rigaku Americas Corp, The Woodlands, TX, United States

A truly global data-collection strategy algorithm must consider a great number of variables.
Ideally one should be able to consider any source, any goniometer and any detector. Then
one must involve the relationships among these components, including the limits of motion
and all possible collisions. A truly flexible routine also allows the user to add his own limits,
such as additional restrictions for temperature, pressure, humidity (etc.) control devices.

An even greater number of experimental variables, or concerns, ensue. The basic

requirements are completeness and redundancy. Then there are the details: a good
"distributed" redundancy (the target redundancy is distributed over most or all unique
reflections), avoid including partial reflections due to factors such as high Lorentz factors or
α 1-α 2 splitting, appropriate detector swing overlap when necessary, and support for ω and/or
φ scans for any geometry, to name a few. The routine must work in automatic mode using
reasonable defaults and also allow "experts" a great deal of flexibility. Then there is a crucial
measure of a useful algorithm - it must be FAST.

An algorithm that implements these ideas will be discussed and demonstrated.

07.10.6

What can a synchrotron do for Chemical Crystallography?

Simon Teat

Lawrence Berkeley National Laboratory, Berkeley, CA, United States

Synchrotrons are not just the domain of protein crystallography. At the Advance Light Source
station 11.3.1 is a dedicated chemical crystallography station. The high intensity of
synchrotron radiation can yield high quality structures on crystals that are otherwise too small
or weakly diffracting to produce anything on a laboratory source. The factors affecting a
crystals ability to diffract will be outlined, along with how the synchrotron can aid and the data
collection strategies that have been used to provide higher resolution data on weakly
diffracting samples. Not all synchrotron data collections serve to determine an unknown
structure, some are to see how a known structure is changed by the external environment. In
many of these cases using a small crystal can make the difference between success and
failure.
07.10.7

Data Collection Planning and Automation for Chemical Crystallography

1 1 2
Joerg Kaercher , Michael Ruf , Rob Hooft
1 2
Bruker AXS Inc., Madison, WI, United States, Netherlands Bioinformatics Centre (NBIC),
Nijmegen, Netherlands

We report on the latest development of expert systems for automating data acquisition and
structure solution for high end research applications. This development was made possible in
part through advances in the analytical software, specifically new and improved algorithms
and decision making expert systems. The technology automates many of the routine aspects
of data collection and analysis but also still allows the expert user to interact or intervene in
more challenging cases. By combining automation with the latest developments in area
detector and X-ray source technologies the productivity of the instrument can be significantly
enhanced.
In this paper we present an overview of the decision tree implemented in a fully automated
system that involves all the individual steps in producing publication quality structures from
single-crystals without user intervention. The system is designed to make all decisions
autonomously, while at the same time keeping the user informed about the progression of the
experiment in an easily comprehensible way. Suitable remedies are suggested if the software
encounters a problem it cannot tackle, such as radiation induced crystal decay or a
temperature induced phase transition.
The expert system proceeds through the following stages: quantify the crystal quality,
determine the unit cell and the crystal symmetry, select a data collection strategy, acquire and
reduce the diffraction data, scale the diffraction data, determine the space group, solve the
phase problem, refine and validate the molecular structure, and finally generate a report. The
results are provided as a Crystallographic Information File (CIF) and as a Hypertext Markup
Language (HTML) report.
07.11.1

Reconstructing RNA folding intermediates from time-resolved SAXS data

Lois Pollack

Cornell University, Ithaca, NY, United States

Small angle x-ray scattering (SAXS) provides information about the size and shape of
macromolecules in solution. In a time-resolved mode, SAXS has the potential to reveal
structure(s) of transient intermediates that occur as molecules function or fold. I will discuss
successes and challenges we have encountered in reconstructing low resolution structures
from time-resolved scattering data. The scientific focus of these studies is on RNA folding to
biologically functional forms, such as ribozymes or riboswitches.
07.11.2

Extended structures in RNA folding intermediates are due to non-native interactions

rather than electrostatic repulsion.

Nathan Baird, Haipeng Gong, Syed Zaheer, Karl Freed, Tao Pan, Tobin Sosnick

University of Chicago, Chicago, United States

RNA folding occurs via a series of transitions between metastable intermediate states for
2+
Mg concentrations below those needed to fold the native structure. In general, these folding
intermediates are considerably less compact than their respective native states. Our previous
work demonstrates that the major equilibrium intermediate of the 154 residue specificity
domain (S-domain) of the B. subtilis RNase P RNA is more extended than its native structure
(1). We now investigate two models with falsifiable predictions regarding the origins of the
extended intermediate structures in the S-domains of the B. subtilis and the E. coli RNase P
RNA that belong to different classes P RNA and have distinct native structures (2). The first
model explores the contribution of electrostatic repulsion, while the second model probes
specific interactions in the core of the folding intermediate. Using small-angle X-ray scattering
(SAXS) and Langevin Dynamics (LD) simulations, we show that electrostatics only plays a
minor role, whereas specific interactions largely accounts for the extended nature of the
intermediate. Structural contacts in the core, including a non-native base-pair, help to stabilize
the intermediate conformation. We conclude that RNA folding intermediates adopt extended
conformations due to short-range, non-native interactions rather than generic electrostatic
repulsion of helical domains. These principles apply to other ribozymes and riboswitches that
undergo functionally relevant conformational changes.

Reference 1. Baird, N. J., Westhof, E., Qin, H., Pan, T. & Sosnick, T. R. (2005). Structure of a
folding intermediate reveals the interplay between core and peripheral elements in RNA
folding. J. Mol. Biol. 352, 712-22.

Reference 2. Baird, N. J., Gong, H., Zaheer, S. S., Freed, K. F., Pan, T., Sosnick, T. R. (in
press) Extended Structures in RNA Folding Intermediates Are Due to Nonnative Interactions
Rather than Electrostatic Repulsion. J. Mol. Biol.
07.11.3

Kinetics of hepatitis B virus core assembly by time-resolved small angle x-ray

scattering
1 2 3
Hiro Tsuruta , Kelly Lee , Adam Zlotnick
1 2
Stanford University, Menlo Park, CA, United States, University of Washington, Seattle, WA,
3
United States, Indiana Unviersity, Bloomington, IN, United States

Hepatitis B virus (HBV) is a major pathogen and one of the most prevalent causative agents
of cancer in humans. HBV is one of the few systems for which interactions between virus and
drugs targeted against its protective capsid have been well characterized both structurally and
biochemically. It is thus an ideal system for exploring the relation of virus structure and
function. We have applied synchrotron time-resolved small angle x-ray scattering (TR-SAXS)
to elucidate the disassembly and assembly of HBV cores. Icosahedral T=4 core capsids
dissociate into 120 dimers of subunits in vitro in the presence of chaotropes such as
guanidine hydrochloride. We first studied the kinetics of chaotrope-induced disassembly
process over a few minutes. We observed an oscillating dissociation/reassociation
multiphasicity instead of a one-way disassembly process. This unusual disassembly process,
however, turns out to be consistent with a theoretical prediction by a computational modeling
study. We recently conducted TR-SAXS experiments on the assembly of the HBV core
capsid. Our solution x-ray scattering studies in equilibrium indicated that a low concentration
of urea keeps the capsid protein in an assembly-ready dimeric form at least for several hours.
We conducted time-resolved studies of the assembly induced by a rapid salt concentration
jump at a mildly alkaline pH value. The assembly reaction is surprisingly fast: the half-life of
the order of a few seconds or shorter, depending on salt concentration. Our recent results
suggest the presence of a transient assembly intermediate which is larger than T=3 or T=4
capsid and undergoes partial disassembly prior to incorporating the dimeric capsid protein to
form the T=4 capsid. The assembly process also involves a slow annealing process after the
formation of a capsid.
07.11.4

A Combined Crystallography, SAXS and Computational Approach to the

structure of Gln4 – yeast glutaminyl tRNA synthetase

1,2 1 1,2 1 1
Edward Snell , Thomas Grant , Joseph Luft , Jennifer Wolfley , Elizabeth Snell , Hiro
3 4 4 4,5 4,5
Tsuruta , Stephanie Corretore , Erin Quartley , Eric Phizicky , Elizabeth Grayhack
1 2
Hauptman Woodward Medical Research Institute, Buffalo, NY, United States, Department of
3
Structural Biology, SUNY Buffalo, Buffalo, NY, United States, SSRL, Menlo Park, CA, United
4
States, Department of Pediatrics, University of Rochester Medical Center, Rochester, NY,
5
United States, Department of Biochemistry and Biophysics, University of Rochester Medical
Center, Rochester, NY, United States

tRNA synthetases play an essential role in protein synthesis by covalently coupling

the correct amino acid to the correct tRNA, prior to its use in translation. Unlike their
prokaryotic homologs, eukaryotic tRNA synthetases often have additional domains appended
to their N or C-terminus. In some cases, these domains are known to have non-specific RNA
binding activity, but their function remains poorly defined in the context of a tRNA synthetase.
In this report, we address the structure of yeast Gln4, the tRNA synthetase that covalently
Gln
couples glutamine to tRNA , and of its N-terminal domain. We have crystallographically
solved the structure of full-length Gln4, but find that 216 residues comprising 95% of the N-
terminal domain is not resolved, although biochemical analysis confirms its presence. The N-
terminal domain appears to be ordered, based on low resolution refinement and comparison
to diffraction data from a C-terminal truncated Gln4. To complement the crystallographic
studies, we used Small Angle-X-ray Scattering (SAXS) to examine the structure of Gln4.
Alignment of full-length Gln4, C-terminal domain and the N-terminal domain with the
crystallographic results shows that the N-terminal domain is ordered and attached to the C-
terminal body by a flexible linker. SAXS studies of a homologous structure from C. glabrata
indicate a similar envelope, suggesting a common but as yet undefined function for this
domain. In addition, we have solved the structure of the truncated N-terminal domain, which
is currently undergoing refinement. We will describe these results, as well as our progress on
the complementary use of crystallography and SAXS to develop and test hypotheses for
structural and functional investigations.
07.11.5

MADMAX: Multi-wavelength anomalous diffraction using medium-angle x-ray solution

scattering
1 1 2 1
Lee Makowski , Diane Rodi , Dave Gore , Robert Fischetti
1 2
Argonne National Lab, Argonne, IL 60439, United States, Illinois Institute of Technology,
Chicago, IL 60616, United States

Anomalous diffraction from proteins in solution has the potential to provide detailed
information about the relative positions of atoms. Its use is hampered by the extreme
weakness of the signal. When x-ray energy is changed, small systematic changes in
scattering occur even in the absence of anomalous diffraction. These are not informative of
the sample structure and complicate observation of the anomalous signal. Knowledge of the
structure of the absorption edge is used to inform the analysis of changes in scattering near
the edge. We used a principal components analysis of patterns taken at x-ray energies near
the absorption edge to isolate the anomalous signal from other effects and measure it to ~ 5 A
spacing. Measured differences compare well to theoretical expectations calculated from the
atomic coordinates of proteins of known structure. Application to Fe-containing proteins;
seleno-met-labelled proteins and membrane proteins has been demonstrated.
07.11.6

Comprehensive structural analyses of proteins, nucleic acids and their complexes in

solution.
1 1 1 1 4
Robert Rambo , Greg Hura , Michal Hammel , Alexei Kazantsev , Susan Lees-Miller , Norm
3 3 1,2
Pace , Robert Batey , John Tainer
1 2
Lawrence Berkeley National Lab, Berkeley, CA, United States, The Scripps Research
3
Institute, La Jolla, CA, United States, University of Colorado at Boulder, Boulder, CO, United
4
States, University of Calgary, Calgary, Alberta, Canada

Synchrotron based small-angle X-ray scattering (SAXS) of biological macromolecules has

developed into a powerful structural tool that complements X-ray crystallography (MX),
Nuclear Magnetic Resonance, and Electron Microscopy. SAXS based measurements of
proteins and nucleic acid complexes provides complementary data on small and large
assemblies, extended conformations, and flexibly linked domains in solution at about 12 Å
resolution. In practice, SAXS investigations of biological samples can show inconsistencies
that suggest limitations to the subsequent computational analyses and modeling. Our results
show that many of the problems with SAXS experiments derive from sample heterogeneity
that can be effectively resolved to provide reliable SAXS data. Furthermore, we provide an
analysis pipeline and suggest an appropriate statistical basis for data interpretation. Our
results show that the combination high resolution information with accurate SAXS data can be
used to effectively model the Ku/DNA-PKcs non-homologous end joining complex, the ligand
free state of the SAM riboswitch and the complete architecture of RNase P.
07.12.1

The First Crystal Structure of an Intron Debranching Enzyme

1 1 2 2 1
Eric Montemayor , Alexander Taylor , Joshua Combs , Scott Stevens , John Hart
1 2
Univ. of Texas Health Sci. Center at San Antonio, San Antonio, United States, University of
Texas at Austin, Austin, United States

The intron debranching enzyme (Dbr1) hydrolyses the unique 2’-5’ phosphodiester bond
found within branched or lariat RNA species. Hydrolysis of this bond is necessary for the
recycling of excised introns and the formation of several small nucleolar RNAs; hydrolysis of
the 2’-5’ phosphodiester bond has also been shown to play a role in the movement of genetic
elements by retrotransposition. Dbr1 enzymes contain a highly-conserved N-terminal domain
that is homologous to the metallophosphatase superfamily of enzymes and a C-terminal
domain with virtually no sequence similarity to any other protein of known structure. Here we
present the structure of the 354 amino acid Dbr1 from the parasite Entamoeba histolytica.
This structure reveals that the N-terminal domain of Dbr1 indeed adopts a
metallophosphatase-like fold; however, a cysteine residue that is conserved among Dbr1
enzymes is in a position usually occupied by an aspartate in other metallophosphatases. The
structure also provides a clear view of the C-terminal domain of Dbr1, a helical structure that
forms an extensive interface with the metallophosphatase domain. This structural information
is currently being used to design new experiments that will elucidate the mechanistic basis for
recognition and hydrolysis of 2’-5’ phosphodiester bonds by Dbr1.
07.12.2

Structure and Regulation of Yeast Glycogen Synthase-2

Sulochanadevi Baskaran, Peter Roach, Anna DePaoli-Roach, Thomas Hurley

Indiana University School of Medicine, Indianapolis, IN, United States

Glycogen is a major energy reserve in most eukaryotes and its rate of synthesis is controlled
by glycogen synthase. The activity of eukaryotic glycogen synthase is regulated by the
opposing effects of glucose-6-phosphate and phosphorylation and a conserved arginine
cluster is responsible for the regulatory control. We solved the crystal structure of yeast
glycogen synthase-2 by multiple isomorphous replacement using two tantalum bromide
cluster derivatives, combined with four-fold molecular averaging and partial model phase
combination. The presence of a unique sequence insertion in the C-terminal Rossmann-
domain forms the majority of the enzyme’s tetrameric interface. This interface is surprisingly
small and underlies the extensive conformational flexibility critical for the regulation of
enzymatic activity. The conserved cluster of arginine residues are localized within a single
alpha-helix (termed the regulatory helix) positioned orthogonally to one of the molecular two-
fold axes. Site-directed mutants based on our initial structure demonstrate that arginines 583
and 587 are necessary and sufficient for activation by glucose-6-phosphate. A screen of new
crystallization conditions compatible with the presence of glucose-6-phosphate was
performed and a new crystal form that only grew in the presence of this activator was
identified. The structure of the activated form was solved using the individual subunits of our
previous structure for molecular replacement calculations. Binding of glucose-6-phosphate to
the N-terminal ends of the regulatory helices induces a large conformational transition
amongst the subunits, akin to the petals of a flower opening, which frees each of the subunits
in the tetramer to open and close their inter-domain clefts in response to substrate binding
and product release. Additional structures containing product UDP and substrate-analogs
further define the catalytic cycle of this complex enzyme and provide detailed insight into the
molecular bases for its activity states.
07.12.3

Crystal structure of the bacteriophage Mu transpososome

Sherwin Montano, Ying Pigli, Phoebe Rice

Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL,
United States

Bacteriophage Mu utilizes DNA-based transposition to integrate its genome into the

genome of the host cell. It encodes a MuA transposase, a member of the DDE
transposase/integrase family, that recognizes the ends of the phage, and brings them
together to form a synaptic complex called the transpososome. Both the cleavage of the
phage DNA and the strand transfer to the host DNA occur within the transpososome and are
catalyzed by the MuA protein. In vitro, the simplest working system for Mu transposition
comprises a tetramer of MuA bound to two right ends. We have crystallized this tetramer
bound to two right ends in the presence of a target DNA, a complex that contains a total of
~240kDa of protein and 135bp of DNA. The crystals diffract anisotropically to 4.5 Å in one
direction and 5.2 Å in the other two. Experimental phases were obtained using MIRAS with
four derivatives which include Ta6Br12 clusters, mersalyl acid, SeMet modified protein, and
brominated DNA where every T on one strand had been replaced with 5-Br-dU. Building the
transpososome model at this resolution was facilitated by the fact that individual structures of
the largest 3 of the 4 protein domains have been previously determined. Anomalous
difference Fourier peaks from Se and Br served as markers in the placement of these
domains as well as in the model building of the DNA. The resulting structure shows a highly
intertwined complex that agrees with a vast array of biochemical data, but also reveals
unexpected interactions.
07.12.4

Structural and biochemical studies of symplekin, a scaffold in pre-mRNA 3’ -end

processing

Kehui Xiang, Takashi Nagaike, Song Xiang, Turgay Kilic, Maia Beh, James Manley, Liang
Tong

Columbia University, New York, United States

The maturation of most eukaryotic messenger RNAs requires extensive processing, including
3’-end cleavage and polyadenylation. The mammalian pre-mRNA 3’-end processing complex
has an essential ~1160kD component symplekin, which was originally identified in tight
junctions. Symplekin mediates interactions with many other 3’-end processing factors and,
like its yeast homolog Pta1, is thought to be a scaffold in the 3’-end processing machinery.
Here we report the crystal structure of the N-terminal domain of symplekin in a ternary
complex with a RNA polymerase II C-terminal domain (CTD) Ser-5 phosphatase Ssu72 and a
CTD phosphor-peptide. Our structure indicates the N-terminal domain of symplekin consists
of 7 pairs of anti-parallel helical repeats in an overall shape of an arc. Ssu72 binds to the
concave face of the arc, with its active site 25 Å away from the contacting surface.
Unexpectedly, we found that the phosphatase activity of Ssu72 can be stimulated by
symplekin, suggesting a potential regulatory role for symplekin rather than simply a passive
scaffold in pre-mRNA 3’-end processing. More strikingly, the CTD phosphor-peptide in the
active site of Ssu72 has a cis pSer5-Pro6 peptide bond configuration, which contrasts to other
known CTD peptide conformations. Our studies provide molecular basis with which to
understand symplekin as a scaffold and its new function in pre-mRNA 3’-end processing.
07.12.5

Crystal structure of an active Hsmar1 transposase in humans that has evolved into a
novel DNA repair protein

Millie Georgiadis, Kristie Goodwin, Tsuyoshi Imasaki, Suk-Hee Lee

Indiana University School of Medicine, Indianapolis, IN, United States

While transposase activity has played an important evolutionary role accounting for half of the
present organization of the human genome, little is known about the role of transposases in
humans today. The Hsmar1 transposon, a class II transposable element, is an ancient
element within the human genome introduced at least 50 million years ago in ancestral
primates. To date, only one example of an intact copy of the Hsmar1 transposase domain has
been identified within the human genome. This “functional” Hsmar1 transposase domain
exists within a chimeric fusion protein, Metnase (also known as SETMAR), which resulted
from insertion of the Hsmar1 transposon downstream of a SET gene encoding a histone
methyltransferase, ultimately fusing the SET and transposase domains. Metnase retains
many of the transposase activities including terminal inverted repeat (TIR) specific DNA-
binding activity, DNA cleavage activity, albeit uncoupled from TIR-specific binding, and the
ability to form a synaptic complex. However, Metnase has evolved as a DNA repair protein
involved in non-homologous end joining. In order to obtain crystals of the transposase
domain, an homology structure-based protein engineering approach was used to introduce
substitutions for several surface residues. Additional protein engineering efforts including
substitution of a Leu residue within the predicted core of the enzyme with a Met in order to
increase the expected anomalous signal for SeMet SAD phasing proved essential for the
structure determination. The structures of two different crystal forms determined at 2.5 Å and
1.9 Å reveal a novel dimeric enzyme with unusual active site plasticity that may be involved in
modulating metal binding. We show through characterization of a dimerization mutant that the
dimeric form of the enzyme is required for its DNA cleavage, DNA-binding, and non-
homologous end joining activities. Our analysis suggests that the structure of the Metnase
transposase has been remarkably conserved through evolution; however, there is a clustering
of substitutions in the modern enzyme within the putative DNA-binding site that may have
resulted in a loss of transposition specific DNA cleavage activity and the acquisition of DNA
repair specific cleavage activity.
07.12.6
+
Mechanism of NADH/NAD Sensing by the Redox Sensing Repressor, Rex.

Krystle J. McLaughlin, Claire M. Strain, Mark S. B. Paget, Clara L. Kielkopf

1
Department of Biochemistry and Biophysics, University of Rochester School of Medicine and
2
Dentistry, Rochester, NY, United States, Department of Biochemistry, University of Sussex,
Falmer, Brighton, United Kingdom
+
The ratio of reduced to oxidized nicotinamide adenine dinucleotide (NADH/NAD ) has
emerged as a sensitive indicator of the intracellular redox state. Yet, how the slight chemical
+
differences between oxidized NAD and reduced NADH are transmitted into signals for DNA
+
binding remains unknown. To elucidate the molecular basis for NADH/NAD -dependent gene
regulation, we present three novel structures of a Rex family repressor (T-Rex), a key sensor
of oxygen availability among Gram-positive bacteria. First, the 2.3 Å resolution structure of T-
+
Rex bound to NAD and a consensus Rex operator site is compared with our previous
structure of the induced T-Rex/NADH complex. NADH releases the T-Rex dimer from DNA by
a remarkable >40° rigid-body closure between the dimeric subunits. Second, structures of
variants in the absence of ligand (at 2.4 Å and 2.5 Å resolution, respectively) reveal that T-
+
Rex is pre-configured for DNA binding in the absence of NAD . Complementary site-directed
mutagenesis experiments establish the importance of highly conserved residues for
+
NADH/NAD sensing. The NAD(H)-sensing mechanism of Rex may serve as a prototype for a
wide-range of redox-sensing proteins whose function is regulated by the intracellular
+
NADH/NAD ratio.
07.12.7

Structural recognition and functional activation of Fc receptors by innate pentraxins

1 2 2 2 2,3
Jinghua Lu , Kristopher D. Margon , Lorraine L. Marnell , Carolyn Mold , Terry W. Du Clos ,
1
Peter D. Sun
1
Structural Immunology Section, Laboratory of Immunogenetics,National Institute of Allergy
2
and Infectious Diseases, Rockville, MD, United States, Department of Internal Medicine and
Department of Molecular Genetics and Microbiology, University of New Mexico, Albuquerque,
3
NM, United States, VA Medical Center, Albuquerque, NM, United States

Pentraxins are a family of ancient innate immune mediators conserved throughout evolution.
The classical pentraxins include serum amyloid P component (SAP) and C-reactive protein
(CRP), which are two of the acute-phase proteins synthesized in response to infection. Both
recognize microbial pathogens and activate the classical complement pathway though C1q.
More recently, members of the pentraxin family were found to interact with cell-surface Fcγ
receptors (Fcγ R) and activate leukocyte-mediated phagocytosis. Here we describe the
structural mechanism for pentraxin’ s binding to Fcγ R and its functional activation of Fcγ R-
mediated phagocytosis and cytokine secretion. The complex structure between human SAP
and Fcγ IIa reveals a diagonally bound receptor on each SAP pentamer with both D1 and D2
domains of the receptor contacting the ridge helices from two SAP subunits. We further
extend these findings in two aspects: 1) High-affinity IgG receptor, Fcγ RI, is also recognized
by pentraxins. The crystal structure of human Fcγ RI reveals a unique architecture of the
three-Ig domains of Fcγ RI, which is reminiscent of the head of a seahorse. Despite the
additional third Ig domain (D3), the first two Ig domains (D1 and D2) of Fcγ RI adopt a similar
structure to that of Fcγ RIIa and also confer the high affinity for the binding of IgG. Therefore,
like Fcγ RIIa, Fcγ RI binds to two diagonally-spanned subunits of each CRP or SAP pentamer
with both D1 and D2 domains of the receptor contacting the ridge helices from two SAP
subunits. 2) We show that pentraxins also recognizes Fcα RI, both in solution and on cells.
Fcα RI bound to the effector face of CRP and SAP in a region close to but not identical with
that of Fcγ Rs. The binding of CRP to Fcα RI transfected RBL cells induced degranulation and
the phosphorylation of Syk kinase. Mutational and binding studies show that pentraxins are
diverse in their binding specificity for FcRs but conserved in their recognition structure.

Taken together, these results establish antibody-like functions for pentraxins in the Fc
receptor pathway and suggest a new crosstalk between the innate and adaptive immune
systems.
07.12.8

Engineering a gp41 epitope-display scaffold protein

Robyn L. Stanfield, Robert Pejchal, Johannes S. Gach, Michael B. Zwick, Ian A. Wilson

The Scripps Research Institute, La Jolla, CA, United States

Currently, only a handful of potent, broadly-neutralizing antibodies against the HIV-1

virus have been discovered. Structural studies of these antibodies in complex with their viral
epitopes have led to a better understanding of how the antibodies achieve their effective
neutralization, and many studies are underway to try to use this structural information to
develop an effective vaccine. One epitope, the gp41 Membrane Proximal External Region
(MPER) is located on the gp41 protein, adjacent to its’ membrane-spanning domain. Crystal
structures of three different broadly-neutralizing antibodies against overlapping sections of the
MPER region suggest that the MPER undergoes conformational change during viral fusion,
and that the antibodies are recognizing different stages of that process. The epitope is
probably only accessible to antibodies for a brief period of time during the pre-hairpin
intermediate phase of viral fusion, adding to the difficulty of eliciting similar antibodies against
this region. To try to stabilize the MPER epitope in a relevant conformation for use as an
immunogen, we are using proteins to act as stabilizing scaffolds for the epitope. Based on
the previously determined crystal structure of the anti-HIV-1 Fab Z13e1 in complex with a
gp41 epitope peptide (1) we have engineered a scaffold protein that contains the Z13e1
epitope and binds Z13e1 with nanomolar affinity. The scaffold protein was developed by first
searching the PDB for proteins with regions of main-chain structural homology to the Z13e1
epitope, and then introducing side chains critical for Fab recognition into that region. We
have cloned and expressed the scaffold protein and determined its structure in complex with
Fab Z13e1. The crystal structure shows almost perfect structural homology between the
scaffold-constrained and peptide epitopes bound to the Fab. This Z13e1 epitope scaffold will
be tested as an immunogen to elicit Z13e1-like antibodies against the HIV-1 virus, and will
also be useful as a reagent to identify or select for antibodies against the same region.

Pejchal, R. et al. A conformational switch in human immunodeficiency virus gp41 revealed by

structures of overlapping epitopes recognized by neutralizing antibodies. J. Virol. 83: 8451,
2009.
07.12.9

Mapping of conformational epitopes in dust mite allergens Der f 1 and Der p 1

1 2 2 2 2
Maksymilian Chruszcz , Martin D. Chapman , Lisa D. Vailes , Jill Glesner , Anna Pomés ,
1
Wladek Minor
1 2
University of Virginia, Charlottesville, VA, United States, INDOOR Biotechnologies, Inc.,
Charlottesville, VA, United States

The Group 1 mite allergens, Der f 1 and Der p 1, are potent allergens excreted by
Dermatophagoides farinae and Dermatophagoides pteronyssinus, respectively. Monoclonal
antibody-based epitope mapping studies identified multiple species-specific epitopes on the
Group 1 mite allergens, and a unique cross-reactive epitope defined by mAb 4C1. Binding of
4C1 to this epitope inhibits human IgE binding to both allergens. In order to determine the
molecular basis of the cross-reactivity, the crystal structures of Der f 1 and Der p 1, both in
complex with 4C1, were elucidated. Structural data reveal the epitope that is common to both
Der f 1 and Der p 1. In both allergens the epitope is not only formed by the same amino acids,
but the conformations of the epitope forming residues are very similar. Moreover these amino
acids have the same conformations whether the allergens are complexed with antibody or
not, in the case of both Der f 1 and Der p 1. The crystal structure of 4C1 alone shows that the
CDR regions of the antibody do not significantly change in conformation upon allergen
binding. Identification of the key amino acids involved in the unique cross-reactive epitope on
the Group 1 mite allergens in combination with site-directed mutagenesis will facilitate
identification of IgE-binding epitopes. This approach will lead to design of modified allergen
molecules that could be used in recombinant vaccines for the treatment of dust mite allergy.
07.12.10

In crystallo posttranslational modification within a MauG/pre-methylamine

dehydrogenase complex
1 1 2 3
Carrie Wilmot , Lyndal Jensen , Ruslan Sanishvili , Victor Davidson
1 2
University of Minnesota, Minneapolis, MN, United States, GM/CA-CAT, Advanced Photon
3
Source, Argonne, IL, United States, University of Mississippi Medical Center, Jackson, MS,
United States

MauG is a highly unusual di-heme enzyme that completes the synthesis of the novel amino
acid derived catalytic cofactor, tryptophan tryptophylquinone (TTQ) found in the enzyme
methylamine dehydrogenase (MADH). TTQ is formed by post-translational modification of two
Trp residues of the β polypeptide chain of MADH during which two atoms of oxygen are
incorporated into the indole ring of β Trp57 and a covalent bond is formed between the indole
rings of β Trp57 and β Trp108. The natural substrate for MauG (preMADH) is a 119kDa protein
precursor of MADH with mono-hydroxylated β Trp57 and no cross-link (Fig 1). MauG
catalyzes a six-electron oxidation to complete TTQ biosynthesis, and it can do this in a H2O2-
dependent or O2/reducing equivalents-dependent reaction. We have solved the X-ray crystal
structure of MauG complexed with preMADH to 2.1 Å resolution (Rwork 13.5%; Rfree 18.9%).
The c-type heme irons and the nascent TTQ site are separated by long distances over which
electron transfer must occur to achieve catalysis. In addition one of the hemes has an atypical
His-Tyr axial ligation. The crystalline protein complex is catalytically competent, as on addition
of hydrogen peroxide MauG-dependent TTQ synthesis occurs. This structure, also to 2.1 Å
resolution (Rwork 14.2%; Rfree 19.4%), identifies the heme to which H2O2 / O2 binds.

Figure 1: TTQ synthesis

M-002

Enhancing protein crystallization success: Exploring additives and crystal detection.

Russell Judge, Sumiko Takahashi, Elizabeth Fry, Kenton Longenecker, Erin Fleck, Mark Chiu

Abbott Laboratories, Abbott Park, IL, United States

In exploring the use of additives to improve crystallization success, we have focused

on the use of ionic liquids. First ionic liquids were tested in the crystallization of model
proteins to determine the effectiveness of these solutions as precipitating agents and as
additives. The ionic liquids produced changes in crystal morphology and mediated significant
increases in crystal size in some cases. Based upon the experiments performed with model
proteins, the ionic liquids were used as additives for the crystallization of the poorly diffracting
monoclonal antibody 106.3 Fab in complex with the B-type natriuretic peptide (5-13). The
ionic liquids improved the crystallization behaviour and provided improved diffraction resulting
in the determination of the structure.

Our second area of interest is in crystal detection, where the challenge in screening is to
quickly identify and distinguish protein crystals from non-protein crystals, which often also
form in crystallization experiments. Here we will discuss our experience with UV fluorescence
imaging and fluorescent probe labelling for crystal detection.
M-005

Crystal Structure of the Stress Response Regulator PerR from Streptococcus

pyogenes

Shu-Ying Wang, Shi-Yu Chao, Chih-Cheng Tsou, Jiunn-Jong Wu

National Cheng Kung University, Tainan, Taiwan

Streptococcus pyogenes is a significant human pathogen that causes life-threatening

diseases such as necrotizing fasciitis and streptococcus toxic shock syndrome. Unlike other
gram-positive bacteria, S. pyogenes does not produce catalase, an oxidoreductase that is
essential in other bacteria for resistance to the damaging effects of growth in an aerobic
environment. Therefore it would be interesting to understand how this organism copes with
such stress. In S. pyogenes, PerR has been identified as a transcriptional repressor of Dpr,
which is an iron-binding protein that confers resistance to multiple stresses. Studies have
shown that Dpr expression is induced by high concentrations of iron, zinc, nickel, or hydrogen
peroxide. In pursuit of understanding the molecular mechanism by which PerR regulates the
expression of Dpr, we have solved the crystal structure of PerR to 1.7Å resolution. The
structure shows that PerR is a homodimer with two zinc binding sites per monomer. It has
been proposed that PerR dimerization is necessary for DNA-binding, and that metal
coordination at the regulatory site causes a protein conformational change that affects the
protein DNA-binding ability. The crystal structure of PerR provides a basis with which to test
these hypotheses and to better understand this entire molecular mechanism.
M-008

Crystal Structure and IgE-epitope Mapping of BG60

1 1 2 2 1
Tse-Hao Huang , Chun-Hsiang Huang , Song-Nan Su , Ho-Jen Peng , Shwu-Huey Liaw
1 2
National Yang-Ming University, Taipei, Taiwan, Taipei Veterans General Hospital, Taipei,
Taiwan

Up to 20% of the population in developed countries suffers from Type 1 allergic diseases
such as rhinitis, bronchial asthma or conjunctivitis. The major cause of outdoor allergy is from
airborne grass pollen such as Bermuda grass (Cynodon dactylon). A 60-kDa isoallergen
mixture of Bermuda grass (BG60) have been isolated and characterized. Here we have
reported a crystal structure of BG60 at 2.15 Å resolution, which is the first flavinylated allergen
with the C and the 8-methyl group of the FAD cofactor cross-linking to Cys
6 177 113
and His ,
respectively. The protein structure belongs to the vanilly-alcohol oxidase (VAO) superfamily,
and possesses an open and large substrate-binding groove compared with other known
o
members. Large BG60 isoforms display a higher Tm value of ~20 C than the small isoforms.
A short N-terminal segment is conserved in the superfamily and presence in the large but not
the small isoforms, forms extensive interactions with surrounding residues hence greatly
enhances the structural integrity. Putative IgE-binding epitopes were then predicted, and
several peptide decamers were designed. A peptide representing residues 396-405 displayed
strong IgE reactivity to four allergic sera and cross-reacted with BG60-binding IgE.
M-011

Structural Basis for Unique Ganglioside Recognition by Botulinum Neurotoxin Type C

Zhuji Fu, Abby Kroken, Andrew Karalewitz, Joseph Barbieri, Jung-Ja Kim

Medical College of Wisconsin, Milwaukee, WI, United States

Botulinum neurotoxins (BoNTs) are comprised of seven serotypes (A through G) and are the
most toxic proteins known, causing rapid paralysis through inhibition of neurotransmitter
release. BoNTs are synthesized as a single 150 kDa polypeptide. The N-terminal ~50 kDa
domain (light chain) is a zinc protease and the C-terminal domain (heavy chain, HC) is
composed of a translocation domain (HCT) and a receptor binding domain (HCR). The HCR
domain of BoNT/A and BoNT/B bind motor neurons via a cell surface ganglioside and a
protein receptor. While earlier studies by Kozaki and Binz and their collaborators showed that
HCR/C binds gangliosides, there is limited information for the identity of the host receptor and
the mode of neuron binding by BoNT/C. We have determined the crystal structure of HCR/C
to 2.5 Å resolution and have studied HCR/C’ s affinities to various gangliosides by
biochemical/cell-based assays. The overall structure of HCR/C is similar to the HCR domains
of BoNT serotypes /A, /B, /E, and /F. However, there are several significant differences, most
notably: 1) the relative orientation of the two sub domains is different, and 2) the “ganglioside-
binding pocket” is different from that of HCR/A, HCR/B, or HCR/T, lacking the signature
Tryptophan, indicating that HCR/C binds gangliosides via a unique mechanism different from
other BoNT serotypes, consistent with our biochemical data. In a solid phase binding assay,
HCR/C binding to gangliosides was dependent upon W1258 (which is located away from the
homologous location of the ganglioside binding pocket of HCR/A), and HCR/C showed the
highest affinity for gangliosides that contained two sialic acid moieties. In addition, unlike
HCR/A and HCR/B, HCR/C bound primary neurons at 4C, independent of synaptic activity,
and mutation of W1258 drastically abolished the binding of HCR/C to the background level.
The structure of the HCR/C, together with the biochemical results, reveals the structural basis
for a unique ganglioside binding mode by BoNT/C and provides insight into the basis for
BoNT/C entry into neurons.
M-014

High-throughput pH Measurements: Seeing is Believing

1 2 2
Roger Sayle , Vincent Fazio , Janet Newman
1 2
NextMoves Software, Santa Fe, NM, United States, CSIRO, Parkville, VIC, Australia

Coaxing macromolecules into crystals suitable for X-ray diffraction analysis is a multivariate
problem, dependent on the type, construct and purity of the macromolecule preparation, and
additionally the chemical nature of the precipitation solution and the physical arrangement
used to crystallise the protein sample. We are interested in characterizing the process of
crystallization so as to understand the biophysical underpinnings, with the long term goal of
making the production of suitable diffraction quality protein crystals a more robust and
reproducible process.
As component of this, we are developing high-throughput techniques to analyse the chemical
properties of the precipitants used in crystallisation. We present here a novel method of
determining the pH of a solution, which uses imaging technology rather than a physical probe.
This allows the pH to be measured rapidly, and with sufficient accuracy, in 96 or higher
density plates.
We use this assay to return the pH values of 96 well crystallization screens, but perhaps more
importantly, as a quality control check during the preparation and storage of crystallization
screens.
M-017

Center for Structural Genomics of Infectious Diseases (CSGID)

1 1 2 3 4
Wayne Anderson , Elisabetta Sabini , Aled Edwards , Daved Fremont , Andrzej Joachimiak ,
5 6 7 8 2
Wladek Minor , Christine Orengo , Zbyszek Otwinowski , Scott Peterson , Alexei Savchenko
1 2
Northwestern University, Chicago, IL, United States, University of Toronto, Toronto,
3 4
Canada, Washington University, Saint Louis, MO, United States, University of Chicago,
5
Chicago, IL, United States, University of Virginia, Charlottesville, VA, United States,
6 7
University College London, London, United Kingdom, UTSW, Dallas, TX, United States,
8
JCVI, Rockville, MD, United States

The Center for Structural Genomics of Infectious Diseases (CSGID, http://csgid.org/csgid/)

has been funded by NIAID with the goal of applying structural genomics approaches to
potential drug targets from NIAID category A, B, and C priority pathogens. The goal of CSGID
is to determine approximately 400 protein structures during a five-year period. The targets
that enter the high throughput pipeline of CSGID are proteins with biomedical relevance and
potential therapeutic benefits and they include drug targets, essential enzymes, virulence
factors, and vaccine candidates. All the steps, from target selection through structure
deposition are available to the scientific community as a free service. Scientists can request
structure determination by CSGID for targets of interest simply by filling an online submission
form. Community requested targets that are considered to be feasible and biomedically
relevant are submitted to NIAID for approval and then entered into the CSGID pipeline. As of
April 2010, 21% of the target proteins selected at CSGID have been requested by the
scientific community. A major focus of CSGID is to determine the structures of complexes of
the target proteins with small molecule ligands such as natural substrates, cofactors and drug
candidates, for drug discovery purposes. In order to identify small molecule ligands, target
proteins are screened using a thermal denaturation shift assay and a smaller number of
possible drug candidates are screened using different CSGID small molecules libraries. The
Center also has access to the Life Sciences Collaborative Access Team (LS-CAT) beamlines
at the Advanced Photon Source (APS) for screening of crystals using the robotic system for
crystal sample changing and the state of the art equipment for crystallographic data
collection. After 2½ years since the beginning of the contract, CSGID has deposited in the
PDB more than 170 structures, with and without ligands bound, of which 24 belong to targets
requested by the community.
In addition to structure determination, CSGID also provides the scientific community with the
protein expression systems (deposited in the Biodefense and Emerging Infections Research
Resources Repository), and the results of ligand screens.
CSGID is a consortium of institutions and investigators that have experience with high
throughput approaches. Each institution contributes to different aspects of the project,
working together to produce a complete, coordinated high throughput structure determination
pipeline.
CSGID has been funded with Federal funds from the National Institute of Allergy and
Infectious Diseases under Contract No. HHSN272200700058C.
M-020

X-ray Crystallographic Studies on the Structure-Function Relationship of UDP-glucose-

4-epimerase from Aspergillus nidulans.

Sean Dalrymple, Inder Sheoran, Amira El-Ganiny, Susan Kaminskyj, David Sanders

University of Saskatchewan, Saskatoon, SK, Canada

Morphologically simple eukaryotes, such as fungi, are becoming increasingly potent human
pathogens because the resulting fungal diseases are quite often therapeutically intractable on
account of their underlying metabolic similarities with animal systems. Even with aggressive
treatment, fungal infections produce high mortality rates and many drugs are starting to lose
effectiveness due to emerging fungal resistance. Typically, drugs are focused against fungal
extracellular carbohydrates, the building blocks for fungal cell walls, because these
components are not found in animal systems. To this effect, we are interested in Aspergillus
nidulans UDP-glucose-4-epimerase, UgeA, which is responsible for the interconversion
between UDP-glucose and UDP-galactose along the galactose metabolic pathway. As UDP-
galactose is a precursor for lipopolysaccharide biosynthesis, UgeA is therefore an important
target for antifungal drug development. Furthermore, UgeA exhibits interspecies variation
and heterogeneity at both structural and functional levels. Subsequently, the structure-
function relationship differences between UgeA of the host and the pathogen can be exploited
for targeted design of potential drugs. As such, detailed structural characterization of UgeA
from Aspergillus nidulans is necessary for identifying potential inhibitors. In the present
study, we report the preliminary structure of A. nidulans UgeA for which synchrotron
diffraction data has been collected at the Canadian Light Source (CLS). The native UgeA
data was processed in P1 with unit cell parameters a = 67.1 Å, b = 68.2 Å, c = 163.2 Å, α =
86.4 , β = 82.4 , and γ = 60.7
the unit cell contains six molecules packed as three sets of dimers. A discussion on the
solution and refinement of the A. nidulans UgeA structure will be presented along with details
of the co-factor binding site as well as the relevant structure-function relationship extracted
from the experimental model.
M-023

Structure of DUF1341 Representative – a Possible Aldose

Norma Duke, Ruiying Wu, Brian Feldmann, Andrzej Joachimiak

Argonne National Laboratory, Argonne, IL, United States

The crystal structure of CAL13803, a protein found in Yersinia enterocolitica, a Gram-negative

coccobacilli, belonging to the family Enterobacteriaceae, has been determined. Y.
enterocolitica infections produce severe diarrhea and other symptoms in humans and a
search for possible antibacterial targets is on going. The CAL13803 protein belongs to a
large family of bacterial proteins of unknown function (DUF1341). Structure was determined
to 1.90 Å, using SAD data collected at the 19-BM beamline of the Structural Biology Center.
The CAL13803 structure is a classical  barrel observed for Class I aldolases with the
putative active site residues (K160, Thr190, Phe162) mapping to Escherichia coli 2-keto-3-
deoxy-6-phosphogluconate (KDPG) aldolase catalytic site template (1FQ0). However, in
contrast with trimeric KDPG aldolase, CAL13803 appears to be a dimer. Sequence and
structural searches have been conducted to further refine function prediction. BLAST
searches find that the sequence is virtually identical to that of the protein ZP_04619510.1,
from Yersinia aldovae, which is annotated as a 4-hydroxy-2-oxoglutarate aldolase/2-dehydro-
3-deoxyphosphogluconate aldolase. The closest structural homologue found, via DALI, is 2-
keto-3-deoxy-6-phosphogalactonate (KDPGal) aldolase from E. coli (2V82). In addition, close
structural homologies are found for pyridoxine 5’ phosphate syntase from Yersinia pestes
(3F4N), and thiamine phosphate pyrophosphorylase from Pyrococcus furiosus (1XI3).
Structural comparisons and co-crystallization trials with various ligands are being initiated to
help identify specific biochemical function of CAL13803 protein.

Acknowledgement: This work was supported by the U.S. Department of Energy, Office of
Biological and Environmental Research, under contract DE-AC02-06CH11357, and by a grant
from the National Institute of Health (GM074942).
M-026

Percentile based spread: a better measure of structural difference

Edwin Pozharski

University of Maryland, Baltimore, United States

Traditional measure of difference between two or more similar structures is the root mean
square difference (r.m.s.d.). This estimate of the variation in atomic positions is sensitive to
the presence of outliers, which contribute disproportionately to the overall r.m.s.d. With
outliers present, the measure does not reflect the average atom shift since the assumption of
normal distribution is violated.

The percentile based spread is proposed as an alternative measure of structural difference. It

is shown that interatomic distances generally follow the theoretically predicted distribution
when isomorphous structures are compared. Multivariate approximation is described and
possible sources of interatomic distance outliers are discussed, such as variation of model
precision, conformational changes and modeling errors. Several examples are analyzed,
including isomorphous crystal structures, proteins undergoing conformational changes and
multiple model ensembles.
M-029

Structures of the Interaction Protein KREPA6 of the Editosome in Complex with VHH
Domains from Llama Antibodies Which Served as Crystallization Chaperones
1 1 2 1 3
Young-jun Park , Meiting Wu , Els Pardon , Stewart Turley , Andrew Hayhurst , Junpeng
1 2 1
Deng , Jan Steyaert , Wim G. J. Hol
1
Biomolecular Structure Center, Department of Biochemistry, School of Medicine, University
2
of Washington, Seattle, WA, United States, Structural Biology Brussels, Vrije Universiteit
3
Brussel, Brussels, Belgium, Department of Virology and Immunology, Southwest Foundation
for Biomedical Research, San Antonio, Texas, United States

Trypanosomes are protozoan parasites several of which are the causative agents of severe
infectious diseases. In trypanosomes, a ~2 M Dalton multi-protein complex called the
“editosome” plays a crucial role in mitochondrial gene expression by extensive U-
insertion/deletion editing of pre-mRNAs. Many key editing steps occur in three different types
of editosomes, which share a core of 12 proteins. This common set of twelve proteins
includes enzymes for uridylyl (U) removal and addition, two RNA ligases, two proteins with
RNase III-like domains, and six proteins with predicted oligonucleotide binding (OB) folds.
Biochemical results indicate that the OB-fold proteins form an extensive protein-protein
interaction network that connects two trimeric subcomplexes that catalyze U removal or
addition and RNA ligation. The key “interaction protein” KREPA6, plays a central role in
interconnecting several editosome subcomplexes.
Crystallization of KREPA6 appeared to be a tremendous challenge but was immediately
successful once VHH domains from llama antibodies were employed as “crystallization
chaperones”. We used both immune VHH libraries and a large semi-synthetic VHH libraries
combined with phage-display techniques to generate and select for VHH domains. Three
different crystal structures of KREPA6-VHH domain complexes were solved. In each case a
KREPA6 dimer occurred in the center of the heterotetramer. Interestingly, biochemical
solution studies showed that the VHH domains dissociate the KREPA6 homotetramers and
form heterotetramers as seen in the crystal structures. Solution studies also indicated that the
C-terminal tail of KREPA6 is involved in the dimerization of KREPA6 dimers to form
tetramers. The crystal structures of two of the VHH domains showed a novel parallel
arrangement of β -strands from antibody and protein antigen. The three structures of KREPA6
in complex with VHH domains show how the antibodies facilitate protein crystallization by
taking care of the majority or all of the crystal contacts.
These studies show that llama VHH domains are versatile tools to crystallize recalcitrant
proteins.
M-032

IMCA-CAT Insertion Device Beamline Upgraded for High-Throughput, Shutterless,

Continuous-Rotation Data Collection

Anne M. Mulichak, Kevin P. Battaile, Joe Digilio, Rong Huang, J. Lewis Muir, Eric Zoellner,
Ann Bertling, Lisa J. Keefe

IMCA-CAT, Argonne National Laboratory, Argonne, IL, United States

The IMCA-CAT insertion device beamline, 17-ID, has been upgraded to a micro-focused
high-flux diffraction beamline for automated high-throughput macromolecular crystallography.
The energy range is 6–20 keV, allowing MAD/SAD experiments at energies for commonly
used derivatives. The full beam is focused to 65 m x 30 m at the sample position and the
GM/CA-CAT mini-quad collimators provide the user with selectable beam sizes of 300, 20,
10, and 5 m. Beam stability is achieved with custom software that automatically positions
the beam with ±2 m positional accuracy. Automated sample mounting is performed with the
Rigaku ACTOR robot, and samples are viewed with the Maatel on-axis viewing system. The
new ALIO goniometer has a small (1.2 m) sphere of confusion, thus maintaining accurate
sample positioning. A new detector, the PILATUS 6M pixel-array from DECTRIS, permits
shutterless, continuous-rotation data collection. The high volume of data is managed with a
64 TB storage system that consists of a highly-available Lustre distributed parallel file system
with Fibre Channel and InfiniBand interconnects. Custom software provides an intuitive
interface for controlling the beamline. Rigaku JDirector software for data collection enables
queuing of data collection jobs for automated data acquisition. Both unattended and remote
data collection modes are supported. The automated and rapid data collection capabilities of
beamline 17-ID are ideally suited for high-throughput crystallography projects such as
pharmaceutical industry drug discovery programs.
M-036

Crystal structure of PAN C-termini bound to 20S proteasome reveals AAA+ ATPase
open the 20S gated channel with a mechanism different from that of 11S activator.
1 2 2 1
Yadong Yu , David Smith , Alfred Goldberg , Yifan Cheng
1 2
UCSF, San Francisco, CA, United States, Harvard Medical School, Boston, MA, United
States

The primary site for protein degradation in eukaryotic cell is the 26S proteasome, which is
composed of the 20S proteolytic core and two 19S regulatory particles that each contains a
hexameric ATPase ring. The ATPases activate proteolysis by docking their C-termini with a
conserved hydrophobic-tyrosine-X (HbYX) motif into pockets in the 20S to stimulate the
opening of a gated substrate entry channel into the 20S. In contrast, 11S activators use C-
termini for binding while the additional structural element, activation-loops open 20S gate. To
understand how PAN relies only on C-termini to activate 20S, we inactivated PA26 by
mutating its activation-loops and replaced its C-termini with those from PAN. The resulting
fusion protein formed tight complex with 20S. Cryo-EM reconstruction and crystal structure of
this complex were solved at 8 and 4Å respectively. Both structures show a rotation in 20S -
ring compared to that in 20S structure itself. The crystal structure defined the detailed
interactions between the critical C-terminal HbYX motif and the 20S -subunits. In particular
the H-bond, cation-π and hydrophobic interactions contributed by the highly conserved
tyrosine residue largely trigger the radial movement of -subunit N-terminal fragments which
lead to gate opening. Upon binding of HbYX motif, 20S pocket undergoes an induced-fit
conformational change and becomes much tigher than those with 11S activitor. Altogether
these 7 tightened pockets transform into a rotation of -ring. The findings imply that AAA+
ATPases use C-termini of higher specificity to activate 20S. It may further help understand
how hexameric AAA+ ATPase can work out the symmetry mismatch with heptameric 20S
proteasome.
M-039

Crystallographic and Cryo-EM Studies of the HK97-like Bacteriophage DNA Packaging

Portal
1 2 1 1 1
Bogi Nocek , Dong-Hua Chen , Adam Stein , Rory Mulligan , James Abdullah , Robert
1 2 1
Jedrzejczak , Wah Chiu , Andrzej Joachimiak
1 2
Midwest Center for Structural Genomics, Argonne, Il, United States, National Center for
Macromolecular Imaging, Houston,Tx, United States

During large dsDNA virus assembly, viral DNA synthesized using bacterial resources is
transferred into preformed empty prohead. The assembly and packaging of viral DNA is
driven by a translocation motor system composed of several proteins and is powered by
hydrolysis of ATP. A key component of the packaging machine is a portal protein. This protein
assembles into a large ~500 kDa ring-shaped portal with a central channel. The motor
connects the head of the phage to its tail and promotes translocation of the dsDNA into the
prohead during packaging. At present, the structural information of phage portals is limited to
two representatives, SPP1 and phi29. Here we present the 2.9 Å crystal structure and low
resolution cryo-EM results of the dsDNA bacteriophage HK97 family portal.
HK97 bacteriophages are widespread and they infect Gram-positive bacterial hosts. The
portal is a 300-residue protein and shares very little sequence similarity to SPP1 and phi29
proteins. It assembles into a dodecamer with a characteristic funnel-like structure and a 40 Å
wide central channel. The surface of the channel is mainly electronegative, but it includes
three positively charged rings that may promote DNA transfer. Details of the HK97 structures
will be presented.
This work was supported by National Institutes of Health Grant GM074942 and by the U.S.
Department of Energy, Office of Biological and Environmental Research, under contract DE-
AC02-06CH11357.
M-042

Capillary-top crystal mounting method for S-SAD phasing and semi-automated

mounting device
1,2 2 2
Nobuhisa Watanabe , Yu Kitago , Isao Tanaka
1 2
Nagoya University, Nagoya, Japan, Hokkaido University, Sapporo, Japan

For the S-SAD phasing that utilizes single-wavelength anomalous diffraction from sulfur
atoms, using longer-wavelength X-rays has advantages for the detection of small anomalous
signals from them. However, the accuracy of the measured diffraction intensity decreases at
longer wavelengths because of the greater X-ray absorption effect. We had improved the
standard cryogenic crystal mounting method with a new tool, the capillary-top mounting
method (formerly the loopless mounting method), by which we can remove the buffer around
the protein crystal just before flash freezing of the crystals. This capillary-top mounting
method makes it possible to eliminate amorphous ice around the protein crystal and reduces
systematic errors in the evaluation of small anomalous differences. However, use of this
method requires a large amount of skill. The processes of harvesting and flash freezing the
crystal are performed using both hands, and the mouth is also used for cryo-solution
aspiration.

In order to reduce its laboriousness, we have developed a new device that can freeze the
protein crystal semi-automatically using a micro-manipulator. Using this device, one can
harvest the protein crystal from the crystallization drop, and further procedures, such as
withdrawal of the solution around the crystal by suction and subsequent flash freezing of the
protein crystal, are carried out automatically. The loop glued to the tip of the glass capillary of
the custom cryo-pin for the capillary-top mounting method was also improved. The
conventional nylon loop was replaced with a microlithography shaped polyimide film.

We have recently designed a new pattern of the polyimide film and made a prototype of an
implementation tool for the gluing process of the polyimide film at the tip of the glass capillary.
These devices make it easy for structural biologists to use the capillary-top mounting method
for S-SAD phasing using longer-wavelength X-rays.

This work was supported by the Targeted Proteins Research Program (TPRP) of the Ministry
of Education, Culture, Sports, Science and Technology, Japan.
M-045

Function Assignment by Catalytic Site Alignment with ProMOL

1 1 1 1 1 1
Paul Craig , Mario Rosa , Scott Mottarella , Greg Dodge , Sean Bourne , Luticha Doucette ,
2
Herbert Bernstein
1 2
Rochester Institute of Technology, Rochester, NY, United States, Dowling College,
Oakdale, NY, United States

The ProMOL plug-in for the PyMOL molecular graphics environment uses the geometry and
measurement tools incorporated in PyMOL to identify and align the catalytic site motifs of
enzymes. The motifs are described in the Catalytic Site Atlas (http://www.ebi.ac.uk/thornton-
srv/databases/CSA/). These motifs were used by Torrance et al., (J. Mol. Biol. 347:565-81,
2005) to create two sets of JESS templates that were distributed broadly across the six
classes of the Enzyme Classification system. The first template set was based on two atoms
(JESS CaCb: C-alpha and C-beta of each catalytic site residue) while the second template
set was based on three atoms (JESS FA: C-alpha, C-beta, and one side chain atom) to
explore enzyme family homology. We have used a subset of the same structures (20 total
PDB entries) to prepare motifs in ProMOL that are based on all side chain atoms of the
catalytic site residues. The performance of the two JESS template sets and the ProMOL
template set were compared for the template structures, their homologs and randomly chosen
structures from the Protein Data Bank. The ProMOL motifs returned an exact identification of
templates and known homologs 74% of the time, while the JESS CaCb templates returned
exact identifications 8% of the time and the JESS FA templates returned an exact
identification 18% of the time. These templates were then used to suggest functions for
multiple PDB entries that are classified as "Structural Genomics, Unknown Function". Of 39
entries that were tested, ProMOL template alignment suggested 28 function assignments
while the JESS FA template alignment suggested 4 function assignments. The library of
ProMOL templates is being expanded and will be used to extensively test PDB structures of
unknown function.

The project is funded in part by NIGMS grants R15 GM078077-01 and 3 R15 GM078077-
01S1.
M-048

How we got the chemical bond wrong (and how we can rescue it)

I. David Brown

McMaster University, Hamilton, Ontario, Canada

How do you define a bond? And why do we need both an ionic and a covalent model?
Neither model provides a satisfactory definition of a bond, and the boundary between the two
models is fuzzy. The concept of a localized bond, which is good for intuitive modeling, is only
found in the covalent picture, but simulations of structures are only successfully performed
using the ionic model. And quantum mechanics sees no clear difference between ionic and
covalent bonds, so why do we still need two incompatible models to describe chemical
structure?

The mistake we have made is to assume that a chemical bond necessarily involves a pair of
electrons. If we abandon this idea we can create a unified localized bond model using only
two atomic properties: the valence (oxidation state) and the coordination number. The
electrostatic field of the ionic model defines localized bonds that combine the intuition of the
covalent model with the quantitative predictions of the ionic model. This results in a simpler,
more revealing and powerful picture of how localized bonding can be used to predict chemical
structure and properties for all types of bond.

A key feature of the unified model is an electronegativity, defined as the valence of an atom
divided by its coordination number. Atoms with electronegativity > 1 have more valence
electrons than bonds. They tend to form saturated bonds with the remainder appearing as
lone pairs. Atoms with electronegativity < 1 have more bonds than valence electrons and
form extended structures.
M-051

Conformational Polymorphism in Crystals of a Novel HCV Inhibitor

1 1 2 1 2 3
Qi Gao , Antonio Ramirez , Fukang Yang , Soojin Kim , Baoqing Ma , Michael Galella
1 2
Bristol-Myers Squibb Company, New Brunswick, NJ 08903, United States, Bristol-Myers
3
Squibb Company, Wallingford, CT 06492, United States, Bristol-Myers Squibb Company,
Princeton, NJ 08540, United States

The subject of this paper is two neat polymorphic forms of bis-HCl salt of a potent Hepatitis C
virus (HCV) inhibitor targeting the virus-encoded nonstructural protein 5A (NS5A).
Crystallographic studies of the polymorphic N-1 and N-2 forms showed two dramatically
different conformations that reflect the C2 symmetry in the chemical structure. In the crystal of
N-1, the molecule sits on a crystallographic 2-fold axis, while in the crystal of N-2 it is found in
general positions with a pseudo 2-fold axis that is perpendicular to the ideal 2-fold axis in the
conformation of N-1. We have not been able to prepare N-1 since the first time when N-2 was
crystallized. The extinct N-1 was reasoned to be probably a metastable kinetic form, although
it was not experimentally confirmed due to the lack of material. X-ray crystal structures of N-1
and N-2 were analyzed to understand the relationship of intramolecular (conformer) and
intermolecular (lattice) energy in the crystallization and stability of polymorphs. Computational
methods were also applied to gain a semi-quantitative assessment of the relative stability
between the two polymorphs. Semiempirical AM1 calculations suggested greater stability of
the N-2 conformation and that this stability does not appear to be related to salt formation,
although salt formation could be crucial for crystal packing and lattice energy. This is in
agreement with our experimental observations that no crystallization has been achieved with
the free base. MMFF coordinate scans for isolated dihedral angles indicated that central aryl-
aryl angle bending in N-1 is an important source of destabilization and the -isopropyl-
carbamate side-chain conformations are unfavorable.
M-054
Crystal structure of phosphoglucosamine mutase from Bacillus anthracis, an enzyme
in the peptidoglycan biosynthetic pathway.

Ritcha Mehra-Chaudhary, Lesa Beamer

University of Missouri, Columbia, MO, United States

The enzyme phosphoglucosamine mutase (PNGM) participates in the biosynthetic pathway of

peptidoglycan, a component of the bacterial cell wall. Enzymes in this pathway are
considered excellent targets for antibacterial compounds. We present here the first structure
of a PNGM, from the bioterrorism agent B. anthracis. Peptidoglycan is a major component of
the protective coat of the bacterial spore, and plays a known role in the pathogenesis of
anthrax. The structure of B. anthracis PNGM (447 residues) was determined by molecular
replacement (MR) at a resolution of 2.7 Å. Limited sequence identity (~30%) between B.
anthracis PNGM and the available models complicated the MR efforts. Conformational
variation between the two molecules in the asymmetric unit (asu) was an additional hurdle to
refinement of the structure. Anomalous difference Fourier maps from a 3.2 Å
selenomethionine data set proved critical for identifying methionine residues and successful
model building. The resulting structure reveals that PNGM shares the overall architecture
common to the large a-D-phosphohexomutase enzyme superfamily, although domain 4 of the
protein has topological differences from other structurally characterized members of the
family. The structure also shows that while key catalytic residues are highly conserved, other
residues within the active site vary, and may be responsible for the distinct substrate
specificity of the PNGM enzymes. A novel observation from the crystal structure is a potential
dimeric interface between the two molecules in the asu, which is also present in the crystal
packing of several related enzymes, and suggests that PNGM enzymes may function as
dimers in solution. The implications of the structure for understanding enzyme function and
its utility for inhibitor design with applications to medicine and bioterrorism will be discussed.
M-057

Structural Basis for Substrate Specificity of Inositol Pentakisphosphate 2-Kinase

Varin Gosein, Gregory J. Miller

McGill University, Montreal, Canada

Inositol phosphate (IP) signaling pathways are critically involved in cell communication and
IPs have been implicated in a number of diseases including cancer and diabetes. IP signaling
pathways are maintained by the IP kinase family of enzymes that sequentially phosphorylate
inositol triphosphate (IP3) to create an array of >30 signaling molecules with up to 8
phosphate groups on the inositol ring. One IP kinase, inositol pentakisphosphate 2-kinase
(IPK1), catalyzes the conversion of inositol 1,3,4,5,6 pentakisphosphate (IP5) to inositol
hexakisphosphate (IP6). Studies have shown that IP6 possesses anticancer activity in vitro
and in vivo against numerous tumors. IP6 treatment of human leukemic cell lines in vitro
significantly reduced the abnormal cell population while leaving normal leukocytes unaffected
while IP6 given to animal models for colon, liver, lung, and skin cancers reveal the antitumor
activity of IP6 in vivo. The precise mechanisms underlying the action of IP6 remain
unresolved, as there are no selective inhibitors for IPK1 to modulate levels of IP6 and
knockouts of IPK1 are embryonic lethal. A high-resolution molecular structure of IPK1 would
facilitate the design of selective inhibitors for IPK1 and would provide the first insights into the
catalytic cycle and mechanism of the enzyme. Studies in yeast have shown mutations in IPK1
can be rescued by IPK1 orthologs, which demonstrates functional conservation of IPK1
despite very low sequence similarity across species. Arabidopsis thaliana IPK1 (AtIPK1)
shows highest conservation with human IPK1 (HsIPK1) within six short regions; however, the
degree of similarity decreases significantly outside of these regions. It is anticipated that
these regions assemble into commonly structured catalytic sites and, in fact, may adopt a
novel fold. HsIPK1 accumulates in inclusion bodies when expressed in E.coli which has
complicated structure determination of this IPK1, but bacterial expression of AtIPK1 has been
proven to be soluble and active and amenable to crystallization. Our overall objective is (1)
develop the first system for investigation of the catalytic cycle of IPK1 during IP6 synthesis,
and (2) to generate a homology model of HsIPK1 using a crystal structure of AtIPK1.
M-060

Resonant Multi-Wave Diffraction Study on Magnetite

Shih-Chang Weng, Cheng-Gang Chen, Yen-Ru Lee, Chia-Hung Chu, Shih-Lin Chang

Department of Physics, Natl. Tsing Hua Univ., Hsihchu, Taiwan

Multi-wave diffraction under resonance conditions, relating to both atomic and electronic
structures, provides site- and element-specific information of the crystal studied. This
characteristic gives an opportunity to investigate the electronic configuration of interesting
materials, such as transition-metal oxides. In this presentation, the resonant multi-wave
diffraction is used to study the charge distribution of magnetite below the Verwey transition
temperature. Due to the three-wave interference and resonant scattering, resonant multi-
wave diffraction could reveal the 3d-electron distribution in magnetite. Two three-wave
diffractions, (002)/(-3-31) and (002)/(311), were measured in the vicinity of Fe K-edge. The
self-normalized relative intensities were recorded for different photon energies and compared
with the calculated results based on the dynamical diffraction theory with Born approximation.
The variation of the diffracted intensity shows the evidence of charge-ordering on the
octahedral iron sites of magnetite below the Verwey transition temperature.
M-063

X-ray Diffraction Measurements of Substrate-Supported Crystals Using a Hexapod

Lin Yang

Brookhaven National Laboratory, Upton, NY, United States

Crystals formed on a planar substrate usually have a common crystal plane parallel to the
substrate while their in-plane orientation undefined. In diffraction measurements of these
structures, it is often required to anchor the X-ray beam on a fixed spot on the sample, such
as an optically visible crystallite or island, while rotating the sample to access different Bragg
peaks, so that the lattice constants as well as the local orientation of the lattice can be
defined. Here, a hexapod is used in place of a traditional multi-circle diffractometer to perform
area-detector-based diffraction measurements on an organic transistor device that contains
6,13-bis(triisopropylsilyethynyl)-pentacene (TIPS-pentacene) crystals. The hexapod allows for
sample rotations about any user-defined rotation center. Two types of complex sample
motions have been programmed to characterize the structure of the TIPS-pentacene crystal:
an in-plane powder average has been performed at a fixed grazing-incident angle to
determine the lattice parameters of the crystal; then the in-plane component of the scattering
vector was continuously rotated in transmission geometry to determine the local lattice
orientation.
M-066

Real space asymmetric units optimized for integer grids

1 2 1
Marat Mustyakimov , Ralf Grosse-Kunstleve , Paul Langan
1 2
Los Alamos National Lab, Los Alamos, NM, United States, Lawrence Berkeley Lab,
Berkeley, NM, United States

Efficient handling of direct-space asymmetric unit information in crystallographic map

calculations can significantly accelerate widely used algorithms, e.g. bulk-solvent correction in
macro-molecular refinement. We have investigated various trade-offs in the choice of
optimized asymmetric unit definitions. It has been found that a number of the established
asymmetric unit definitions in the International Tables (IT, Vol. A) are not optimal for the
handling of grids as used in map calculations. More suitable definitions are suggested. A
major property of the alternative definitions is that the interior of each face or edge of the
asymmetric unit polyhedron has a constant multiplicity. We observe that such an asymmetric
unit exists for each space group type. The new definitions and related algorithms have been
added to the cctbx library.
M-069

Portable Temperature Controlled Microplate for Optimization of Protein Crystals

Gabriela Juarez-Martinez, Philipp Steinmann

Centeo Biosciences Ltd, Dumbarton, United Kingdom

Using temperature as an additional crystallization variable can increase space search and
help finding better crystallisation conditions. The correct management can aid in controlling
crystal nucleation, growth and dissolution of defects on the surface of the crystal; can modify
the solubility and super saturation of the sample in a reversible manner; can prevent
denaturation of temperature sensitive proteins and in improving the reproducibility of results.

Currently the crystallography community is restricted to use fridges/incubators and

temperature controlled rooms, however these do not always offer the best solution. Problems
arise when the samples are taken out of the fridge/incubator e.g. for inspection under the
microscope. At that moment the temperature control is lost which in some cases can affect
the systems in a negative manner (dissolution of the crystal, protein denaturation,
destabilization of the system). Even if the unintended temperature fluctuation result in the
formation of a crystal, without knowing anything about this temperature fluctuation, the results
are often difficult to reproduce.

In response to this problem, Centeo has designed an electronic portable temperature

controlled microplate. The system consists of 5 rows with 8 wells per row, allowing screening
up to 40 different experiments simultaneously. Each row can be independently controlled
allowing to screen up to 5 different temperatures from 4°C to 60°C at once. When in use, the
microplate resides in a docking station, from which it can be removed for portable, battery
powered operation. This work will show the performance of the system including
crystallisation results.
M-072

High-throughput Crystallization: What have we Learned?

Ievgeniia Dubrovska, Ludmilla Shuvalova

Northwestern University, Chicago IL, United States

Structural genomics has been around for about 10 years and produced numerous important
new methods, automated procedures and inspired design of robotic instruments. These new
methods and tools were rapidly incorporated into the structural genomics center’s pipelines
and adopted by structural biology labs. One of the critical stages of protein structure
determination, crystallization, was not an exception. With all that new technology do we have
to change the strategy of the experiment?
At Northwestern University group of Center for Structural Genomics of Infectious Diseases
(CSGID) we have tried to analyze our observations of crystallization experiments
accumulated in 2.5 years of our project. How does one decide when protein is ready for
screening? Which screens and how many to use? How to best balance time and effort
involved with sample requirements? Crystals growing “like mushrooms”…do we want them
to? Does ligand screening help? For how long to keep the plate? These and some other
questions addressed in our poster presentation.
M-075

Synthesis, Structures and Characterization of Organotin complexes

S. M. Lee, H. Mohd Ali, K. M. Lo

University of Malaya, Kuala Lumpur, Malaysia

Based on literature review, metal complexes are widely prepared and have been successfully
applied in the treatment of numerous human diseases including cancer. Among the many
metal complexes, organotin complexes have been widely studied for their biological activities
such as anticancer, antihistamine, antifungal, biocides and anti-fouling. Schiff base derived
from substituted salicylaldehyde has been widely used as polydentate ligands in the
preparation of metal complexes. In the present studies, a series of Schiff base ligands were
prepared by reacting 3-hydroxy-2-naphthoic hydrazide with substituted salicylaldehydes. The
diorganotin complexes were subsequently prepared by adding the ligands with diorganotin
dichloride or oxide in 1:1 molar ratio and were characterized by various spectroscopic
methods including IR, NMR spectroscopies. The x-ray structures of some of the diorganotin
complexes were reported. All the complexes were found to be isostructures and the tin atom
in each of the complexes is in a distorted cis-C2NO2Sn trigonal-bypyrimidal coordination.
The deprotonated ligand was coordinated as tridentate via the azomethine nitrogen and two
phenoxo oxygens. The tridentate 5-bromosalicylideneaminato(3-hydroxy-2-
naphthohydrazidate) and 5-chlorosalicylideneaminato(3-hydroxy-2-naphthohydrazidate)
dianions of each of the complexes were stabilized by an intramolecular hydrogen bonding O-
H—N. The distortion from the trigonal-bypyrimidal coordination was influenced by the
presence of the R groups.
M-078

A Structural Study of Second-order Jahn-Teller Distortions in CuX5 Complexes (X=Cl,

Br).

Bryan Reynolds, Marcus Bond

Southeast Missouri State University, Cape Girardeau, MOI, United States

While Jahn-Teller distortions in CuX6 and CuX4 complexes are well known and studied, the
second-order Jahn-Teller distortions in CuX5 complexes have received less attention. Reinen
and Astanasov (Chem. Phys. (1989) 136, 27) have mapped out the second-order Jahn-Teller
2+
active distortion modes for trigonal bipyramidal Cu following the discovery by Reinen and
Friebel (Inorg. Chem. (1984) 23, 791) that [Co(NH3)6]CuCl5 contains disordered, distorted C2v-
3-
symmetry CuCl5 rather than a trigonal bipyramidal complex. Since that time many new
pentacoordinate halocuprate(II) structures have been reported, some of which show
distortions outside the direct Berry rotation pathway that interconverts square pyramidal and
trigonal bipyramidal geometries. We have examined distortion modes, as defined by Reinen
and Astanasov, of CuX5 complexes (both isolated and not) found in structures from the
Cambridge Structural Database and from our own laboratory, and map out the frequency with
which these modes are observed experimentally. In addition we examine the structural data
to seek an empirically defined distinction between five-coordinate CuX5 complexes and 4+1
coordinate complexes in which one Cu-X bond is semicoordinate.
M-081

Increasing accuracy in low-resolution structures

Bradley Hintze, Christopher Williams, Vincent Chen, Dave Richardson, Jane Richardson

Duke University, Durham, NC, United States

Structural biology provides an unparalleled view of the molecular world, allowing researchers
to uncover important mechanisms that give rise to a protein’s function, provided there is
sufficient detail in the data (which is generally a function of the data’s resolution). Currently,
much research in structural biology is devoted to structure determination of large proteins and
complexes, which tend to yield low-resolution data and present many challenges to
crystallographers. Despite a variety of computational techniques that can be employed to
assist low-resolution structure determination,
validation typically shows an order of magnitude
more errors (such as atom-to-atom clashes and
highly irregular secondary structure) in 3-4 Å
models than in 1-1.5 Å models. The figure shows a
squashed surface helix at 3.5 Å resolution with the
sidechain density as small nubbins; fitting such
density often leads to Ramachandran and rotamer
outliers. The research presented here aims to
identify systematic patterns of such errors and create techniques to diagnose and correct
them. We will utilize more thorough restraints on the regularity of geometry, torsion-angle
patterns, and secondary structure and will rely on the power of consistently satisfying both all-
atom contacts (with explicit hydrogens) and the diffraction data. Finding such solutions will
require new calculation methods informed by analysis of how low-resolution systematically
distorts electron data. Significantly better accuracy would lead to great improvements in the
biological utility of low-resolution structures.
M-084

Apex2 and Proteum2 Suites – Hints and Tips

Garold L. Bryant, Jr., Bruce C. Noll, Michael Ruf, Charles F. Campana, Matthew M. Benning,
Joerg Kaercher

Bruker AXS Inc., Madison, WI, United States

The Apex2 and Proteum2 suites represent at least 40 man-years of programming. Several
online venues exist for finding help and even though every effort has been made to make the
suites intuitive, new features and enhancements are sometimes overlooked. Many helpful
features such as report and CIF file generation are now available. Helpful hints and tips from
application scientists will cover a wide variety of questions usually encountered by customers.
A helpful list of do’s and don’ts for Proteum2, Apex 2 and BIS will be presented. Getting the
most from various configurations of Bruker instruments will be presented.
M-087

Screening Based on Proximity to Crystallization Conditions, Not Crystals.

Marc Pusey

iXpressGenes, Huntsville, AL, United States

Current crystallization screening protocols involve exposure of the macromolecule to a range

of cocktail solutions, with the endpoint being the appearance of a crystal. Conditions resulting
in clear or precipitated outcomes are dropped from subsequent consideration. We suggest
that a more advantageous approach would be a method that indicates proximity to
crystallization conditions, not one that relies on randomly getting lucky. We are developing
such an approach based upon fluorescence anisotropy, a measure of the rotational rate (and
thus volume or mass) of the protein in solution. The method involves measuring a series of
dilutions of the protein in the crystallization solution. Lead conditions are optimized using the
capillary counter diffusion method. Experiments with model proteins, where the plate-based
outcomes were already known, indicated that ≥ 80% more crystallization conditions could be
obtained on average. We are now using this as the sole screening method with test proteins
and to date has found crystallization conditions for all of them. The data also indicates that
the signal intensity as a function of concentration is also a significant lead indicator.
Advantages are: Speed, screening results can be had within 6-30 hrs after having pure
protein in hand; more leads, many conditions that would have been discarded are found to
be leads; reduced protein consumption, fewer screens and assay drop volume reduction can
reduce the protein required to ≤ 0.3 mg/96 condition screen. Finally, the data is obtained as
numerical values, facilitating subsequent analysis.
M-090

Crystallographic Studies of a Fluorescent Phytochrome

Michele Auldridge, Kenneth Satyshur, David Anstrom, Katrina Forest

University of Wisconsin, Madison, WI, United States

The three dimensional structure of phytochrome from Deinococcus radiodurans provided an

opportunity to design structure-based variants of this red light photoreceptor with altered
photocycle properties. The most dramatic of these are a family of fluorophores with
substitutions at the Asp207 position. Given that these novel proteins use a tetrapyrrole
chromophore available within cells and have emission maxima above 700 nm, they are
extremely promising biotechnology tools. We have solved the crystal structure of the
Asp207His variant of the chromophore-binding domain (CBD) of D. radiodurans phytochrome,
and at 1.24 Å this structure is the highest resolution DrBphP structure to date. An altered
hydrogen bond network within the biliverdin-binding pocket is observed. His207 forms a direct
hydrogen bond to the chromophore A ring oxygen, and an interaction between Tyr263 and
the now-missing Asp207 is altered. Additionally, with the goal of decreasing the size of this
fluorophore and facilitating its use as a fluorescent tag in vivo, we changed several residues
in the native coiled-coil dimer interface. Most significantly, Leu311 and Leu314 were replaced
with glutamate to introduce charge repulsion. Interestingly, recombinantly expressed Dr
phytochrome with the Asp207His substitution surreptitously binds the biliverdin precursor,
protoporphyrin IX. Structural studies of phytochrome with this cyclic tetrapyrrole are currently
underway.
M-093

The SMART X2S: Reports from the field. Experiences in Research and Teaching.

Bruce C. Noll, Michael Ruf, Joerg Kaercher

Bruker AXS Inc, Madison, Wisconsin, United States

The Bruker SMART X2S automated benchtop diffractometer for single crystal diffraction has
been installed in a number of facilities for teaching and for research. It is showing itself to be
fully capable of accommodating both tasks. After some time in the field, the SMART X2S is
producing publications and training students in the science of crystallography. The instrument
has been used for undergraduate education, graduate research, and as a principal instrument
at the ACA Summer School. At the 2009 Summer School, eleven structures were determined
in nine days. A summary of these varied experiences will be presented.
M-096

Progress on the Structure of the Essential Yersinia pseudotuberculosis Type III

Secretion System Membrane Protein YscU
1 2 2 2 1
Xiaoming Zhao , Innokentiy Maslennikov , Christos Tzitzilonis , Roland Riek , Partho Ghosh
1 2
University of California San Diego, La Jolla, United States, Salk Institute for Biological
Studies, La Jolla, United States

YscU is an essential inner membrane protein of the Yersinia pseudotuberculosis type III
secretion system, which is responsible for translocating bacterial proteins from the bacterial
cytosol into the cytosol of host mammalian cells. YscU is predicted to contain four
transmembrane domains and a large, C terminal cytosolic domain. Intact YscU and two
different truncation fragments of YscU, all containing the transmembrane regions, have been
cloned as TEV protease-cleavable fusions to the protein MISTIC. Intact YscU and the
truncation fragments of YscU were found to be expressed to levels required for
crystallographic studies. These proteins were extracted from membranes with detergent and
2+
purified by Ni NTA chromatography, after which MISTIC was removed by cleavage with
2+
TEV protease. Cleaved proteins were reapplied to the Ni NTA column and lastly purified by
size-exclusion chromatography. Detergent concentrations of purified protein samples were
analyzed by NMR, and oligomerization states by static light scattering. Crystallization trials
are underway.
M-099

Enhancements in Motif Preparation and Identification in ProMOL

1 2 1
Mario Rosa , Herbert Bernstein , Paul Craig
1 2
Rochester Institute of Technololgy, Rochester, NY, United States, Dowling College,
Oakdale, NY, United States

The ProMOL plug-in for PyMOL enables users to create motifs based on catalytic sites and
then identify the function of protein structures by comparison to these motifs. Significant
upgrades have been made in the functionality and coding of the plug-in.

The Motif Maker function was rewritten to eliminate some earlier bugs and to expand its
capabilities. It can now build a motif with up to 10 residues (rather than 5) and repeated
residues in an active site (e.g., 3 histidines) are now accepted. Now it is possible to explore
larger motifs such as those found in protein-protein interaction sites. Once testing is
complete, the code written by Motif Maker will be readily incorporated directly into ProMOL,
which will expand the capabilities of the Motif Finder. The code for the Motif Finder in
ProMOL was expanded to include an expanded set of motifs (based on the JESS motif set)
that more systematically covers the different enzyme classes. The results generated by Motif
Finder can now be viewed on the screen directly or exported as a simple table with comma
separated values in a format that can be read by most spreadsheet programs. In the future,
we plan to change the motif finder to compare exact residue matches (rather than the atom
counting currently in place) and to handle batch processing of structure submissions to the
Motif Finder.

As an open source plugin, ease of modification of ProMOL is important. The code used of
ProMOL was originally stored in a single file, which hampered efforts at further development.
Therefore, the file was split into several files. The original file retains the functions necessary
to hook ProMOL to PyMOL and to initiate the user interface of ProMOL. The other files are
stored within a directory 'ProMol_dir' and grouped according to their function in the overall
program. For example, files that hold the graphic user interface are stored within the 'Tabs'
folder, while files used in the methodology of the program are stored in the 'Methods' folder.

The project is funded in part by NIGMS grants R15 GM078077-01 and 3 R15 GM078077-
01S1.
M-102

Copper Binding and Dynamic Behavior in Doped Cadmium-Histidine

1 2 3
Michael Colaneri , Kristin Kirschbaum , Jacqueline Vitali
1 2
SUNY at Old Westbury, Old Westbury, NY, United States, University of Toledo, Toledo, OH,
3
United States, Cleveland State University, Cleveland, OH, United States

Electron Paramagnetic Resonance (EPR) spectroscopy and X-ray crystallography was

employed to gain a better understanding of copper binding and dynamic behavior in doped
histidine crystals. Recent EPR measurements on copper-doped Cd-histidine showed a
significant temperature dependence that can be explained by copper site dynamics. At
temperatures lower than 150K, the EPR spectra exhibit two copper patterns, each related by
the crystalline two-fold axis that runs through the host cadmium ion and between the two
coordinating histidines. At a temperature higher than 200K and up to room temperature, the
EPR spectra consist of only a single copper species that is characterized by rhombic g and A
hyperfine tensor values, which are close to the averaged values of the corresponding low
temperature, symmetry site related tensors. Subsequent EPR experiments display a 1:1
conversion from the low to high temperature copper species within a relatively narrow
temperature range near 175K. These observations differ from those found in a previous EPR
and crystallographic study of the dynamic behavior of copper in doped Zn- histidine (Dalosto
et al., J. Phys. Chem. 2001, A105, 1074). Here the authors propose that the copper
undergoes a continuous 4-state dynamic Jahn-Teller distortion, having a transition
temperature near 268K, and further suggest that the copper site dynamic behavior is
correlated with a fluctuating disorder of a water found in the host structure. In order to
investigate the possibility of a similar type of host structural explanation, the crystallographic
analysis of Cd-histidine was performed at temperatures that straddle the transition
temperature (at 130K and 200K). The results of this study as well as an interpretation of the
77K EPR spectral superhyperfine splittings will be discussed.
M-108

A tale of two polymorphs: macroscopically observed phase changes and atypical

hydrogen-bonding in four uranium-bearing supramolecular materials

Nicholas P. Deifel, Christopher L. Cahill

The George Washington University, Washington, DC, United States

2+
Synthetic efforts to explore coordination polymers of uranyl (UO2 ) systems have often
focused on exploiting metal-ligand coordination to create new families of materials. Hydrolysis
of the uranyl ion in these systems commonly results in the formation of polynuclear building
units and thus accurate prediction of the coordination environment of inorganic building units
remains difficult. It is now known that highly acidic aqueous reactions in high chloride and
2-
bromide media favours the formation of the uranyl tetrahalide anion ([UO2X4] ) over
polynuclear building units. This structural unit has been shown to interact with protonated,
linear dipyridyl amines and 1,4-diazabicyclo[2.2.2]octane (DABCO) in a predictable
supramolecular synthon (NH· · · X2M).

Presented will be a systematic overview of our success with the aforementioned system and
two examples of polymorphism that contrast with the ‘rules’ determined thus far. The first
example of these anomalous phases involves a pseduopolymorph of [UO2Cl4](H2DABCO)
that results from hydration. This material, [UO2Cl4(H2O)](H2DABCO), does not form the
predicted uranium-bearing building unit. Exposed to air, it undergoes a solid-state
transformation to the dehydrated form which shows markedly different fluorescent properties.
The second example is a true packing polymorph of [UO2Br4](C10H10N2). The first phase
crystallizes in the triclinic P-1 space group with both the uranium and organic species sitting
on an inversion center: the bifurcated NH· · · X2M synthon is observed. A second phase has
recently been discovered that crystallizes in a monoclinic space group (P21/c) with two
crystallographically distinct uranium and organic sites. The expected NH· · · X2M hydrogen-
bonding synthon is only observed in one of these pairings; in the other, atypical hydrogen-
bonding between the pyridyl group and the uranyl oxygen is observed.
M-115

Ribosomal Protein Structures and Sequences Define the Prokaryotic Tree of Life

1,2 2 1 1 1 1
William L. Duax , Robert Huether , David Dziak , Lukas Klein , Kevin Gibas , Brian Riefler ,
1 1
Fiona Henning , Rasheen Powell
1 2
Hauptman Woodward Medical Research Inst., Buffalo, NY, United States, State Univ. of New
York at Buffalo, Buffalo, NY, United States

Search vectors composed primary of Gly, Ala, Arg, and Pro residues (GARP) distributed across
the entire protein sequence retrieve 98% of each of the ribosomal proteins in prokaryotic species
with no false ³hits². Different combinations of G, A, R and P and insertions or deletions
differentiate each ribosomal protein from all others. Specific combinations of amino acids in two
sequence positions in perfectly aligned L1 ribosomal proteins from 1600 different prokaryotic
species in the gene bank separate all Gram positive from Gram negative bacteria. We are able to
identify site mutations that subdivide each ribosomal protein ensemble into the individual phylum
of bacteria. Further subdivision into orders, families, genus, and species is trivial. For example,
specific residues in three positions in the alignment of prokaryotic L1 ribosomal proteins isolate
44 L1 proteins from cyanobacteria and 17 L1 proteins from chloroplasts unequivocally supporting
the postulated evolution of the latter from the former. While there are significant differences
between the sequences of the ribosomal proteins in different classes and orders of prokaryotes,
within each order the amino acid sequences have remained highly conserved since divergence
and speciation. We have found that the total GARP content of the ribosomal proteins of each
class and order is a marker of the order of evolution and that the last universal common ancestor
(LUCA) appears to have been an Actinobacteria. Perfect alignment of thousands of members of a
protein family is essential to determining the molecular level details of its evolution, the evolution
of protein fold and function and the evolution of bacterial species. Three dimensional structural
information played an essential role in developing a new GARP based technique to achieve
perfect sequence alignment. In retrospect it is possible to understand why GARP residues are
100% conserved in specific positions in families of proteins present in all species. Support in part
by: Mr Roy Carver, Stafford Graduate Fellowship, Caerus Forum Fund and The East Hill
Foundation.
M-118

In Pursuit of Chemical Matter for Blood Coagulation Factor XIa by Fragment-Screening

Technology
1 1 1 1 1
Melissa Harris , Francis Rajamohan , Seungil Han , Tracy Brown-Phillips , John Bryant ,
1 1 1 2 4
Dave Cunningham , Andrew Butler , Boris Chrunyk , Ronald Sarver , Joel Morris , Rick
5 1 6 2 1 1
Sciotti , Dave Hepworth , Ing-Kae Wang , Shengwu Wang , Jane Withka , Ravi Kurumbail
1 2
Pfizer Global R&D, Groton, CT, United States, Pfizer Global R&D, La Jolla, CA, United
3 4
States, Neogen Corp., Lansign, MI, United States, National Cancer Inst. NIH, Washington,
5
DC, United States, Walter Reed Army Inst. of Research, Washington, DC, United States,
6
Novartis Inst., Cambridge, MA, United States

Cardiovascular diseases remain as the leading cause of death in most of the world. Despite
technical advances, very few novel drugs have been approved for the treatment of acute
coronary syndrome and atrial fibrillation, two of the more prevalent disabilities that result in
high morbidity and mortality. The pursuit for novel medicines to alleviate these symptoms has
targeted both the platelets and the coagulation system that together maintain hemostasis.
Blood coagulation factor XIa (fXIa) functions upstream of factor Xa in the intrinsic coagulation
cascade. Based on human genetics and animal studies, fXIa appears to be an attractive
target for the development of safe anti-thrombotics. In an attempt to identify potential starting
points for medicinal chemistry, we employed several fragment-screening techniques based on
biochemical assays, isothermal denaturation, surface plasmon resonance, nuclear magnetic
resonance spectroscopy (NMR) methods and crystallography. We also evaluated the
druggability of the target by ‘de-construction’ of a known fXIa inhibitor. The poster highlights
some of the key learnings and challenges we faced during our multi-disciplinary approach to
identify suitable chemical matter for this challenging coagulation protease.
M-121
®
Automating Microseeding Protein Crystallography Set-Ups Using Mosquito

Ben Schenker, Joby Jenkins, Rob Lewis, David Smith

TTP LabTech, Cambridge, MA, United States

Crystallising proteins, required for structure determination by X-ray diffraction, is a difficult and
labour-intensive task. One of the many challenges facing the protein crystallographer is
growing crystals of sufficient size and quality to successfully determine the protein’s structure
(this typically requires crystals of around 100-300 µm). For structure-based drug design a
further challenge is being able to generate a sustainable crystal system capable of producing
liganded structures iteratively to support active chemistry. Microseeding, where small crystals
are crushed and suspended in a slurry of crystallisation buffer to produce new nucleation
sites, is a recognised technique to improve crystal quality as well as promote the growth of
larger, single crystals. However, it requires experimentation with varying concentrations of
solutions to achieve successful results and as a manual process this can be very consuming.

One approach to increase the speed and efficiency of microseeding set-ups is through the
automation of the seeding process. However, this is not a simple process because of the
®
problems that crystallisation robots have with dispensing low volumes. The mosquito liquid
handler (TTP LabTech) is ideally suited to automating the complex set-ups required for
microseeding due to its ability to perform multiple aspirations and dispenses with each pipette
and the precise handling of nanolitre volumes of solutions, regardless of their viscosity. Here
we describe an automated approach to setting up microseeding protocols in 96-well plates
using mosquito.
M-124

BioCAT as a National Center for X-ray Solution Scattering of Biological Samples

liang guo, thomas irving

BioCAT Illinois Inst. of Technology, Chicago IL, United States

The BioCAT beamline 18ID is a NIH Biotechnology Research Resource operated by the
Illinois Institute of Technology at the Advanced Photon Source, Argonne National Laboratory.
It is one of the premier x-ray facilities in the world offering researchers opportunities to
perform solution scattering studies on samples of proteins and RNAs/DNAs, and their
complexes. Both static and time-resolved small angle scattering (SAXS) and wide angle
scattering (WAXS) experiments can be used to obtain high data quality suitable for structural
modeling with currently available software packages. With high beam flux and efficient
detectors at the beamline, typical sample consumption for static experiments is ~60 ul at a
concentration of 2 mg/ml for small proteins or 0.5 mg/ml for RND/DNA samples. Time-
resolved experiments can achieve 1 ms time resolution with a Pilatus 100K photon counting
detector (Dectris) coupled with a Bio-Logic SFM-400 stopped-flow instrument, at a sample
consumption around 8 mg per experiment for protein samples or 2mg for RND/DNA samples.
Time-resolved experiments can be performed with a short dead time of 0.5 ms. Data quality
is high enough to allow reliable shape and size determination, offering the capability to follow
the structural change of biological molecules during their functioning process.

Acknowledgments:

Use of the Advanced Photon Source was supported by the U.S. Department of Energy, Basic
Energy Sciences, Office of Science, under contract No. W-31-109-ENG-38. BioCAT is a
National Institutes of Health-supported Research Center RR-08630.
M-127

A Nonlinear Optical Approach to Imaging for Crystal Centering

1 1 2 2 2
David Kissick , Ellen Gualtieri , Kevin Bataille , Michael Becker , Robert Fischetti , Steve
2 2 2 3 1
Ginell , Lisa Keefe , Anne Mulichak , Vadim Cherezov , Garth Simpson
1 2
Purdue University, West Lafayette, IN, United States, Advanced Photon Source, Argonne,
3
IL, United States, The Scripps Research Institute, La Jolla, CA, United States

Second order non-linear optical imaging of chiral crystals (SONICC) was used to image
protein crystals at cryogenic temperatures. The need for automated crystal centering has
motivated the development of many techniques that determine the position of protein crystals
frozen in loops (e.g. brightfield image analysis, birefringence, intrinsic UV fluorescence and X-
ray diffraction based centering). These techniques have been limited by a lack of contrast,
minimum detectable crystal size, and sample damage respectively. SONICC provides the
necessary contrast by effectively eliminating the background from centrosymmetric materials
(e.g. amorphous water and cryoprotectants). Images of crystals grown in lipidic cubic phase
(LCP) demonstrate the effectiveness of this approach in turbid environments. The locations of
the crystals from LCP were confirmed with subsequent mini-beam, raster scanning diffraction
experiments. In addition, preliminary diffraction studies showed no evidence of structural
damage to crystals that were exposed for fifteen minutes with ~500mW laser power, where a
typical image requires less than one minute of exposure at ~100mW. Further experiments are
planned to assess the suitability of SONICC for automated crystal centering, especially with
respect to crystal damage.
M-130

CATS & G-ROB: 6-AXIS ROBOTIC-ARM-BASED AUTOMATED SYSTEMS FOR

CRYSTALLOGRAPHY
1 2 3
JEAN-LUC FERRER , NATHALIE FERRER , JEAN-LOUP RECHATIN , XAVIER
1 1 1 2 2
VERNEDE , JACQUES JOLY , FLORIAN BOUIS , PIERRE MAZEL , PIERRICK ROGUES ,
3
MATTHIEU PRIVAS
1 2
INSTITUT DE BIOLOGIE STRUCTURALE, GRENOBLE, FRANCE, NATX-RAY,
3
GRENOBLE, FRANCE, IRELEC, GRENOBLE, FRANCE

CATS and G-Rob systems were developed on protein crystallography beamline FIP-
BM30A at the ESRF. CATS (Jacquamet et al., JSR 16, 2009, 14-21) is a sample changer
currently now installed on various synchrotrons (SLS, BESSY, DLS, APS, ...). G-Rob, also a
6-axis robotic arm based system, is a fully integrated device for crystallography beamlines
and laboratories. G-Rob is an “all in one” system, since it integrates the following functions:

- sample changer,

- goniometer for frozen samples, capillaries, … (Jacquamet et al., Acta Cryst. D60, 2004,
888-94),

- crystallization plates/micro-chips screening for in situ analysis of diffraction condition and

data

collection (Jacquamet et al., Structure 12, 2004, 1219-25),

- goniometer for non-classical sample environments (high pressure cells, …),

- beam monitoring.

G-Rob provides unique features. It is automated: thanks to its tool changer, it goes
automatically from one application to another. CATS and G-Rob are also highly flexible: if a
new application or a new sample format emerges in the community, a new tool can be
designed to implement it. They are highly reliable systems, based on well-known, industrial
quality equipments, with reduced maintenance.

They are currently in use on beamline FIP-BM30A. It was made available to the research
community in 2005 and up to now, users have expressed an unprecedented high degree of
satisfaction. The crystallization plates screening capability for example appears to be a
precious tool in several cases (crystals too small to be fished, or too fragile, of when there is
no good cryoprotectant).

Several results obtained on FIP-BM30A are presented, such as in situ screening of

membrane proteins, ribosome, high pressure protein diffraction, etc. Recent experiments
demonstrated also the possibility of the automated structural screening for the Fragment
Based Drug Design strategy: the same crystal was reproduced in presence of a library of
fragments. Systematic in situ data collection has shown some of the fragments present in the
active site, without having to manipulate the crystals individually. Movies are available on
www.natx-ray.com.
M-133

SCrALS: Challenging Samples, Straightforward Solution.

1 2
Allen Oliver , Jeanette Krause
1 2
University of Notre Dame, Notre Dame, IN, United States, Univeristy of Cincinnati,
Cincinnati, OH, United States

The Service Crystallography at the Advanced Light Source (SCrALS) project provides
chemical crystallography synchrotron access for samples considered too small or poorly
diffracting to collect on a regular laboratory-based system. Synchrotron sources generate a
more brilliant beam with a significantly higher X-ray photon flux allowing such samples to
have data collected. SCrALS is a mail-in service with data collected during regularly
scheduled beam-time. This presentation will highlight selected projects that would otherwise
not have been possible without access to the more intense beam provided by a synchrotron
source. Contact the authors for more information on the SCrALS project: aoliver2@nd.edu or
jeanette.krause@uc.edu.
M-136

High-Throughput Automation and Remote Access at SSRL Protein Crystallography

Beam Lines: Novel Tools for Training, Education and Collaboration

Aina Cohen, Clyde Smith, Graeme Card, Tzanko Doukov, Thomas Eriksson, Ana Gonzalez,
Scott McPhilips, Pete Dunten, Irimpan Mathews, Jinhu Song, Mike Soltis

SSRL/Stanford University, Menlo Park, United States

The ultimate goal of synchrotron data collection is to get the best possible data from the best
available crystals and the combination of high-throughput automation and remote access at
SSRL has revolutionized the way in which scientists interact with synchrotron beam lines to
achieve this goal. This has also triggered a shift in the way crystallography students and
novices are introduced to synchrotron data collection and trained in the best methods to
collect high quality data. SSRL provides expert crystallographic and engineering staff, state-
of-the-art crystallography beam lines, and a wide range of accessible tools to facilitate data
collection and in-house “remote training”, and encourage the use of these facilities for
education, training, outreach and collaborative research. Hands-on workshops are also
offered by SSRL User Support, both at SSRL and at remote locations, to facilitate the
education of the next generation of protein crystallographers.
The SSRL Structural Molecular Biology group operates 7 crystallography beam lines on the
SPEAR3 storage ring, BL1-5, BL7-1, BL9-1, BL9-2, BL11-1, BL12-2 and BL14-1. All of the
beam lines are MAD-capable, with three of the stations (BL7-1, BL9-1 and BL11-1) using a
single-crystal side-scattering monochromator with a limited energy range (typically 3000-4000
eV) and the other four using double crystal monochromators giving a much wider energy
range capability (over 10000 eV). BL12-2 is a new undulator station optimized for data
collection with microcrystals. This station has an in-house designed microdiffractometer and
a newly-installed Pilatus 6M pixel array detector, which will result in data collection times on
the order of only a few minutes. All of the beam lines are fully automated, with samples being
mounted using the Stanford Automated Mounting system (SAM) and controlled with the Blu-
Ice/DCS software system. Images collected during sample screening are automatically
analyzed and the results, including the number of spots, Bravais lattice, unit cell, estimated
mosaicity and resolution, are visible almost immediately through Blu-Ice, and also via the
internet through Web-Ice. The availability of this real-time analysis enables researchers to
then make informed choices as to which samples and experimental parameters should be
used to collect the best possible data.
M-139

Automated data collection software and hardware for small angle scattering at the
Cornell High Energy Synchrotron Source.

Soren S. Nielsen, Richard E. Gillilan

Cornell University, Ithaca, NY, United States

Data collection software is becoming an increasingly important factor in high throughput

protein structure analysis on modern SAXS beamlines. As acquisition time decreases and the
number of processed samples increases, it becomes important to obtain instant feedback on
measurements to avoid errors. Automation of as much of the data analysis as possible
dramatically reduces the workload. For researches that want to push the boundaries of
automated SAXS data analysis, the source code for the basic tools is often either difficult to
obtain, too specialized for a particular beamline, or developed in widely diverse programming
languages, which means algorithms will have to be reimplemented all over again. BioXTAS
RAW is a free and open source software project that is aimed at developing tools for high
throughput data reduction and analysis, not only intended to be used as an integrated part of
the data collection software on a SAXS beamline, but also by users at home. This Python-
based software, available on SourceForge, is being developed as an alternative to current
closed source and commercial software for isotropic scattering data. BIOXTAS RAW is an
outgrowth of the BioXTAS project, a collaboration between The Technical University of
Denmark and The University of Copenhagen and has been used at the I711 beamline at the
MAX-lab synchrotron in Sweden and other sources worldwide. It is currently being integrated
as the primary data reduction and analysis software on the BioSAXS beamlines G1 and F2 at
CHESS. In addition to software and algorithmic details, this presentation will also discuss the
redesign of CHESS F2 beamline as a dedicated BioSAXS station with robotic sample loading,
including plans for automated microfluidic mixing.
M-140

Structure of the reassembled Venus

Masami Isogai, Yoshihiro Kawamoto, Kazuto Inahata, Kenji Sugimoto, Toshiji Tada

Osaka Prefecture University, Sakai, Osaka, Japan

Bimolecular fluorescence complementation (BiFC) assay based on the association of a

fluorescent protein fragments is widely employed to study protein-protein interactions in cells.
Non-specific interaction is a significant problem, because it interferes with the BiFC assay. To
understand the mechanisms of reconstitution, we have attempted to determine the structure
of the fluorescent protein, Venus, complemented between the N-terminal and C-terminal
fragments. The fragments were co-expressed in E. coli and purified by the affinity and ion-
exchange chromatographic techniques. The fluorescent fraction was crystallized by the
sitting-drop vapor diffusion method. The diffraction data were collected to 2.5 Å resolution at
100 K using an ADSC CCD detector at the NW12 station of PF, Japan. The crystal belongs to
P212121, with unit-cell parameters of a = 58.97, b = 116.02, c = 156.62 Å. Assuming three
molecules of the reassembled Venus in the asymmetric unit, the VM value was calculated to
3 -1
be 2.9 Å Da . The crystal structure was solved by molecular replacement using the program
MOLREP with the structure of Venus (PDB: 1MYW) as a search model. Completion of model
building and structure refinement are underway.
M-142

The wwPDB Common Annotation and Deposition Tool Development

1 1 1 1 2
John Westbrook , Martha Quesada , Jasmine Young , Zukang Feng , Tom Oldfield , Sameer
2 2 3 4 4
Velankar , Jawahar Swaminathan , Takanori Matsuura , Eldon Ulrich , Steve Madding ,
2 3 4 1
Gerard Kleywegt , Haruki Nakamura , John Markley , Helen Berman
1 2
RCSB PDB, Rutgers Chemistry, Piscataway, NJ, United States, PDBe, European
3
Bioinformatics Institute, Hinxton, United Kingdom, PDBj, Osaka University, Osaka, Japan,
4
BMRB, University of Wisconsin-Madison, Madison, WI, United States

The Worldwide Protein Data Bank (wwPDB) is committed to using the highest standards of curation
and processing for experimentally-determined 3D biomolecular structure data. The wwPDB Common
Deposition and Annotation Tool project was initiated to produce a set of common deposition and
annotation processes and tools across the wwPDB that will serve to support the goals of quality and
dependability over the next 10 years. The new tools make use of interactive interfaces and best of
breed visualization tools to ensure the quality of data curation and communication with the originating
scientists. To date, the project has delivered a sequence processing workflow, supported by a
graphical user interface and managed by a workflow engine. The annotation processes are described in
XML and are accessed by the workflow engine through a Python API. The resulting architectural
foundation will be used to complete the full deposition and annotation workflow. The new processes
were defined using business process re-engineering methodologies resulting in process-driven
requirements and the incremental design and delivery of products. The wwPDB Common Deposition
and Annotation suite of tools are built on Python, the Boost.Python Library and C++, supporting work
automation, as well as computationally intensive tasks.

wwPDB members are: RCSB PDB (supported by NSF, NIGMS, DOE, NLM, NCI, NINDS and
NIDDK), PDBe (Wellcome Trust, EU, BBSRC, NIH and EMBL), PDBj (BIRD-JST) and BMRB
(NLM).
M-145

Rapid Automated Processing of Data (RAPD) Software Package

Jonathan Schuermann, David Neau, Kanagalaghatta Rajashankar, Frank Murphy

Cornell University (NE-CAT), Argonne, IL, United States

RAPD (Rapid Automated Processing of Data) is a software package aimed to help users in
collecting and processing meaningful crystallographic data from a synchrotron beamline. The
package is coded in Python as independent but interacting modules which are designed to
run with minimal user input. RAPD monitors the beamline for collection of a snapshot (or
pair), automatically autoindexes the collected images and generates an optimal data
collection strategy. RAPD runs Labelit for autoindexing, Mosflm for integration, Raddose for
radiation damage calculation, and Best for data collection strategies. Error correction is
incorporated automatically so programs are rerun adjusting certain parameters if errors are
detected. RAPD takes advantage of multi-core CPU's by parallelizing certain tasks. Typical
time from data collection to appearance of the strategy in the user interface is 25 seconds on
an Intel Core i7. Collection of a sweep of data triggers automated processing using Xia2. All
results are accessible on a secure AJAX-based website which displays results in variable
levels of detail. Users may log in remotely and view, download or reprocess data from current
or historic data collection trips.
Current and future work involves several new features. First, implementing STAC to take full
advantage of the MK3 mini-kappa, allowing users to continue data collection across different
crystals. Second, near real-time processing of data from multiple sweeps. Third, modify the
code to take further advantage of a computer cluster. Fourth, automated structure solution
pipeline.
M-148

Automated (and non-automated) data processing with autoPROC

Clemens Vonrhein, Claus Flensburg, Peter Keller, Wlodzimierz Paciorek, Andrew Sharff,
Thomas Womack, Gerard Bricogne

Global Phasing Ltd., Cambridge, United Kingdom

The processing of macromolecular diffraction data can often be quite challenging, especially
to novice users. Extracting the best possible data from a large collection of different datasets,
such as occur in binding studies or in complex protocols for MAD phasing (involving multi-axis
goniometers, multiple and possibly interleaved wavelengths, inverse-beam strategies and
combinations thereof), requires making systematic and thorough use of all available
information. This includes prior knowledge about the sample (likely crystal forms, known or
expected behaviour of crystals in the X-ray beam) as well as the relationships (e.g.
goniometric) between the various datasets that should eventually contribute to making up the
scaled (and optionally merged) dataset from which to carry out phasing or refinement.

The autoPROC toolbox helps the unexperienced and the expert user alike to deal with a large
variety of data collection scenarios, both by providing a collection of advanced tools for fine-
tuning the processing steps and by producing easily understood feedback and diagnostics. It
is centred around the processing programs XDS [1] and MOSFLM [2], and uses
POINTLESS/SCALA [3] and other programs from the CCP4 [4] suite for the later stages. It
has been in constant use and development for nearly 5 years and a public release is planned
within 2010.

[1] Kabsch, W. J. (1993). Appl. Cryst. 26, 795-800.

[2] Leslie, A.G.W. (1992). Joint CCP4 + ESF-EAMCB Newsletter on Protein Crystallography,
No. 26.

[3] Evans, P. R. (2005). Acta Cryst. D62, 72-82.

[4] Collaborative Computational Project, Number 4 (1994). Acta Cryst. D50, 760-763
M-151

Automated in situ Diffraction Screening at Beamline X06DA at the Swiss Light Source

Vincent Olieric, Rouven Bingel-Erlenmeyer, Meitian Wang, Roman Schneider, Claude

Pradervand, Wayne Glettig, Takashi Tomizaki, Ezequiel Panepucci, Vincent Thominet, Joerg
Schneider, Jose Gabadinho, Xiaoqiang Wang, Andreas Isenegger, Clemens Schulze-Briese

Paul Scherrer Institut, Villigen PSI, Switzerland

X06DA is the third macromolecular crystallography beamline at the Swiss Light Source. It has
been designed to fulfill the requirements of both academic and industrial users. To achieve
maximum efficiency, high degree of automation was implemented from the optics to the
experimental environment. A Bartels dual channel cut monochromator (DCCM) ensures rapid
energy changes with a true fixed exit. The obtained X-ray beam has a focal spot size of 80 x
45 microns at the sample position and a total photon flux of 5E11 photons/sec, which is
comparable to an undulator beamline. The mini-hutch end-station allows both rapid manual
mounting and robotic sample exchange.

In addition, a crystallization facility, directly adjacent to the X06DA mini-hutch, has been
implemented. Crystallization experiments are performed using nano-dispensing robots and
drops inspection is done via an automated imaging system. The unique feature of this facility
is the possibility to test the crystals for diffraction directly in the plates (in situ screening) by
transfering them from the crystal hotel to the mini-hutch in an automated manner. Without any
manipulation to the crystals, this gives users a rapid feedback on important parameters such
as diffraction limit, anisotropy, cell parameters or mosaicity, and aids to prioritize subsequent
optimization steps. Moreover, users are welcome to bring any kind of SBS standard
TM
crystallization containers, including microfluidic chips and the CrystalHarp which yield a
particularly low background in the diffraction image.

First results obtained at the crystallization facility and future improvements will be presented.
Other methodological developments such as a new type of multi-axis goniometer and phasing
with weak anomalous scatterers will be described as well.
M-154

Minstrel™ HT UV: A Fully Automated and High Performance Imaging System for
Protein Crystallization with UV Fluorescence

Jian Xu, Michael Willis

Rigaku Automation, 5999 Avenida Encinas, Suite 150, Carlsbad, CA, United States

Rigaku introduces the Minstrel HT UV™, custom engineered to meet the increased demand
for a high-throughput ultraviolet and visible crystal imaging and protein crystal monitoring
system. We have focused our research and development efforts and the combined
knowledge of experts in optics, photochemistry, illumination, and automation to develop a
custom solution that provides the highest sensitivity, the highest optical resolution, and the
least photo damage to a protein sample. The result of this effort provides a substantial leap
forward in imaging technology in both UV and visible color spectrum. The optimal balance of
high resolution and depth of field for the crystallographic application seamlessly images
hanging drop, sitting drop, and microbatch experiments for all UV suitable plates. The Minstrel
HT UV together with Gallery™ 700 Incubators provide around the clock unattended
incubation with user specified temperature, retrieval, temperature controlled imaging, and
analysis. Developed by crystallographers for high throughput labs, Rigaku’s automated high-
throughput incubation and imaging solution delivers exceptional reliability with versatility and
scalability into the future. In addition, the new Minstrel HT UV software provides scientists
with an easy to use and intuitive flow. Validation studies with various proteins prove that the
Minstrel HT UV provides researchers with a significant advantage in the protein crystallization
process.
M-157

BLUICE-EPICS: a modern control system with classical look for macromolecular

crystallography at the Advanced Photon Source
1 1 1 1 1
Sergey Stepanov , Oleg Makarov , Mark Hilgart , Sudhir Pothineni , Derek Yoder , Michael
1 1 1 1 2
Becker , Craig Ogata , Ruslan Sanishvili , Nagarajan Venugopalan , Janet Smith , Robert
1
Fischetti
1 2
Argonne National Laboratory, Argonne, IL 60439, United States, University of Michigan, Ann
Arbor, MI 48109, United States

The trio of macromolecular crystallography beamlines constructed by General Medicine and

Cancer (GM/CA) Institutes at the APS has been in growing demand due to their outstanding
micro crystallography capabilities. To raise the efficiency of these beamlines a significant
effort has been put into designing fast, convenient, intuitive and robust beamline controls that
could easy accommodate new beamline developments and provide high level of automation.
This resulted in a system combining the widely praised user interface of SSRL BLUICE as a
frontend and the industrial power of EPICS as a backend. While the GM/CA controls have
the look and feel of BLUICE, their software design is very different making them faster,
simpler and more flexible than most similar systems. First, the software consists of only two
layers: the BLUICE clients and the EPICS servers. The BLUICE clients are designed to
operate in parallel with other beamline controls streamlining the tasks performed by staff such
as beamline preparation, maintenance, auditing and user assistance. Then, BLUICE-EPICS
deploys multiple plugins that can be written in any programming language thus involving more
staff into the development. Further on, it makes use of unified motion controls allowing for on-
the-fly scanning and optimization of any beamline component. Finally, even the graphical
frontend is made language-flexible so that the old Tcl/Tk controls inherited from BLUICE are
being gradually replaced by more modern Java controls and the two seamlessly coexist.
From the users’ perspective BLUICE-EPICS provides: one-click change between 5, 10 and
20μ m beam sizes; one-click beamline energy change that may involve switching undulator
harmonics, mirrors lanes and beam realignment; automated diffraction rastering for finding
small crystals and ’sweet’ spots on poorly diffracting crystals with automated scoring of raster
cells by the number of reflections; data collection along a vector; automated on-the-fly
fluorescent rastering, a faster and lower-irradiation compliment to the diffraction rastering;
fully automated fluorescence measurements for MAD that include signal optimization, fast on-
the-fly energy scanning and automated adaption of scan range to chemical shifts; fly-scan
minibeam realignment; automated loop and crystal centering, controls for sample
automounter, automated screening, data collection auditing, remote access and a lot more.
M-160

Exploring the path towards autonomous protein crystal harvesting: First experiences
with an operator-assisting crystal harvesting robot.
1,3 1 1 2 1
Bernhard Rupp , Jace Walsh , Alex Melka , Sean Murphy , Robert Viola
1 2
Square One Systems Design, Jackson, WY 83002, United States, Johns Hopkins University
3
Applied Physics Lab, Laurel, MD 20723, United States, q.e.d. life science discoveries,
Livermore, CA 94551, United States

Robotic crystal harvesting has become reality with the installation of the first demonstration
unit in mid-2010. The Universal Micromanipulation Robot (UMR) has advanced from an
operator-assisted prototype to a complete operator-assisting crystal harvesting work cell. We
discuss the challenges that had to be overcome and which still lie ahead for the design of a
platform-integrated system capable of fully autonomous harvesting of protein microcrystals.

During the transfer of manual harvesting protocols into robotic processes, a number of novel
and generally applicable technologies have evolved. Tape cutting and re-sealing has been
simplified and improved, and a universal and reliable cryo-protection procedure has been
developed. Combining robotic low-viscosity oil cryo-protection with reliable hyper-quenching
allows standardization of cryo-protocols. In addition, optional room-temperature diffraction
characterization the same crystal prior to flash-cooling will allow consistent analysis and
comparison of cryo-protocols, providing information about native versus flash-cooled
diffraction properties.

Algorithmic crystal localization and UMR control remain two significant hurdles in deploying a
fully automated solution. While significant advancements have been made in the application
of machine vision to protein crystal harvesting, real time image processing interfaced with
mechanical control and feedback are at the cutting edge of technology, and the design of
autonomous systems represents a research frontier in mechatronics. Advantages in overall
harvesting success rate of a robotic platform will very likely lead to market acceptance of fully
automated and integrated crystallization platforms. In addition to general throughput and
reliability advantages, we believe that advanced micro-manipulation robotics will open the
field to further new science and emerging crystallization technologies of potentially far
reaching impact.
Work sponsored by NIH STTR Phase II Grant No. 2 R42 GM073278-02A1.
M-164

Remote access data collection at SPring-8 protein crystallography beamlines

1 2 2 1 2
Kazuya Hasegawa , Go Ueno , Takaaki Hikima , Yukito Furukawa , Daisuke Maeda ,
1 2
Takashi Kumasaka , Masaki Yamamoto
1 2
SPring-8/JASRI, 1-1-1, Kouto, Sayo, Sayo, Hyogo, Japan, RIKEN SPring-8 Center, 1-1-1,
Kouto, Sayo, Sayo, Hyogo, Japan

High intensity X-ray beams available at synchrotron beamlines are now indispensable for
protein crystallography, because they enable not only rapid data collection but also data
collection using micro size crystals. However, one inconvenience of using synchrotron
beamlines is a time consuming and expensive trip to distant facilities. Remote access
beamline control and data collection via the internet are solutions for this issue. At SPring-8,
we have developed the remote access system and have started user operation at protein
crystallography (PX) beamlines.

Our remote access system makes use of beamline automation system consisting of
[1] [2]
integrated beamline control software BSS and sample auto-changers SPACE . For the
secure and stable operation from the distant places, three network sessions are provided,
which correspond to a device-control command path, a streaming video image of samples,
and a data management link, respectively. The first two sessions have been developed as a
common platform of SPring-8 network, which can be used at all beamlines in the future. For
data and sample management for PX users, the third session has been provided based on
[3]
database server D-Cha , which has been used originally for mail-in data collection at PX
beamlines.

The test use of the new system has started and is now open for users at RIKEN beamlines
BL26B1 and BL26B2. Further implementation of this system at public beamlines, where
anybody can apply a proposal, is also planned. The remote access data collection at SPring-8
will enhance the usability of beamlines and expand the opportunity of applications.

[1] Ueno G. et al. (2005) J. Synchrotron Rad., 12, 380-384

[2] Ueno G. et al. (2004) J. Appl. Cryst., 37, 867-873

[3] Okazaki N. et al. (2008) J. Synchrotron Rad., 15, 288-291

M-169

Automated crystal centering implementation at GM/CA beamlines at the Advanced

Photon Source using XREC
1 2 1 1
Sudhir Babu Pothineni , David Watts , Sergey Stepanov , Mark Hilgart , Nagarajan
1 1 1 3 2
Venugopalan , Ruslan Sanishvili , Craig Ogata , Janet Smith , Victor Lamzin , Robert
1
Fischetti
1 2
Argonne National Laboratory, Argonne IL 60439, United States, European Molecular
3
Biology Laboratory, Hamburg 22603, Germany, University of Michigan, Ann Arbor MI 48109,
United States

Automated crystal centering software XREC, developed at EMBL, Hamburg [S. Pothineni,
et.al. Acta Cryst. D62, 1358 (2006)] has been made operating within BluIce-EPICS, the
General Medicine / Cancer Institutes (GM/CA) beamlines control system. XREC processes a
series of optical images recorded while a crystal is rotated on the goniometer, determines the
crystal center and provides an estimate of solution reliability. Automated crystal centering is
the main bottleneck of unattended screening. An additional challenge at the GM/CA
beamlines is the small sizes of crystals and X-ray beams putting more stringent requirements
for crystal centering. Automated 'loop' centering is regularly used at GM/CA beamlines while
the automated 'crystal' centering is still under evaluation for crystals upto 20 microns. At the
GM /CA beamlines the automated loop/crystal centering is a two step process. In the first step
automated loop centering is based on the low resolution camera images, which puts the loop
into the field of view of on-axis high resolution camera. In the second step the loop/crystal
centering is based on high resolution camera. The process normally takes up to 30 sec.
Occasionally the initial sample position may not be in the field of view of low resolution
camera and in these cases the first step is automatically repeated after analyzing the XREC
centering parameters and automatically moving the sample into field of view of low-res
camera. The images used for loop centering are stored in a database for future comparison of
crystal centering success rate against the crystal sizes.

The condition for automated crystal centering is that the crystal must be visible for human
eye. When this is not the case, the GM/CA control system offers automated X-ray diffraction
and/or fluorescence rastering for centering tiny or otherwise invisible crystals. This process is
planned to be optimized by defining a loop bounding box (region of interest) using the XREC
algorithms. The GM/CA software also provides an option to collect the X-ray diffraction data
from predefined, multiple segments rather than at a single point on the crystal. This is also
planned to be improved by stretching the XREC algorithms to define the crystal as a whole
(size and shape). The proof of principle of these applications will be presented.
M-180

Experimental Charge Density Study of 7-(4-trifluoromethyl)coumarin propargyl ether, a

Potent P450 Inhibitor
1 1 2
Cheryl Stevens , Naijue Zhu , Edwin Stevens
1 2
Xavier University of Louisiana, New Orleans, Louisiana, United States, University of New
Orleans, New Orleans, Louisiana, United States

7-(4-trifluoromethyl)coumarin propargyl ether was synthesized as a potential therapeutic for

treating nicotine addiction by inhibiting P450 metabolism. A total of 127,735 reflections were
measured on a Bruker Kappa APEX II CCD X-ray diffractometer at 150(2) K to a 2θ max of
116.3°. Integration of these intensities resulted in 11, 800 unique reflections with Rint = 0.0267.
Unit cell dimensions were determined to be a = 5.01500(9) Å, b = 10.2382(2) Å, c =
11.9391(2) Å, α = 114.8696(3)°, β = 93.4358(4)°, and γ = 94.0350(4)° for space group P1bar.
A conventional refinement of 19 non-hydrogen and 7 hydrogen atoms resulted in R = 0.0466.
A fit of the data to a radial model gave an R = 0.0449 while a full multipole refinement gave R
= 0.0291. Deformation density maps of the propargyl ether group shows a buildup of density
in the triple bond. Electrostatic potential maps have been calculated and show negative
potential associated with regions of reactivity near the trifluoromethyl substituent and the
ketone group. Critical points show the presence of C-H...F and C-H...O hydrogen bonds.
NIH/MBRS-SCORE (SC1GM084722) support is gratefully acknowledged.
M-183

The crystal structure of the C-terminal domain of Gcn2 reveals a novel 3-D domain
swapped dimer

Hongzhen He, Lakshmi Palam, Ronald Wek, Millie Georgiadis

Department of Biochemistry and Molecular Biology, Indiana University School of Medicine,

Indianapolis,Indiana, United States

In response to amino acid starvation, the protein kinase Gcn2 phosphorylates eukaryotic
initiation factor 2 (eIF2). Phosphorylation of eIF2 then prevents the exchange of GTP for GDP
on eIF2B thereby blocking initiation of protein translation. Gcn2 is itself regulated in part by its
C-terminal domain, which facilitates interaction of Gcn2 with the ribosome and contributes to
the activated and inhibitory conformations of Gcn2. The C-terminal domain is also involved in
dimerization of Gcn2, which has been shown to be important for mediating its response to
metabolic stress. Although the kinase domains are conserved in other eIF2 kinases, the C-
terminal domain is found only in Gcn2. At the sequence level, the C-terminal domain of Gcn2
has no obvious homologues in other proteins. Limited proteolysis and mass spectrometric
methods were used to identify domain boundaries within the C-terminal part of Gcn2.
Following expression and purification in E. coli, diffraction quality crystals were then obtained
of this domain. The crystal structure was determined at 1.9 Å by using single wavelength
anomalous dispersion (SAD) phasing from a mercury derivative. The structure determination
was facilitated by the use of HKL3000 at the GM/CA 23ID beam line at the APS. A 3-D search
for related structures reveals no significant structural matches with our structure of the C-
terminal domain of Gcn2 suggesting that it is a novel structure. An interesting feature of the
structure is the extensive 3-D domain swapping within the dimer. Dimerization is mediated by
formation of a beta sheet involving the polypeptide chains of each subunit (the 3-D swap) and
associated hydrophobic interactions as well as a central interaction between the two subunits.
In addition, a potential RNA/ribosome binding site has been identified based on our structural
analysis. Delineating these structural features of Gcn2 will provide insight into the underlying
mechanisms activating this eIF2 kinase in response to amino acid starvation.
M-189

The Crystal Structure of the Secreted Chlamydial Protein Pgp3

Ahmad Galaleldeen, Alexander Taylor, Ding Chen, Guangming Zhong, P. John Hart

UTHSCSA, San Antonio, United States

Chlamydiae are gram negative intracellular pathogens that are known to cause various health
problems in humans. Little is known about the molecular mechanism(s) through which
Chlamydiae interact with the host cells and manipulate their cell signaling and immune
response because they replicate, differentiate, and perform all biosynthetic processes within
host cytoplasmic vacuoles, and this sequestration makes them very difficult to manipulate. It
is generally accepted that pathogenesis is related to inflammatory responses caused by
chlamydial infection. Several chlamydial species carry a 7.5 Kb cryptic plasmid that encodes
eight putative open reading frames (pORFs). pORF5 encodes Pgp3, a 27 KDa protein that is
first detected in the cytosol of Chlamydia-infected cells 12 hours post-infection. Pgp3 interacts
with host cell signaling pathways, including the activation of host pro-inflammatory genes.
Here we present the crystal structure of Pgp3, which reveals a trimeric protein comprised of 2
globular domains connected by a coiled-coil triple helix. The C-terminal domain is an all β
structure with a jelly-roll fold that resembles members of the tumor necrosis factor (TNF)
family of cytokines, a fold rarely observed in bacteria, while the N-terminal domain has a
novel fold.
M-192

Diphthamide Biosynthesis Requires a Novel SAM-dependent [4Fe-4S]-containing

Enzyme.
1 1 1 2 1
Andrew T. Torelli , Yang Zhang , Xuling Zhu , Michael Lee , Boris Dzikovski , Rachel M.
1 1 1 2,3 1 1
Koralewski , Eileen Wang , Jack Freed , Carsten Krebs , Hening Lin , Steven E. Ealick
1
Department of Chemistry & Chemical Biology, Cornell University, Ithaca, NY, United States,
2
Department of Biochemistry & Molecular Biology, The Pennsylvania State University,
3
University Park, PA, United States, Department of Chemistry, The Pennsylvania State
University, University Park, PA, United States

Diphthamide is a unique, posttranslationally modified histidine residue that is absolutely

conserved in archaebacterial and eukaryotic translation elongation factor 2 (EF2). This
modification is the target of irreversible ADP-ribosylation by lethal eubacterial factors, such as
diphtheria toxin, exotoxin A and cholix toxin, which interferes with the ability of EF2 to
catalyze tRNA translocation during peptide synthesis. Diphthamide represents one of the
most complex posttranslational modifications and its biosynthesis occurs in three steps. The
first step requires four gene products in eukaryotes and results in formation of a C-C bond
between the poorly nucleophilic C-2 atom of the target histidine and the 3-amino-3-
carboxypropyl group of S-adenosylmethionine (SAM). Here we present the crystal structure
of Dph2, the sole protein required for this reaction in the archaeon Pyrococcus horikoshii.
Each monomer of the PhDph2 homodimer binds a [4Fe-4S] cluster using an unusual
arrangement of three cysteines that are each contributed by structurally-conserved domains
related by pseudo-three-fold symmetry. Biochemical evidence suggests the [4Fe-4S] cluster
is used to homolytically cleave SAM to generate a 3-amino-3-carboxypropyl radical capable of
modifying the target histidine in the first step of diphthamide biosynthesis. This is distinct from
enzymes in the radical SAM superfamily, which use [4Fe-4S] clusters to cleave SAM
molecules and produce 5`-deoxyadenosyl radicals. Thus, our findings suggest PhDph2 is a
novel SAM-dependent [4Fe-4S]-containing enzyme that catalyzes unprecedented chemistry.
M-195

Ultrasensitive protein crystal detection by second harmonic generation microscopy

Garth Simpson, David Kissick, Ellen Gualtieri, Vadim Cherezov

1 2
Purdue University, West Lafayette IN, United States, Scripps Research Institute, La Jolla
CA, United States

Second order non-linear optical imaging of chiral crystals (SONICC) is demonstrated as a

sensitive and selective detection method for protein crystallization, with detection limits for the
onset of crystallization corresponding to crystal dimensions well below the optical diffraction-
limit. Validation of SONICC was performed in lipidic cubic phase crystallization trials of G-
protein coupled receptors through direct comparison with conventional image analysis
methods currently used in high-throughput screening (e.g., bright-field, birefringence, uv-
excited fluorescence, and trace fluorescent labeling). In cases scored highly for crystallization
hits by manual expert image inspection of lipidic mesophase trials, a simple automated
algorithm for analysis of the SONICC images typically also yielded positive hits. However,
SONICC also identified almost twice as many possible hits than manual inspection of images
obtained lipidic mesophoase trials using conventional approaches, suggesting a false
negative rate as high as ~40% that is overcome by nonlinear optical imaging.


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M-201

Characterization of novel LCP matrices for membrane protein crystallization by high-

throughput SAXS
1 1 2 2 1
Thomas Weiss , Ping Liu , Wei Liu , Vadim Cherezov , Hiro Tsuruta
1 2
Stanford University / SSRL, Menlo Park, CA, United States, Scripps Research Institute, La
Jolla, CA, United States

Membrane proteins are essential components of living cells. They perform a variety of
functions including transport of ions and nutrients, energy transformations and transduction of
signals across the cell membrane. Involvement of membrane proteins in many crucial cellular
and physiological processes and their location at the cell surface makes them important
pharmaceutical drug targets. High-resolution structural studies of membrane proteins depend
on the availability of crystals diffracting to sufficiently high resolution. Crystallization in lipid
mesophases (in meso), such as the lipid cubic phase (LCP), has proven to yield high quality
crystals for an increasing number of membrane proteins. Broader application of this in meso
crystallization approach requires identification of novel lipidic matrices with specific phase
properties capable of stabilizing proteins with a wide range of sizes and architectures. We
have developed an integrated method to assess the lipid phase behaviour at conditions
closely mimicking crystallization trials in a high-throughput manner. In this method the lipid
samples are prepared using an in meso crystallization robot in specially designed 96-well
LCP sandwich plates, in which 50nL droplets of lipidic mesophase are overlaid with 1uL of
screening solution and held between two x-ray transparent synthetic films with a thin spacer
to form a 12x8 grid. After incubation at 20 ˚C the position of the lipid sample within each well
on the LCP plate is recorded into a file using a crystal screening imager. The LCP plate is
then transferred to a temperature controlled holder for the x-ray scattering measurements on
the BioSAXS beamline BL4-2 at SSRL. A specifically developed software interface
implemented as part of the data collection software Blu-Ice at the beam line reads in the
sample position file, aligns each LCP sample in the beam and automatically collects SAXS
data. The obtained two-dimensional scattering data is then fed into a newly developed
software pipeline that automatically integrates the data radially, determines the peak positions
and identifies the underlying lipid phase and its basic structural parameters. This high-
throughput approach allows us to study effects of detergents, additive lipids, proteins as well
as great variety of precipitants on the lipid mesophases in order to understand the phase and
structural behaviour of novel lipidic matrices and their compatibility with in meso crystallization
trials.
M-204

Crystal Structure of Bacillus Subtilis Histidine Kinase KinD Sensor Domain in Complex
with Pyruvate

Ruiying Wu, MinYi Gu, Andrzej Joachimiak, Marianne Schiffer

Argonne National lab, Argonne, IL, United States

Sporulation in most Gram-positive bacilli is an example of adaptation of bacteria to starvation.

In Bacillus subtilis, pyruvate is secreted into the extracellular medium; the external
concentration of pyruvate could be acting as an important signal for the initiation of sporulation.
In B. subtilis, the sporulation initiation is tightly controlled by the sporulation transcription factor,
Spo0A through its phosphorylation by kinases. Here we provide the first crystal structure of
kin-kinase family: KinD sensor domain complexed with pyruvate. The diffraction data for KinD
(3fos) without the pyruvate was reprocessed and the electron density was improved. KinD
sensor consists of two domains PAS-like first domain and a second domain that also has the
characteristic beta-sheet of the PAS-like domains. Pyruvate was found bound to the first
domain mainly through interactions with a pair of arginine residues (Arg95 and 116). Glycerol
molecules, previously assigned as waters were also identified on the solvent accessible
surfaces. The structural details, domains role and possible mechanism underlying signal
reception will be discussed.
This work was supported by National Institutes of Health Grant GM074942 and by the U.S.
Department of Energy, Office of Biological and Environmental Research, under contract DE-
AC02-06CH11357.
M-207

Proposed amino acid sequence and crystal structure determination of a chitinase at

1.49 Å from tamarind (Tamarindus indica) seeds

Pravindra Kumar, Deepak Patil, Manali Datta, Shaily Tomar

Indian Inst. of Technology, Roorkee, India

Chitinases are hydrolases involved in plant defense against a variety of pathogens. A protein
with chitinase (CHT) activity has been isolated and purified from tamarind (Tamarindus indica)
seeds. N-terminal amino-acid sequence analysis of this protein confirmed it to be an
endochitinase, which belongs to the acidic class III chitinase family. The protein was
crystallized by the vapour-diffusion method using Polyethylene glycol (PEG). The crystal data
was collected at home source (Bruker MicrostarH rotating anode and Mar345 dtb detector) at
1.49 Å resolution and structure was determined by molecular replacement method. Based on
high resolution electron density map, all amino acids were identified and the complete primary
structure of the CHT is proposed. The final refined model, consisting of 270 amino acid
residues, 320 water molecules, and a single glycosylation site containing one N-
acetylglucosamine unit, has a crystallographic R-factor of 17.8% and a free R-factor of 19.2%
.
M-210

Using Single Crystal X-ray Diffraction to Determine Equations of State: an Example of

Ordered and Disordered Plagioclase Feldspars.
1 1 2 3 3
Lindsay Sochalski-Kolbus , Ross Angel , Fabrizio Nestola , Emiliano Bruno , Piera Benna
1 2 3
Virginia Tech, Blacksburg, United States, University of Padua, Padua, Italy, Univeristy of
Turin, Turin, Italy

Advances in single crystal X-ray diffraction with lab sources allow very small differences in the
equations of state (the pressure variation of volume) of crystalline materials to be determined.
Our work focuses on a group of framework aluminosilicates which show unusual structural
and thermodynamic properties at high pressures, and in which bulk moduli differences of 2%
can be readily distinguished.

The combination of a full 4-circle Eulerian diffractometer with unfiltered MoKα radiation (to
provide a stable platform and stable profiles of rocking curves), point detector, full-profile
fitting of the rocking curves, and 8-position centering was used to collect the cell parameters
for the samples up to maximum pressures above 8.5 GPa. The samples were individually
loaded into an ETH-designed diamond anvil cell with beryllium seats and 408 half-opening
angle. Along with the samples, a quartz crystal and ruby chip were loaded as pressure
calibrants. To ensure hydrostatic pressures, the cell was loaded with a 4:1 methanol/ethanol
mixture. The unit-cell volumes of the samples and quartz crystals were measured at each
pressure increment with errors of 1 part in 10,000. Given that quartz has a low bulk modulus,
the pressure in the cell is determined through its equation of state with a typical precision and
reproducibility of 0.01 GPa or better. This is a significant improvement over the use of ruby as
a pressure marker, which has a typical reproducibility of the order of 0.03-0.05 GPa, in part as
a result of the sensitivity of the frequency of the ruby fluorescence line to temperature
fluctuations.

Our data for the aluminosilicate framework mineral group plagioclase feldspars show unusual
th
compressional behaviour that requires fitting with a 4 order Burch-Murnaghan EoS. The
introduction of Al/Si disorder in the Na end-member reduces the bulk modulus from 52.3(9)
GPa (ordered) to 50.4(5) GPa (disordered). In a sample in which 20% of the Na is substituted
by Ca, the bulk modulus changes from 61.2(5)GPa to 59.7(7)GPa on disordering, and for a
sample with 77%Ca, there is no significant difference in the bulk moduli of ordered and
disordered samples.
M-213

Single-crystal neutron diffraction study of hydrous wadsleyite: hydrogen positions for

H2O incorporation into Earth’ s deep interior
1 2 3 4 5
Steven D. Jacobsen , Joseph R. Smyth , Matthias Gutmann , Daniel J. Frost , Erik Hauri ,
1
Craig R. Bina
1
Department of Earth and Planetary Sciences, Northwestern University, Evanston, IL, United
2
States, Department of Geological Sciences, University of Colorado, Boulder, CO, United
3
States, Rutherford Appleton Laboratory, ISIS Facility, Chilton Didcot, United Kingdom,
4 5
Bayerisches Geoinstitut, University of Bayreuth, Bayreuth, Germany, Department of
Terrestrial Magnetism, Carnegie Institution of Washington, Washington DC, United States

Wadsleyite (9 -Mg2SiO4) is a nominally anhydrous, high-pressure polymorph of olivine and

considered one of the major mantle phases at 410-520 km depth. Wadsleyite
(orthorhombmic, Imma) has a remarkable ability to include H at very high pressures and
temperatures through hydroxyl defects associated with O1, an underbonded oxygen site that
is coordinated to five octahedral-Mg sites and not to Si (e.g. Smyth 1987). At conditions of the
upper mantle, wadsleyite can incorporate up to several weight percent of H2O into its
structure, making it potentially one of the largest H2O reservoirs in the global water cycle. X-
ray crystallographic studies of H in wadsleyite have inferred proton positions from R(O…O)
interatomic distances in conjunction with polarized infrared spectroscopy, however no direct
refinement of hydrogen positions in wadsleyite has been possible because of limited
hydrogen concentrations (2 wt% H2O in wadsleyite represents only about 2 H atoms per unit
cell) and limited crystal sizes from high pressure synthesis above 12 GPa and 1200C. We
report synthesis of a hydrous wadsleyite crystal about one mm in size using the large-volume,
5000 tonne multi-anvil press at Bayerisches Geoinstitut in Bayreuth, Germany. The sample
contains 1.77 wt% H2O, as measured by secondary ion mass spectrometery. The structure
was investigated on the single-crystal diffraction (SXD) beamline of the ISIS pulsed-spallation
neutron source at Rutherford Appleton Laboratory, UK. We integrated over 3000 time-of-flight
reflections from three 24-hour exposures and carried out GSAS and Maximum Entropy
Method refinements of the structure. Two hydrogen positions in the structure have been
refined from the data. Methods and hydrogen-bonding environments in wadsleyite will be
presented in detail.
M-216

Chem_BLAST – a rule-based method to develop advanced structural ontologies for

chemical bioinformatics and the PDB, the PubCheM

T.N. Bhat

NIST, Gaithersburg MD, United States

: Today’s Chemical Bioinformatics community must interact with a variety of information

standalone applications and ontologies. This limitation promotes the need to define and
develop rule-based stringent ontologies for information processing and sharing. Chemical
Block Layered Alignment of Substructure Technique (Chem-BLAST) first recursively
dissects chemical structures into blocks of substructures using rules that operate on
atomic connectivity and then aligns them one against another to develop first Chemical
Resource Description Framework (RDF) and then chemical ontologies in the form of a
‘tree’ made up of ‘hub-and-spoke’. The technique was applied for (a) both 2-D and 3-D
structural data for AIDS (http://bioinfo.nist.gov/SemanticWeb_pr2d/chemblast.do ); (b) to
~60000 structures from the PDB which is now available from the RCSB/PDB Web site
(http://www.rcsb.org/pdb/explore/externalReferences.do?structureId=3GGT) and
advanced features at http://xpdb.nist.gov/chemblast/pdb.html . Full description of the
Chem_BLAST along with recent results and illustrations including those for approximately
a million compounds from the PDB and PubChem will be presented.
M-222

Refined Crystallization Screens

Hao An, Gyorgy Babnigg, Hui Li, Andrzej Joachimiak

Argonne National Laboratory, Argonne, IL, United States

A number of commercially available crystallization formulations and screens have been

evaluated in the Midwest Center for Structural Genomics’ structure determination pipeline.
These formulations were applied, using standard protocols, to thousands of unique proteins
and domain constructs. The initial analysis of our results showed that many crystallization
screens conditions are redundant, and some screens pointed to several less populated
regions of crystallization space. To reduce the number of crystallization conditions and
maximize their effectiveness, new refined crystallization screens (ANL-1 and ANL-2) were
designed. These screens, ANL-1 and ANL-2, complement the Index very well. Although Index
shows a slightly higher success rate (0.47), ANL-1 and 2 screens (both show combined
success rate 0.74) contain a more diverse set of conditions and cover a larger crystallization
space. Both of them are now available commercially.

Recently, we analyzed data for more than 5000 successful crystallization trials stored in our
LIMS and indentified the top 384 most successful crystallization conditions. Four new
crystallization screens were designed (MCSG1-4) and are now available on the MCSG web
site (http://www.mcsg.anl.gov). Recently, our LIMS also added the high throughput crystal
data collection system. Using a rich client platform, the LIMS can display and update the
whole data set from a 96-well screen plate. Users can report all the crystal results on one
screen plate in a quick and easy way. This new on-line report system significantly increases
the efficiency of data entry, which will lead to a faster and more precise selection of the best
crystallization conditions.

This work was supported by NIH Grant GM074942 and by the U.S. DOE, OBER contract DE-
AC02-06CH11357.
M-228

autoBUSTER re-refinement reliably reveals interesting features in newly-released PDB

structures

Thomas Womack, Oliver Smart, Andrew Sharff, Claus Flensburg, Peter Keller, Wlodek
Paciorek, Clemens Vonrhein, Gerard Bricogne

Global Phasing, Cambridge, United Kingdom

The BUSTER-TNT refinement program has long shown consistent capabilities to produce
superior maps from a given model and a given X-ray dataset. Recent improvements have
been made to improve its ability to refine models. In particular autoBUSTER now has a
powerful optimizer, good molecular geometry restraint function, TLS and fully automated NCS
setup. Refinement with autoBUSTER typically leads to lower Rfree, smaller Rfree-Rwork
gaps, and improved Molprobity scores even without the explicit representation of hydrogen
atoms.

On every week of the last three years, Global Phasing has re-refined with autoBUSTER every
PDB structure released that week, and analysed the results are with increasingly automated
tools. Typically, every week there is a structure with a difference-density peak at +14 sigma or
more, and it is rare for there to be fewer than ten structures with highest difference-density
peak above 10 sigma.

autoBUSTER routinely shows extra interpretable density at N- and C-termini, ranging from
omitted OXT atoms to substantial extensions of the chain. About one deposition a week has a
claimed ligand which just isn't there, and several will have a sugar or buffer molecule in a
clearly wrong conformation. A gallery of typical errors will be presented, including ones where
autoBUSTER density shows issues more clearly than EDS.

This work was funded by Phases IV and V of the Global Phasing Consortium, and by the
VIZIER FP6 Project under EC Contract LSHG-CT-2004-511960.
M-231

SrReal - an open-source software library for local structure analysis

1 1 1,2
Pavol Juhas , Christopher Farrow , Simon Billinge
1 2
Columbia University, New York, NY, United States, Brookhaven National Laboratory, Upton,
NY, United States

The atomic pair distribution function (PDF) and other total scattering techniques are essential
tools for probing the local structure of nanomaterials, crystals with local distortions, or
randomly oriented molecules. While there are several free programs available for PDF
modeling (PDFgui/PDFfit2) and local structure determination (RMC suite, Liga algorithm), it is
extremely hard for outside developers to customize them (e.g., use special particle shape
damping) or combine with additional structure criteria. SrReal is an open-source library for
Python and C++ that provides highly customizable calculators for various structure quantities
such as PDF, Ewald sums, bond valence sums or overlap of empirical ionic radii. The SrReal
library has been developed as part of DANSE, the distributed data analysis for neutron
scattering experiments. The library can be used at both Python and C++ levels, where its
Python interface was designed for ease of use and the C++ level for speed. The objects in
the library were designed for maximum code reuse, clarity and flexibility. The PDF calculation
can be easily modified by incorporating custom PDF scaling functions, peak width models or
peak profile functions, at either Python or C++ levels. The library calculators can be used
with arbitrary structure representations in Python or C++ by providing a simple adapter class.
The SrReal calculators share a common recipe of iterating over all atom pairs and summing
their pair-wise contributions. A new calculator, for example of pair potential, could be readily
implemented by reusing the recipe and changing only the pair contribution function. The
SrReal library will be demonstrated with several short python and C++ programs and on
actual PDF simulation problems.
M-234

MAIN 2010: finalizing the structure by validation driven structure improvement

Dusan Turk, Martin Turk

Jozef Stefan Inst., Ljubljana, Slovenia

At the final stages of crystal structure determination conformations of side chains, peptide
bond orientations considering electron density maps as well as hydrogen bonding networks
and electrostatic stability and packing of conformations need to considered before the
structure can be considered final and ready for deposition in PDB.

In MAIN a procedure has been developed for automated improvements and completion of the
structure. The procedure includes side chain and peptide bond density fitting combined with
flipping in a combinatorial manner. At first the current state of the model, termed starting
model, is validated towards density maps. Dead end elimination, exhausted, rotational search
is used to fit atoms into electron density maps followed by the energy minimization. Next side
chains of branched residues ILE, VAL, THR and LEU are flipped and adjusted to density by
fragment and side chain fitting, each followed by minimization. Each state is validated and
compared to the starting model. When local improvement is achieved, the geometry of the
modified part replaces that of the starting model. A similar procedure considering peptide
bond orientation follows. After an optimal fit to density maps is achieved combinatorial search
considering packing of short range (below 4A) electrostatic and vdw interactions as well as
hydrogen bond network is considered. To enable this explicit hydrogens are used. Side chain
and residue flipping is at this stage applied to electrostatically asymmetric residues HIS, ASN,
GLN, and solvent molecules in a combinatorial manner. Each of the states is saved together
with their packing energy. The lowest energy state is at the end of procedure transfered to the
working model, which is agaain energetically minimized - as always using real space
refinement procedure. The structure can then be refined against crystallographic targets and
the cycle repeated unless the structure is considered done.
M-237

Multi-crystal Anomalous Diffraction for Low Resolution Macromolecular Phasing

1 2 2
Qun Liu , Zhen Zhang , Wayne Hendrickson
1 2
New York Structural Biology Center, New York, United States, Columbia University, New
York, United States

Anomalous diffraction experiment from single crystal is one of most commonly used methods
for macromolecular phasing. Here we show an alternative way for anomalous phasing with
anomalous diffraction data extracted from multiple crystals. Our study demonstrates that
multi-crystal anomalous data can significantly benefit low resolution phasing in both heavy
atom substructure determination as well as electron density maps interpretation at low
resolution. We propose that the multi-crystal strategy may help solve crystal structures of
large macromolecular complexes and membrane proteins which are prone to be damaged by
X-ray radiation.
M-240

Structure of nanoparticles: frontiers in PDF modeling

Christopher Farrow, Simon Billinge

1 2
Columbia University, New York, NY, United States, Brookhaven National Laboratory, Upton,
NY, United States

Nanoparticles are poised to become vital in the future of energy, medicine, computing and
countless other fields. Despite their current and potential usefulness, there are no robust
methods for determining the structure of nanoparticles with atomic resolution. Furthermore,
the process of nanoparticle growth is still not well understood. These obstacles stand in the
way of high precision design and fabrication of nanoparticles for industrial applications.

The atomic pair distribution function (PDF) has proved to be a powerful tool for investigating
the structure of nanoparticles. In this talk I will describe established and new methods for
modeling nanoparticles with the PDF. I will discuss recent work where these methods have
been applied to model noncrystalized nanoparticle precursor molecules. Finally, I will give an
outlook for how we plan to combine structural information from various sources with the PDF
in order to model complex strained, segregated and disordered nanoparticles.
M-243

A Structural View on Drug Design for an Acyl-Carrier Protein Synthase from

Staphylococcus aureus, Bacillus anthracis and Vibrio cholerae
1,5 2,5 1,5 1,5
Andrei Halavaty , Youngchang Kim , Ludmilla Shuvalova , George Minasov , Ievgeniia
1,5 1,5 2,5 3,5 4,5
Dubrovska , James Winsor , Min Zhou , Keehwan Kwon , Tatiana Skarina , Olena
4,5 3,5 2,5 3,5
Onopryenko , Leka Papazisi , Andrzej Joachimiak , Scott Peterson , Alexei
4,5 1,5 1,5
Savchenko , Wayne Anderson , Center for Structural Genomics of Infectious Diseases
1
Feinberg School of Medicine, Northwestern University, Chicago, Illinois, United States,
2 3
Computational Institute, University of Chicago, Chicago, Illinois, United States, J. Craig
4
Venter Institute, Rockville, Maryland, United States, University of Toronto, Toronto, Ontrario,
5
Canada, Center for Structural Genomics of Infectious Diseases, Chicago, Illinois, United
States

Viability of multi-drug-resistant strains of pathogenic microorganisms is determined by many

complex and interconnected factors. The bacterial type II fatty acid synthesis (FAS) is a well-
organized and regulated pathway with individual soluble proteins that produce diverse array
of vital intermediates, and hence, it represents a valuable source for drug design. While a
number of enzymes of FAS II have been shown as important candidates for inhibition, a
scientific and medical quest for new targets stimulates research in the field. In order for FAS II
to function without a failure, a central component of the system, acyl carrier protein (ACP),
has to constantly deliver the thioester intermediates to the discrete partners of the pathway.
This cannot occur without an initial posttranslational modification by a holo-(acyl-carrier-
protein) synthase (AcpS), which transfers the 4;-phosphopantetheine prosthetic group from
coenzyme A (CoA) to a conserved serine residue of apo-ACP. In this respect, AcpS is
important for keeping FAS II working, and therefore, finding inhibitors of AcpS activity can
destabilize the system. Bacterial AcpSs function as trimers making them distinct from the host
AcpSs, which can be a pseudo dimer or a single domain in the complex multidomain
eukaryotic FAS I synthases. This substantial architectural difference suggests a strategy for
inhibition. While there are a number of 3D structures of bacterial AcpSs available, the
mechanism of the transferase activity and its inhibition are not entirely understood. The high-
resolution X-ray structures presented for apo-AcpS from Staphylococcus aureus, holo-AcpS
from Bacillus anthracis and 3;, 5;-ADP-bound AcpS from Vibrio cholerae will contribute to
understanding of the structural and mechanistic details of the AcpS-reaction also providing
possible alternative approaches to its inhibition.
M-246

Determination of Vault Symmetry by Strictly-Crystallographic Means

1 1 2 2 1,2
Daniel Anderson , Michael Sawaya , Valerie Kickhoefer , Leonard Rome , David Eisenberg
1 2
HHMI at UCLA, Los Angeles, CA, United States, Dept. of Biol.Chem., UCLA, Los Angeles,
CA, United States

Can the symmetry of crystalline ribonucleoprotein vaults be determined with low resolution
diffraction data, using only crystallographic methods and omitting external information?
Previous crystallographic symmetry tests seemed to support the hypothesis that the vault
shell contained 96 copies of major vault protein (48 NCS-rotated copies of 98kDa in the
asymmetric unit). This report contains results and observations from Patterson-based and
density modification-based analyses, expanded from the previous analyses, and using
diffraction data from two crystal forms. This crystallographic re-examination suggests that the
vault shell can form with at least two symmetries, and corrects our previous symmetry
assessment.

48-fold (2QZV): Anderson, et al (2007) PLoS Biology 5, 2661-2670.

39-fold (2ZUO, 2ZV4, 2ZV5): Kato, et al (2008) Acta Crystallographica D64, 525-531;
Tanaka, et al (2009) Science 323, 384-388.
M-251

Cystal Structure of PCSK9 with Different Length Variants of the EGF-A Domain of LDL
Receptor.

1 1 1 1 1 1
Javed Khan , Joseph Myers , Gerald Duke , Steven Sheriff , Mark Witmer , Yaqun Zhang ,
1 1 1 2 2
Claudio Mapelli , Brian Carpenter , Mian Gao , Doree Sitkoff , Michael Lawrence , Rex
2
Parker
1 2
Bristol-Myers Squibb, Princeton, NJ, United States, Bristol-Myers Squibb, Hopewell, NJ,
United States

Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates extracellular levels of low-

density lipoprotein receptor (LDLR) by binding to the epidermal growth factor precursor
homology domain A (EGF-A) of LDLR. This leads to degradation of cell surface LDLR in
hepatocytes and elevation of plasma LDL cholesterol levels. To determine the residues of
PCSK9 that interact with EGF-A, we have determined co-crystal structures of PCSK9 in
complex with 2 different length variants of the EGF-A domain, a longer 40-mer and a shorter
27-mer form of the EGF-A domain peptide. These co-crystal structures reveal the binding
mode and key interacting residues of PCSK9 and the EGF-A peptide, and can be used
towards the discovery and optimization of therapeutic agents to disrupt the PCSK9-LDLR
interaction.
M-254

TITLE MISSING

Daniel Camac, Joseph Myers, Javed Khan, Yaqun Zhang, Vandana Hegde, Mian Gao,
Yongmi An, James Tamura, Malcolm Davis, John Somerville, David Weinstein, John Sack

Bristol-Myers Squibb, Princeton, NJ, United States

ABSTRACT MISSING - abstract was not processed because the template was not used or
was altered
M-257

Lithium Ion Pathways in Lithium Phthalocyanine Electrolytes

1 1 1 2
David Grossie , William Feld , John Kelley , Lawrence Scanlon
1 2
Wright State University, Dayton, OH, United States, AFRL, PRPS, WPAFB, Dayton, OH,
United States
-
The lithium phthalocyanine anion (LiPC ) has been found to show promise as part of the
dielectric for lithium ion batteries. The structures of three structures have been determined in
which the LiPC anion form similar channeled networks through the unit cell. All three
compounds form small, flakey crystals from the few solvents in which they can be dissolved.
Experimental evidence indicates that the lithium atoms within the solid structure migrate in the
presence of an electric field. The determination of the crystal and molecular structures were
initiated so as to determine the pathways through which the lithium ions move.
-

N Y+

N N
N Li+ N
N N

Where Y is Li(H2O)2(acetone)2 or bis-adamantylimidizolium cations

M-260

A new mini-beam device for protein crystallography

Randy Alkire, Michael Molitsky, Frank Rotella

Argonne National Laboratory, Argonne, IL, United States

In recent years there has been a steady increase in the use of smaller diameter beams
(<30<m) for protein crystallography. This has been brought forth mainly thru the use of beam
limiting collimators, like the one developed by GMCA CAT at the Advanced Photon Source
and by the introduction of mini-beam collimators in commercial instruments like the Bruker-
ASC MD2 diffractometer. These instruments have similar designs in that their small beams
result from narrowly defined apertures inside long tubes with guard apertures. Alignment of
these tubes requires several degrees of freedom, including yaw, pitch and vertical and
horizontal translations. Unfortunately for the end user of these devices, yaw and pitch are
manually controlled, making initial installation a very time consuming process.

At the Structural Biology Center we have developed a new mini-beam apparatus that
separates the beam defining aperture from the guard aperture, allowing independent
positioning of each pinhole with simple translation stages, eliminating the need for the time
consuming yaw or pitch adjustment. Modular design of the encased pinholes allows for quick
change-out if different size apertures are required. This design currently has room for two
sets of pinhole/guard pairs. Because these motions are fully motorized, movement from one
pair to the other after setup requires only a single command. If desired, any pinhole/guard
pair can be lowered completely out of the beam path. If a change in experimental setup
requires the mini-beam hardware to be removed, this can be accomplished in less than 5
minutes, with a similar time required for re-installation. A description of the helium filled,
motorized assembly will be presented, along with diagrams of the pinhole assemblies.
M-263

Feasibility Study of Nano-scale Wide-Angle Incidence X-Ray Waveguides Using

Surface Diffraction

Hsin-Yi Chen, Jhih-Ren Huang, Li-Sin Cai, Chia-Cheng Lin, Yan-Zong Zheng, Yung-Shih
Fang, Shih-Lin Chang

Department of Physics, National Tsing Hua University, Hsinchu, Taiwan

A wide-angle incident waveguide with nano-scale structures is designed using crystal

asymmetric surface diffraction. The waveguides are composed of Au/Si/Au sandwiches with
Si stripes as the guiding layers and Au thin films as the cladding layer. The photon energy
8.8785 keV for Si (113) as a surface diffraction, is determined from the crystal orientation and
diffraction geometry. The effects of x-ray guiding in both lateral and vertical directions are
observed if an angle between the surface diffracted beam and the Au layer inside a
waveguide is smaller than the critical angle of total reflection for the Au/Si interface. The
energy tenability is about 300 eV.
M-266

X-Ray Back Reflection from Multi-Plate X-ray Crystal Cavity

1 1 2 2 3
Ying-Yi Chang , Sung-Yu Chen , Mau-Tsu Tang , Yu. Stetsko , M. Yabashi , Shih-Chang
1 1 1 1 1 1
Weng , Chia-Hung Chu , Yi-Wei Tsai , Po-Yu Liao , Yu-Hsin Wu , Chien-Chung Chang ,
1
Shin-Lin Chang
1 2
Department of Physics, National Tsing Hua University, Hsinchu, Taiwan, National
3
Synchrotron Radiation Research Center, Hsinchu, Taiwan, REKEN/Spring-8, Hyogo, Japan

High-resolution X-ray back reflection of (12 4 0) from multi-plate monolithic silicon crystal
cavities has been carried out to investigate the possibility of enhancing the cavity resonance
effect by increasing the reflectivity of the back reflection. According to the theoretical
calculations based on the dynamical theory, the reflectivity can be raised by increasing the
number of crystal plates. Hence, several 2-, 4-, 6-, and 8-plate crystal cavities of a gap of 100
μ m and the plate thickness of 20-70 μ m are designed and fabricated using the
microelectronic lithographic technique. The diffraction experiments are then performed by
using the synchrotron X-rays of 14.4388 keV with the energy resolution Δ E = 0.36 meV.
Interference fringes due to the cavity resonance for various multi-plate cavities are observed
and the corresponding finesse of each cavity is measured from the fringe spacing. It is found
that the reflectivity, namely the finesse, increases as the number of crystal plates increases,
as the calculations predicted. The best result is from the 8-plate cavity where the finesse is
about 3.2, compared with 2.0 for a 2-plate cavity. This result is useful for designing better
finesse crystal cavity for diffraction experiments.
M-269

Diagnostic Tools used for Calibration and Verification at Protein Crystallography

Synchrotron Beam Lines

F. J. Rotella, R. W. Alkire, N. E. C. Duke, M. J. Molitsky

Argonne National Laboratory, Argonne, IL, United States

A number of tools have been adapted and developed for use at the bending-magnet and
insertion-device beam lines (19BM and 19ID) of the Structural Biology Center (SBC) at
Argonne’s Advanced Photon Source (APS). These tools are used diagnostically in the
calibration and operating verification of these synchrotron x-ray beam lines and constituent
equipment. Examples of diagnostic tools used at the SBC are presented.

Hen egg-white lysozyme crystals are a de facto standard that are widely used at protein
crystallography synchrotron beam lines. Diffraction data from lysozyme single crystals are
used to verify the operating fitness of the SBC beam lines at the beginning of every APS user
beam run and during user beam runs to verify the beam lines after equipment is replaced.
The calibration of the multi-module CCD area detectors used at SBC beam lines is verified
using powder diffraction from frozen polycrystalline slurries of lysozyme. Powder diffraction
from standard reference materials such as Si and LaB6 are used to calibrate the precise
position – sample-to-detector distance and detector swing angle (2=) – of the area detectors
used at SBC beam lines. A bench top digital thermometer and type K thermocouple are
employed to measure the temperature profiles and verify the operating fitness of the cold-
streams that cool frozen samples at SBC beam lines. Diffraction from silicon single crystal
3
cubes, which are 0.8 mm in volume and mounted on sample loops, is a sensitive probe of
sample motion induced by improper positioning of the cold-stream or other motion in the x-ray
source or beam line apparatus. Sources of parasitic scattering from beam line components
(e.g., beam collimators) observed in area detector x-ray images can be identified from
knowledge of the materials comprising the components and the x-ray wavelength and
sample-to-detector distance used to measure the diffraction images.

This work was supported by the U.S. Department of Energy, Office of Biological and
Environmental Research under contract DE-AC02-06CH11357.
M-272

The Cardiac Ryanodine Receptor Exon3 Deletion Is Rescued By Beta Strand Switching

Filip Van Petegem, Paolo Lobo, Lynn Kimicka, Ching-Chieh Tung

Univ. of British Columbia, Vancouver, Canada

2+
The contraction of heart muscle requires the rapid release of Ca from the sarcoplasmic
reticulum into the cytoplasm. This event is mediated by opening of the cardiac Ryanodine
2+
Receptor (RyR2), a large Ca selective ion channel. Many mutations in RyR2 are known to
lead to devastating genetic conditions, including catecholaminergic polymorphic ventricular
tachycardia (CPVT). The latter is characterized by triggered cardiac arrhythmias and may
lead to sudden cardiac death.

The most extreme form of CPVT is caused by deletion of the entire third exon of RyR2. This
small exon encodes an α helix and a β strand that is part of a β trefoil core in the N-terminal
domain of the channel. Such a deletion would be predicted to cause misfolding of the protein,
raising the question of why this disease deletion is not lethal.

Surprisingly, thermal melt analysis shows that the Δ exon3 N-terminal domain is not misfolded,
but instead has even gained thermal stability. We solved the crystal structures of both the wild
type and Δ exon3 N-terminal domain at 2.5 Å and 2.2 Å, respectively. The deletion causes a
very dramatic rearrangement, in which an otherwise flexible loop becomes a β strand and
thus rescues the β trefoil domain. Other β strands partially rearrange to accommodate the
rescue segment. These events underscore the unusual structural plasticity of the RyR2 N-
terminal domain.

Despite the rescue, the deletion still causes a very severe disease phenotype, which can be
explained by the loss of key domain-domain interactions within the intact channel. The study
provides rare detailed insights into a severe channelopathy. It raises the question for a
functional role of the rescue segment in wild type RyR2.
M-275

Synthesis of Nanoporous IsoReticular Metal Prophyrin Frameworks for Hydrogen

Storage
1,2 1 1 1 1
Claudia Rawn , Michelle Everett , Patrick Ward , Ruichang Xiong , David Keffer
1 2
University of Tennessee, Knoxville, TN, United States, Oak Ridge National Laboratory, Oak
Ridge, TN, United States

A hypothesized structure, analogous to IsoReticular Metal Organic Frameworks (IRMOFs) but

with the organic component being replaced by a porphyrin, has been used to model the
hydrogen adsorptive capacity. Various techniques have been employed for synthesizing an
IsoReticular Metal-Porphyrin Framework (IRMPF) structure made up of a functionalized
carboxylic tetraphenylporphyrin as the ligands and an oxygen coordinated zinc tetrahendron
as the vertices. Early synthesis attempts in a sealed heated test tubes with the porphyrin and
Zn(NO3)2 > 6(H2O) dissolved in various solvents resulted in a variety of low yield crystals.
None of these crystals when examined by single crystal X-ray diffraction provided adequate
data for a crystal structure solution. Later attempts used solvothermal synthesis with a variety
o
of solvents in various ratios. One in particular experiment was carried out at 100 C using
DMF and CHCl3 resulted in suitable crystals for characterization and single crystal X-ray
diffraction data revealed a Zn oxopolymer structure. The coordinated Zn compound was a
promising result, however, there was no evidence of the porphyrin in the structure. The
adsorption of H2 in five IRMPFs has been calculated using Path Integral Grand Canonical
Monte Carlo (PI-GCMC) simulations using standard force fields. For comparison the
adsorption isotherms of H2 in IRMOF-1 and IRMOF-10 have also been calculated. Liquid
nitrogen temperature (77 K) and room temperature (300 K) were chosen for the temperature
of the simulations and all calculations indicate that all but one of the IRMPFs adsorb a higher
weight fraction of H2 than both IRMOF-1 and IRMOF-10, but are still well short of practical
goals. We observe that IRMPFs provide additional adsorption sites for hydrogen due to the
metal center of the porphyrin. Some, but not all, of the additional functional groups on the
porphyrin enhance adsorption as well.

This work is supported by the STAIR (Sustainable Technology through Advanced

Interdisciplinary Research) program funded by the National Science Foundation through
contract DGE-0801470.
M-278

Use of Complementary Methods to Determine the Quaternary Structure of Proline

Utilization A (PutA)

John Tanner

University of Missouri-Columbia, Columbia, MO, United States

Proline utilization A (PutA) proteins are large (1000-1300 residues) bifunctional enzymes that
catalyze the two-step oxidation of proline to glutamate. We recently determined the first
crystal structure of a PutA, but the quaternary structure was not obvious from the crystal
structure. We therefore turned to the complementary techniques of small-angle X-ray
scattering and analytical ultracentrifugation to study PutA in solution. Surprising, we
discovered that the enzyme forms a donut-shaped, tetrameric assembly in solution. This work
provides a good example of the power of combining complementary biophysical techniques
with high resolution X-ray crystallography.

Reference: Srivastava et al., PNAS, (2010) 107(7):2878-2883.

M-287

Mystery Solved: X-ray Crystallography Explains the Cross-reactivity Between

Structurally Diverse Immunodominant MART-1 epitopes

Oleg Borbulevych, Brian Baker

University of Notre Dame, Notre Dame, IN, United States

Adaptive immunity mechanisms are based on antigen recognition by cytotoxic T cells.

T-cell receptors (TCR) interact with certain peptide epitopes presented by class I MHC
molecules, leading to an intracellular signaling cascade and a subsequent immune response.
The MART-1 antigen is overexpressed by the majority of melanoma affected cells, and hence
represents an attractive target for cancer immunotherapy using cytotoxic T cells.
Immunodominant epitopes of MART-1 presented by the class I MHC HLA-A2 proteins
comprise the decameric MART-1 26-35 epitope EAAGIGILTV and the nonameric MART-1 27-35
epitope AAGIGILTV. Our previous crystallographic study indicated that the conformations of
those peptides in the HLA-A2 peptide binding groove differ significantly. Paradoxically, most
MART-1 specific cytotoxic T cells can still cross-reactively recognize both MART-1 epitopes.

In the present communication we report, for the first time, the crystallographic structures of
the clinically relevant TCR DMF5 in the complex with MART-127-35/HLA-A2 and MART-126-
35/HLA-A2 to 2.3 Å and 2.7 Å resolution, respectively. These structures allow us to unravel
the mechanism of cross-reactivity. Notably, recognition of the MART-127-35 nonamer peptide
by DMF5 is accompanied by a significant rearrangement of the peptide backbone. In contrast,
the conformation of the decameric peptide remains unchanged upon recognition, indicating
that cross-reactivity occurs via a “induced molecular mimicry” mechanism.
M-296

Mechanism of IL-10 Neturalization Elucidated by the Crystal Structure

of an IL-10/anti-IL-10 Fab Complex

Brandi Jones, Laura Baker Smith, Mark Walter

University of Alamba at Birmingham, Birmingham, United States

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M-303

THE STRUCTURE OF ADENO-ASSOCIATED VIRUS SEROTYPE 3B (AAV-3B), A GENE

THERAPY VECTOR

Thomas F. Lerch, Qing Xie, Michael S. Chapman

Oregon Health & Sci Univ., Portland, OR, United States

Adeno-associated viruses (AAVs) are leading candidate vectors for gene therapy
applications. The most widely characterized AAV is serotype 2 (AAV-2), which enters cells
through interactions with heparan sulfate proteoglycan (HSPG). The development of
recombinant AAVs that target specific tissues has been accelerated since the determination
of the AAV-2 atomic structure, which provided a roadmap for vector design. As much as 80%
of the population has been exposed to AAV-2, and the presence of neutralizing antibodies to
this serotype limits the efficacy of AAV-2 based vectors.

The AAV-3b capsid is closely related to AAV-2 (87% identity), but sequence, and
presumably structural differences lead to distinct properties in cell entry and immune
recognition. Like AAV-2, AAV-3b requires heparan sulfate for cellular entry, yet key AAV-2
residues involved in heparan interactions are not conserved in AAV-3b. In an effort to
understand these differences, and perhaps harness them, the structure of AAV-3b has been
determined by X-ray crystallography. The crystals display varying levels of merohedral
twinning that in earlier times would have rendered them unsuitable, but here is shown to be a
tractable complication in structure determination. Structural comparisons of AAV-3b with AAV-
2 and other serotypes of known structure provide insights into how these viruses have
naturally evolved to preserve receptor interactions while avoiding immune neutralization.
M-305

The Structural Basis of 5 Triphosphate Double-stranded RNA Recognition by RIG-I

CTD

Pingwei Li, Cheng Lu

Texas A&M Univ., College Station, TX, United States

RIG-I is a cytosolic sensor of viral RNA that plays crucial roles in the induction of type I
interferons. The C-terminal domain (CTD) of RIG-I is responsible for the recognition of viral
RNA with 5Z triphosphate (5Z ppp). However, the mechanism of viral RNA recognition by RIG-I
is still not fully understood. Here, we show that RIG-I CTD binds 5Z ppp dsRNA or ssRNA, as
well as blunt-ended dsRNA, and exhibits the highest affinity for 5Z ppp dsRNA. Crystal
structures of RIG-I CTD bound to 5Z ppp dsRNA with GC- and AU- rich sequences revealed
that RIG-I recognizes the termini of the dsRNA and interacts with the 5Z triphosphate through
extensive electrostatic interactions. Mutagenesis and RNA binding studies demonstrated that
similar binding surfaces are involved in the recognition of different forms of RNA with or
without 5Z triphosphate. Mutations of key residues at the RNA binding surface afffected RIG-I
signaling in cells.
M-307

POWGEN: A New TOF Neutron Powder Diffractometer at the SNS

1 1 1,2 1
Jason Hodges , Ashfia Huq , Olivier Gourdon , Luke Heroux
1 2
Oak Ridge National Laboratory, Oak Ridge, Tennessee, United States, Forschungszentrum
Juelich GmbH, Juelich, Germany

POWGEN is a fundamental departure from previous designs for a time-of-flight powder

diffractometer at a spallation neutron source and may be considered a third-generation
design. The instrument is optimized for both parametric studies of materials under a wide
range of conditions (T, P, H, flowing gases, etc) and ab-initio crystal structure determinations
3
of complex solid-state materials with asymmetric unit-cells of the order ~1500 Å . The
geometric design of the instrument allows for all detected scattered neutrons to be focused
onto a single diffraction profile yielding high count rate while preserving good resolution [d/d
= 0.0015 at d = 1 Å. The new time-event mode for data acquisition will permit stroboscopic
experiments and filtering of the incoming data, neutron by neutron, allowing very high
resolution diffraction profiles to be generated with [d/d ~ 0.0007 at d = 1 Å. SNS is operated
with the support from the Division of Scientific User Facilities, Office of Basic Energy
Sciences, US Department of Energy, under contract DE-AC05-00OR22725 with UT-Battelle,
LLC
M-311

Synchrotron powder diffraction simplified: the high-resolution 11-BM diffractometer at

the Advanced Photon Source

Matthew Suchomel, Lynn Ribaud, Brian Toby, Robert Von Dreele

Argonne National Laboratory, Argonne, IL, United States

Synchrotrons have revolutionized powder diffraction. They enable the rapid collection of high
quality powder diffraction patterns with tremendous resolution and superb signal to noise. In
addition, the high penetration and exceptional data sensitivity possible at high-energy light
sources like the Advanced Photon Source (APS) allows synchrotrons to explore trace
containment levels, in-situ sample environments and crystallographic site occupancies.
Despite all these advantages, relatively few scientists today consider using a synchrotron for
routine powder diffraction studies.

To help address this, the new high resolution synchrotron powder diffractometer beamline 11-
BM at the APS now offers rapid and easy mail-in access for routine structural analyses with
truly world-class quality data. This instrument offers the highest resolution available in the
Americas and is a free service for non-proprietary users. The instrument can collect a superb
pattern suitable for Rietveld analysis in less than an hour, is equipped with a robotic arm for
automated sample changes, and features variable temperature and in-situ sample
environments. Users of the mail-in program typically receive their high-resolution data within
two weeks of sample receipt. The diffractometer is also available for on-site user experiments
requiring more specialized measurements.

Our presentation will describe this instrument, highlight its capabilities, explain the types of
measurements currently available, and discuss plans to improve access and available sample
environments. We are particularly interested in seeking input from potential users within the
crystallography community.

More information about the 11-BM diffractometer and its associated mail-in program can be
found at our website: http://11bm.xor.aps.anl.gov.
M-315

SER-CAT’ s mission of "Light When YOU Need It" for Member Institutions and the APS
General
Zhongmin Jin, John Chrzas, Jim Fait, Zheng-Qing Fu, John Gonczy, Andrew Howard, Rod
Salazar, Unmesh Chinte, Gerd Rosenbaum, John Rose, B.C. Wang

University of Georgia, Athens, Georgia, United States

SER-CAT’ s mission: To provide "Light When YOU Need It" to its member institutions and
the APS General Users.
12-hour blocks and 16-hours/day on-site user support: Since summer of 2009, SER-CAT
users can reserve time in 12-hour blocks if desired. In addition, SER-CAT is currently
providing 16-hours/day on-site user support.
Remote Access & Participation with Advanced Robotics: Advanced beamline control
hardware, and software integration, makes it possible for SER-CAT users to have a “virtual
synchrotron” at their home institutions. About 80% of SER-CAT users collect and process
data remotely. The sample Dewars on 22ID and 22BM can hold 160 and 96 crystals,
respectively.
Micro Beam Capability: SER-CAT offers a set of highly machined pinhole collimator inserts
ranging from 5 to 300 microns on both its beamlines. A new MD2 micro-diffractometer will be
integrated into 22ID during the August - September shutdown.
Sulfur SAD Phasing: Recent improvements to 22ID have opened up the possibility of protein
structure determination using soft X-ray (6-8kev) based sulfur-SAS methods.
MAD/SAD Experiments: The SER-CAT 22ID and 22BM lines were designed and optimized
to provide tunable X-rays (6-15 keV) for MAD and optimized SAD experiments.
Mail-in Crystallography Services: SER-CAT provides a mail-in data collection service to its
members. Researchers can mail their crystals to SER-CAT and SER-CAT staff will collect
data for them on a first come first serve basis. This is a special perk for SER-CAT institutional
members.
Work supported by the SER-CAT Member Institutions, the University of Georgia Research
Foundation and the Georgia Research Alliance.
M-319

IMAGINE, a Quasi-Laue Single Crystal Neutron Diffractometer At The High Flux Isotope
Reactor
1,2 3 4 2 2
Flora Meilleur , Tibor Koritsanszky , Robert Blessing , Bryan Chakoumakos , Dean Myles
1 2
Oak Ridge National Lab, Oak Ridge, TN, United States, North Carolina Univ, Raleigh, NC,
3 4
United States, Middle Tennessee State Univ., Murfreesboro, TN, United States, Hauptman-
Woodward Med Research Inst., Buffalo, NY, United States

The acquisition, installation and operation of IMAGINE at he High Flux Isotope Reactor
(HFIR) was proposed to the National Science Foundation by a group of researchers from
Physics, Chemistry, Biology, Biochemistry and Geological and Earth Sciences at Middle
Tennessee State University, North Carolina State University, Hauptman-Woodward Medical
Research Institute and Oak Ridge National Laboratory, with 13 additional participants from
US industry and academic facilities. The objective of the program, which received funding
from NSF in July 2009, is to develop a state-of-the-art facility and user-access program for
neutron-diffraction analysis of advanced, complex and functional materials at the HFIR.

IMAGINE will have broad scientific impact and community use, providing new tools,
capabilities and methods for the analysis of light atom positions in materials that will be of
interest across the diverse fields of structural biology, pharmacology, chemistry, condensed
matter physics, nano-structured materials, and in environmental, biomedical and geological
sciences. The instrument will enable the neutron structure of supra and macro-molecules to
3
be determined at or near atomic resolutions (1.5 Å) from crystals with volume < 1mm and
unit cell < 100 Å.

IMAGINE will be commissioned in spring 2011. The IMAGINE team welcomes discussion
and interaction with the community through the installation and commissioning phase of the
instrument, and is excited to start working with the community to build an excellent education
and science program.

The poster will give an overview of the IMAGINE project at the HFIR.

Oak Ridge National Laboratory is managed by UT-Battelle, LLC, for the U.S.
Department of Energy under Contract DE-AC05-00OR22725.
M-321

Crystal structure of the novel PaiA N-acetyltransferase from Thermoplasma acidophilum

involved in the negative control of sporulation and degradative enzyme production
1,2 1,2 1,2 1,2
Ekaterina V. Filippova , George Minasov , Ludmila Shuvalova , Olga Kiryukhina ,
2 2 1,2
Shonda Clancy , Andrzej Joachimiak , Wayne F. Anderson
1
Department of Molecular Pharmacology and Biological Chemistry, Northwestern University,
2
Feinberg School of Medicine, Chicago, IL, United States, Midwest Center for Structural
Genomics, Argonne National Laboratory, Argonne, IL, United States

The crystal structure of a putative Gcn5-related N-acetyltransferase (GNAT) Ta0374 from

Thermoplasma acidophilum, a hyperacidophilic bacterium, has been determined in an apo-form, in
complex with its natural ligand (acetyl coenzyme A) and in complex with a product of reaction
(coenzyme A) obtained by co-crystallization with spermidine. Sequence and structure analysis reveals
that N-acetyltransferase Ta0374 belongs to a novel protein family PaiA involved in the negative control
of sporulation and production of degradative enzymes such as subtilisin, neutral protease,
levansucrase, α -amylase, and alkaline phosphatases. The crystal structure of Ta0374 confirms that it
binds acetyl coenzyme A in a way similar to other GNAT and is capable of acetylating spermidine.
Based on structural and docking analysis we suggest that Glu53 and Tyr93 are the key residues for the
spermidine recognition. In addition, we find that part of the purification His-Tag in the apo-form
structure of Ta0374 blocks binding of acetyl coenzyme A in binary complex and affects a chain-flip
rotation of “motif A” which is the most conserved sequence motif among canonical acetyltransferases.
The details of the Ta0374 structure, conformational changes, ligand-binding site provide
insights into the biological function of Pai regulation.
M-323

Impact of conserved 16S rRNA methylation by KsgA on the structure of the 30S
ribosomal subunit
1 2 3 4 4
Hasan Demirci , Frank Murphy IV , Riccardo Belardinelli , Ann C. Kelley , V Ramakrishnan ,
1 1 1
Steven T. Gregory , Albert E. Dahlberg , Gerwald Jogl
1 2
Brown University, Providence, RI, United States, NE-CAT/Cornell University, Argonne, IL,
3 4
United States, University of Camerino, Camerino, MC, Italy, MRC, Cambridge, United
Kingdom

The most highly conserved ribosome modification is the N6, N6-dimethylation of the
universally conserved adenosines A1518 and A1519 in helix 45 of the small ribosomal
subunit. Dimethylation of both adenines is catalyzed by the KsgA methyltransferase. The
absence of these post-transcriptional modifications enhances initiation from non-AUG codons,
increases decoding errors, and confers resistance to the antibiotic kasugamycin.

To examine the function of A1518 and A1519 dimethylation, we determined the X-ray
crystal structure of the unmodified 30S ribosomal subunit from Thermus thermophilus. We
observe significant changes in helix 45 resulting from lack of methylation and conformational
adjustments in neighboring helices 24a and 44. The unmodified tetraloop assumes a
conformation similar to a canonical GNRA tetraloop fold, consistent with previous NMR
studies predicting a function of adenine dimethylation in the stabilization of the extended
tetraloop conformation observed in the wild-type ribosome. Conformational shifts in helices
24a and 44 explain the varied phenotypic properties of ksgA mutants and reveal the impact of
loss of dimethylation on kasugamycin binding. Overall, our data show, for the first time, the
significance of a post-transcriptional rRNA modification for ribosome structure and suggest a
role for these modifications in the final stages of 30S subunit assembly.
M-325

Structural basis for p300 Taz2:C/EBP interactions.

Maria Miller, Zbigniew Dauter

1 2
Protein tructure Section, MCL, NCI-Frederick, Frederick, MD, United States, Synchrotron
Radiation Research Section, MCL, NCI, Argonne, Illinois, United States

CBP and its paralogue p300 are histone acetyltransferases that regulate transcription by
interaction with multiple transcription factors. The crystal structure of the zinc finger Taz2
domain of the human p300 transcriptional coactivator was determined using an anomalous
diffraction signal of the bound Zn ions. The structure comprises a helical bundle held by three
Zn ions and is very similar to the solution structures determined for the shorter peptide
corresponding to the evolutionarily conserved Taz2 domain from CBP [1] and p300 [2].
Residues 1813-1834 from the current construct form a helical extension of the C-terminal
helix and make extensive crystal contact interactions with the peptide binding site of Taz2.
Based on the analysis of these contacts, we previously proposed a hypothetical model of the
Taz2:p53 binding [3]. In the current study we use the crystal contact interactions to investigate
Taz2 binding to the transactivation domain of the C/EBP\ protein.

[1] De Guzman R.N., Liu H.Y., Martinez-Yamout M., Dyson H.J., Wright P.E. J. Mol. Biol,
2000, 303, 243.

[2] Feng H., Miller Jenkins L.M., Durell S.R., Hayashi R., Mazur S.J., Cherry S., Tropea J.E.,
Miller M., Wlodawer A., Appella E., Bai Y. Structure 2009, 17, 2002.

[3] Miller M., Dauter Z., Cherry S., Tropea J.E., and Wlodawer A., Acta Cryst. D65, 2009,
1301-1308.

Key words: transcription regulation, zinc finger protein, anomalous diffraction

M-327

Crystal Structure of a Dimeric Glycosylated IL-22/IL-22R1 Complex

Ashlesha Desphpande, Brandi Jones, Mark Walter

University of Alabama at Birmingham, Birmingham, AL, United States

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M-329

Mechanism of the UvrA•UvrB DNA Damage Sensor

David Jeruzalmi, Danaya Pakotiprapha, Martin Samuels, Greg Verdine

Harvard Univ., Cambridge, MA, United States

There are several pathways in cells that monitor DNA and mount responses when
damage is detected. One of these, the nucleotide excision repair (NER) pathway, processes
bulky DNA lesions. We study the NER pathway in bacteria where the first steps are
performed by three proteins: UvrA, UvrB, and UvrC. The UvrA•UvrB ensemble monitors DNA
and recognizes damage. On encountering damage, UvrA exits the complex, leaving UvrB
stably bound. Damage searching, formation of the DNA complex and dissociation of ‘A’ are
regulated by ATP. ‘B’ then recruits the endonuclease UvrC, which catalyzes incisions on
either side of the lesion. Additional processing reactions lead to restoration of the original
DNA sequence.

In order to better understand the initial steps of bacterial NER, we have determined
two crystal structures, 1) full-length UvrA bound to ADP and 2) a complex between the two
isolated interaction domains of UvrA and UvrB.

The structure of isolated UvrA revealed its overall architecture and arrangement of its
four-nucleotide binding sites. Structure-guided biochemical studies were used to identify
surfaces that interact with UvrB and DNA.

Our second structure focuses on the UvrA•UvrB complex. From the structure of the
isolated interacting domains and the structures of the isolated components, we deduced a
model for the complete damage-sensing complex.

This work was supported by the National Institutes of Health (CA100742, GV) and the
National Science Foundation (MCB 0918161, DJ).
M-333

N-terminal Domain of Nop56/58 is Critical for Assembly and Methylation Activity of

Archaeal Box C/D sRNP Complex
1 1,2 1 1 1
Shyamasri Biswas , Keith Gagnon , Xinxin Zhang , Carla Mattos , Bernard Brown II , Stuart
1
Maxwell
1
Department of Structural Biochemistry, North carolina state University, Raleigh, NC, United
2
States, Departments of Pharmacology and Biochemistry, University of Texas Southwestern
Medical Center, Dallas, Texas, United States

Box C/D RNP complexes guide the 2’O-methylation of rRNA nucleotides which are critical for
ribosome assembly and function. The archaeal sR8 box C/D RNP complex consists of L7Ae,
Nop56/58 and fibrillarin core proteins and the sR8 sRNA. Although extensive structural
studies have been carried out on the ‘kink turn' RNA-binding protein L7Ae and S-Adenosyl
Methionine -binding methyltransferase protein fibrillarin, little is known about Nop56/58.
Methylation of rRNA is initiated by L7Ae binding to kink-turn RNA motif followed by binding of
the Nop56/58 and fibrillarin proteins. Here we describe the crystal structure of the N-terminal
domain of the Methanocaldococcus jannaschii Nop56/58 core protein. This N-terminal domain
interacts with fibrillarin and this complex exhibits exceptional stability resisting denaturation
and melting temperatures above 100°C. Mutations in the N-terminal domain have identifi ed a
single point mutant and a five amino acid deletion mutant that significantly reduce or abolish
box C/D sRNP-guided nucleotide methylation in vitro, suggesting that these amino acids play
a critical role in catalysis of 2’-O-methylation. Thus, the Nop56/58 core protein, either alone or
in concert with fibrillarin, plays a functional role in nucleotide modification guided by the
archaeal box C/D sRNP complexes.
07.14.1

Harnessing structural tuning parameters in a class of layer ordered CMR materials

1,2 1,2 1 2
Omar Chmaissem , Bogdan Dabrowski , Stanislaw Kolesnik , Yang Ren , Dennis E
1 1
Brown , James Mais
1 2
Northern Illinois University, DeKalb, IL, United States, Argonne National Laboratory,
Argonne, IL, United States

In this talk, I will briefly review the main structural and physical properties of A-site layer
ordered and disordered La1-xAxMnO3 (A = Ba, Sr, Ca; x~0.5) manganites and identify the
characteristics they have in common and those that are distinctly different. Recent theoretical
work successfully explained the colossal magnetoresistance (CMR) properties of the
manganites in terms of parameters that include ferromagnetic (FM) double-exchange and
antiferromagnetic (AFM) superexchange couplings, electron-phonon coupling, quenched
disorder, etc. Here, I will discuss our observation of charge ordering (CO), orbital ordering
(OO) and the presence of a multicritical point in the A-site layer ordered La1-xBa1+xMn2O6
(x~0.04) class of materials and the existence of a strong competition developing between
three distinctly different magnetic ground states (FM, CO AFM and OO AFM). At the right
composition and below some temperature, the three phases start to separate and eventually
they freeze in their respective domains; thus giving rise to spin glass clusters. Neutron and x-
ray data taken as a function of temperature and magnetic fields agree very well with the
complex magnetic and resistive data. On the other hand, disordered La1-xBaxMnO3 (0.2 ≤ x ≤
0.52) does not exhibit any such properties and the materials remain ferromagnetic at all
temperatures. Furthermore, we find that the deliberate introduction of a small structural
disorder on the otherwise perfectly ordered La or Ba layers results in modifying the system’s
property to either move it closer or away from the multicritical point depending whether the
disorder is on the La or the Ba sites. Our asymmetric results suggest that disorder is not the
primary parameter controlling phase separation and the CMR properties. In fact, it may be
used as a fine tuning tool to enhance/reduce the electron-phonon coupling near the
multicritical point; thus, resulting in phase separation. Finally, in this class of materials,
charge ordering and the observed phase separation (responsible for the CMR properties) can
be suppressed under the application of low magnetic fields of 1-2 Tesla. This represents a
huge improvement of an order of magnitude when compared with the large fields (10-60 T)
required to suppress CO, structural transitions, and phase separation in the disordered 3D
counterparts.
07.14.2

The Cosubstitution Reaction of In2O3 by ZnO and SnO2 as Characterized with X-ray
Absorption Spectroscopy

Cathleen Hoel, Jean-Francois Gaillard, Kenneth Poeppelmeier

Northwestern University, Evanston, IL, United States

Transparent conducting oxides (TCOs) are important materials to optoelectronics, such as

flat-panel displays and photovoltaics, with tin-doped indium oxide (ITO) having the high
electrical conductivity and optical transparency required for maximum device performance.
The limited global supply of indium metal coupled with the increased demand has
necessitated the need to develop indium-free TCOs. The cosubstitution of ZnO and SnO2
into In2O3 (ZITO) has demonstrated conductivities comparable to those of ITO, while
1,2
reducing the percentage of In by 40 mole %. The local structural changes that occur in
In2O3 upon substitution with Zn and Sn have been investigated with X-ray absorption
spectroscopy. Zn and Sn were observed to induce first shell oxide bond shortening owing to
the smaller cationic radii compared to In. Zn was six-coordinate with oxygen and the Zn and
Sn next nearest neighbor cation environment was consistent with the bixbyite framework.

1. G. Palmer, et al., Chem. Mater. 9 (1997) 3121

2. S. Harvey, et al., J. Am. Ceram. Soc. 91 (2008) 3683

07.14.3

Effect of lattice distortion via Phosporus doping in Fe-based Oxypnictides

1 3 3 4 4 4
Clarina dela Cruz , W.Z. Hu , Shiliang Li , Qing Huang , Jeff Lynn , Mark Green , Miaoyin
2 2 5 3 1 6 2,1
Wang , Meng Wang , G.F. Chen , N.L. Wang , Herbert Mook , Qimiao Si , Pengcheng Dai
1
Neutron Scattering Science Division, Oak Ridge National Laboratory, Oak Ridge, TN, United
2
States, Department of Physics and Astronomy, University of Tennessee, Knoxville, TN,
3 4
United States, Institute of Physics, Chineses Academy of Sciences, Beijing, China, NIST
5
Center for Neutron Research, NIST, Gaithersburg, Maryland, United States, Department of
6
Physics, Renmin University, Beijing, China, Department of Physics and Astronomy, Rice
University, Houston, TX, United States

We use neutron diffraction to study the structural and magnetic phase diagram of P doped
Fe-based oxypnictide superconductors and their corresponding parent compounds. First, we
find that replacing the larger arsenic with smaller phosphorus in CeFeAs1-xPxO
simultaneously suppresses the antiferromagnetic order and orthorhombic distortion near
x=0.4, thus suggesting the presence of a magnetic quantum critical point. In this system, P
doping drives the system through the quantum critical point to a paramagnetic state. Our
detailed structural analysis reveals that the pnictogen height is an important controlling
parameter for their electronic and magnetic properties, and may play an important role in
electron pairing and superconductivity of these materials. Similar studies were done in the
LaFeAs1-xPxO where P-doping drives the system into a superconducting state. Comparisons
between the two systems were done to identify the correlation of lattice distortion on the
resulting transport properties and magnetism.
07.14.4

Targeting New Materials

Kenneth Poeppelmeier

Northwestern University, Evanston, IL, United States

An example of a new transition metal oxide fluoride, which was synthesized recently, is the
high silver density material Ag4V2O6F2 (SVOF). Ag2V4O11, or silver vanadium oxide (SVO),
is used commercially as the cathode material in primary lithium batteries for high rate
applications, such as those used in implantable cardioverter defibrillators (ICDs). A long-term
goal of the medical battery industry is to increase the capacity of the cathode above 3 V while
maintaining electrode stability. Owing to the high mole fraction of silver and the replacement
of oxide with fluoride, SVOF has a higher capacity above 3 V of 148 mAh/g in comparison to
100 mAh/g in SVO and the upper discharge plateau at 3.5 V is 300 mV higher than the silver
reduction potential of SVO. The electrochemical behavior of SVOF and the significant impact
new materials such as SVOF may have on the future generation of primary lithium batteries
for ICDs will be highlighted.
07.14.5

Single Crystals of Functionalized Nanoparticles as a Medium for Structural and

Spectroscopic Studies of Photosensitizer Dyes on Semiconductor Surfaces

Jason Benedict, Philip Coppens

University at Buffalo, Buffalo, NY, United States

Single crystals of semiconductor nanoparticles functionalized with technologically relevant

ligands offer the opportunity to obtain structural information that can be combined with
theoretical calculations. The latter are based on the experimental geometry and used to
interpret spectroscopic and other information. A series of Ti/O clusters with diameters up to
approximately 2.0 nm have been synthesized and functionalized with a variety of small
molecule ligands which exhibit distinct binding modes. DFT calculations complement
spectroscopic measurements which examine the band gap of the nanoparticles pre- and post-
functionalization and elucidate the influence of adsorbate geometry as well as size
quantization effects on the band structure.The detailed structure of the nanoparticles deviates
significantly from that of anatase, which is commonly used as a structural model for the
substrate used in photovoltaic cells. The structures for the first time unambiguously reveal the
†
different binding modes of the adsorbant molecules. l

Work was funded by the Division of Chemical Sciences, Geosciences, and Biosciences,
Office of Basic Energy Sciences of the U.S. Department of Energy through Grant DE-FC02-
02ER15372
†
J. B. Benedict & P. Coppens, JACS 2010, 192, 2938-2944.
07.14.6

Local structure and its relationship to the properties of “ReO 3” type frameworks
showing positive, low and negative thermal expansion
1 1 1 2 3
Angus Wilkinson , Benjamin Greve , Andrew Jupe , Kenneth Martin , Karena Chapman ,
3 4
Peter Chupas , Peter Lee
1 2
Georgia Institute of Technology, Atlanta, GA, United States, Berry College, Mount Berry,
3 4
GA, United States, Argonne National Laboratory, Argonne, IL, United States, Department of
Energy, Germantown, MD, United States

The cubic “ReO3” framework structure is simple, and yet it has all the structural features
necessary for negative thermal expansion (NTE) due to the transverse thermal motion of
bridging anions. While ReO3 itself does not display negative thermal expansion at room
temperature, materials with this structure display a variety of properties, ranging from
pronounced NTE through to strong positive thermal expansion. We will discuss the thermal
expansion and compressibility of several fluorides and oxyfluorides with this structure, and
examine the role that O/F disorder plays in determining their properties.
07.15.1

Non-merohedral twin with partially known composition

1 2 2
Ilia Guzei , Indika Arachchige , Sergei Ivanov
1 2
UW-Madison, Madison, WI, United States, MPA-CINT, Los Alamos National Lab, Los
Alamos, NM, United States

The hexagonal (space group P61) crystal structure of an ionic multinuclear gold cluster
[Au6S2P(Ph2Cy)6][anion].(solvent) proved to be a non-merohedral twin. The twin components
are related by a 180° rotation about the [110] axis with the minor compo nent contribution of
49.4(6)%. The composition of the cationic hexanuclear gold cluster was reliably established,
however only the heavy atoms could be refined with anisotropic displacement coefficients.
The Ph and Cy groups had to be refined with an idealized geometry taken from the newly
created Idealized Molecular Geometry Library. There were several diffusely diffracting
species in the lattice which could not be reliably identified and even their corresponding
electron count could not be approximated with the PLATON/SQUEEZE option due to the
twinning. Publication prospects for this structure remain unclear because the charge has not
been balanced and possible solvents identified.
07.15.2

The cis-dioxo uranyl cation remains at large

1 1 2
Christopher Cahill , Paula Cantos , Richard Wilson
1 2
The George Washington University, Washington, DC, United States, Argonne National
Laboratory, Argonne, IL, United States
2+
Hexavalent uranium forms almost exclusively as the uranyl cation, [UO2] , a linear triatomic
species. Although predicted to be stable, the cis- form of this species has not been formed
reliably. Reports of its formation and subsequent crystallographic characterization have been
scrutinized and labeled as suspect. A recent result from our group wherein the uranyl cation
was reacted with 2,5-pyradinedicarboxylic acid appeared to yield such a cis-bearing material:
UO2(C7H3O4N), P21, a = 7.0014(6), b = 8.1293(7), c = 7.8051 (7) Å, ‚ = 104.317(1)°, R1 =
2.92%. This was a relatively satisfactory refinement, save for one anomalous thermal
parameter and a suspiciously long C-O bond. Standard CIF checks were not condemning,
either. Careful reexamination (and subsequent re-collection of the data) revealed a P21/n cell:
a = 9.0141(11), b = 8.1137(10), c = 11.6729(14) Å, ‚ = 96.197(2) °, R1 = 2.55%. This larger
cell did not display the anomalous features, nor did it contain a cis-oxo uranyl! The trans form
continues to reign and we cite the need to exercise restraint when encountering such a
combination of results and relatively minor red flags. Did we publish it? No way. The trans? It
is in review.
07.15.3

Porphyrin Polymorphism … do we need more structures?

Allen Oliver, W. Robert Scheidt

University of Notre Dame, Notre Dame, IN, United States

Chloro (octaethylporphyrinato)iron(III) [Fe(OEP)Cl] has two reported polymorphs; one of

which is archived in the Cambridge Structure Database. A short discussion on a third
polymorph and its merits to the literature will be presented.
07.15.4

The Best Way to Solve the Big R Problem?

Kenneth Haller

Suranaree University of Technology, Nakhon Ratchasima, Thailand

A reportedly 1:1 cocrystal adduct of [SnCl2(C2H4COOCH3)2] and 1,10-phenanthroline that had

remained on the shelf for 25 years was recrystallized from ethanol for an X-ray study. The
space group was found to be triclinic P−1 with Z = 6. A strong vector in the Patterson function
2 1 1
of ( /3, /3, /3) suggested pseudo-translational symmetry, and a direct methods solution
consistent with this suggested pseudo symmetry was obtained. However, the refinement
failed, ending with R = 0.32. There is more than one way to skin a cat, and this structure has
been published.
07.15.5

Is a 1.3Å resolution structure only useful for joining the dots?

Gary Nichol

The University of Arizona, Tucson, AZ, United States

Crystallography is often used in organic chemistry to consider questions of conformation or

chirality only after hours spent poring over various correlated nmr spectra have failed to point
to a definitive answer. In many cases a perfectly reasonable set of crystals (they are usually
excellent!) can be easily grown and a structure obtained in a timely fashion. Then there are
cases where compounds stubbornly refuse to yield good crystals, but the crystals which are
obtained can still be used in a diffraction experiment and provide useful information for the
chemist. The question then is – what next? Two recent structures of poorly-diffracting (1.1Å
& 1.3Å resolution) chiral compounds, along with the pros and cons of each structure, will be
presented for audience debate and comment.
07.15.6

Common problems encountered with submissions to Acta Cryst. E

Jim Simpson

University of Otago, Dunedin, New Zealand

As a section editor and co-editor of Acta Cryst. Section E, which in 2009 published 4116
papers, I get to see a reasonable cross-section of small molecule structures that fall within the
proposed ambit of this session. I would like to take the opportunity to share information on the
most common problems that we encounter in Acta E and how they are generally dealt with. It
would be fair to say that problems – even some resulting in A alerts in the CheckCIF process
– do not necessarily condemn a paper to rejection. The use and reliance on validation
procedures as the ultimate determinant of the fate of a publication will also be discussed.
07.16.1

The National Synchrotron Light Source Facility at the Brookhaven National Laboratory

Marc Allaire, et. al.

Brookhaven National Lab, Upton NY, United States

The National Synchrotron Light Source (NSLS) at the Brookhaven National Laboratory is
located on Long Island, New York. The NSLS continues to be extremely active having more
than 2200 users reporting nearly a thousand publications a year. The NSLS provides access
to over 60 dedicated synchrotron beamlines with a wide range of applications targeting many
areas of life, materials, and physical sciences. Synchrotron techniques as diverse as UV/IR/x-
ray (micro)-spectroscopy and imaging, x-ray reflectivity and scattering, powder and single
crystal diffraction, and x-ray footprinting are commonly available at the NSLS. The NSLS has
a long tradition of strong user support. In macromolecular crystallography, a sea of photons is
readily available, including two undulator-based MX beamlines X25 and X29, the latter being
the second most productive beamline worldwide. A recent upgrade to beamline X26C allows
correlated studies of single crystal diffraction with optical absorption and Raman
spectroscopy. Responding to a growing demand, the newest beamline at the NSLS is the
undulator-based beamline X9, which is dedicated to small/wide-angle x-ray scattering
including the study of biomolecules in solution. Beamline X9 has been specially designed to
provide simultaneous SAXS/WAXS data collection. In an effort to expand and educate the
SAXS/WAXS user community, a hands-on training course is now frequently available to new
users of this beamline.

The ƒ„…„ is supported by †‡¡? ˆ‰„‰ Š‹Œ‒†Ž‹†? ‘? ’‹“”•. Supports for specific user programs
are funded by the National Institute of Health.
07.16.2

Neutron Total Scattering and Powder Diffraction Capabilities at the Lujan Neutron
Scattering Center

Thomas Proffen

Los Alamos National Laboratory, Los Alamos, NM, United States

The Lujan Neutron Scattering Center at Los Alamos National Laboratory offers a number of
diffractometers in the user program: NPDF is a dedicated and user friendly total scattering
instrument allowing one to obtain the atomic pair distribution function (PDF) of disordered,
nano-crystalline and amorphous materials. The PDF capabilities were recently extended with
a series of successful PDF measurements on HIPD using the same user friendly web
interface a NPDF. Data obtained on both instruments also allow Rietveld analysis of the
materials – NPDF provides high resolution and HIPD high flux. The instrument HIPPO is a
high flux instrument mainly used for high pressure and texture measurements but it can also
be used as ‘regular’ powder diffractometer. Finally the instrument SMARTS is a engineering
diffractometer at the Lujan Center.

Curious about a specific instrument and experimental capability – come and join us at this
special session and learn more.
07.16.3

Neutron Scattering Opportunities at the SNS and HFIR

Dean Myles

Oak Ridge National Laboratory, Oak Ridge, TN, United States

With the United States' highest flux reactor-based neutron source for condensed matter
research (the High Flux Isotope Reactor) and the world's most intense pulsed, accelerator-
based neutron source (the Spallation Neutron Source), ORNL is becoming one of the
foremost center for neutron science. Research at these facilities encompasses the physical,
chemical, materials, biological, and medical sciences and will provide opportunities for up to
2000 researchers each year from industry, research facilities, and universities all over the
world. Opportunities for crystallography, small angle scattering and reflectometry at ORNL will
be discussed.
07.16.4

Harness Advanced Photons at their Source to Further Your Research

Brian Toby

APS, Argonne National Labe, Argonne, IL, United States

The Advanced Photon Source (APS) at Argonne National Laboratory is the country's brightest
high-energy synchrotron. Each year over 5,000 scientists use the unique x-ray
instrumentation at the APS. This presentation will provide a very brief overview of the diverse
capabilities available to users and how to obtain more detailed information. Also to be
discussed will be how scientists who have not used the APS can get access to the facility.
07.16.5

The General-Purpose Small Angle Neutron Scattering instrument on the High Flux
Isotope Reactor HFIR at Oak Ridge National Laboratory

Ken Littrell

HFIR/NSSD Oak Ridge National Laboratory, Oak Ridge, TN, United States

In May, 2007, the High Flux Isotope Reactor (HFIR) at Oak Ridge National Laboratory
resumed routine operation and service to the scientific user community with a number of
significant upgrades. Among the most important of these is a new supercritical hydrogen
moderator (T ~ 20 K) that is the “brightest” cold source currently available. While this will
eventually provide neutrons to a whole suite of scattering instruments through four cold
neutron guides (CG1-4), the two flagship instruments, new small-angle neutron scattering
(SANS) instruments on CG2 and CG3 have been installed and commissioned.

The CG2 SANS (General Purpose SANS, funded by the Department of Energy (DOE) Office
of Basic Energy Sciences) is a 40m maximum total flight path pinhole SANS instrument
2 5 2
variable wavelength and a large area (1m ) high count-rate, (> 10 Hz) high-resolution (5mm
pixels) detector that can translate from 0 to 45 cm off-axis to increase the dynamic Q-range (<
-1 7 2
0.001-1 Å overall). With a measured flux on sample of 10 /sec/cm and beyond in high-
throughput configurations, this instrument is comparable to the best worldwide. This
dramatically improves both the quantity and quality of data that we can collect from samples
from a variety of systems, enabling us to better serve the neutron scattering community. This
instrument has successfully operated and been fully available to users through an open, peer-
reviewed proposal system since December 2006. In this presentation we will discuss the
present performance of the HFIR GP-SANS instrument, its available sample environment,
gaining access to the instrument through the general user program (and what kind of support
to expect), and our plans for upgrades to both sample environment and the instrument itself.
07.16.6

VIVALDI – the thermal-neutron Laue diffractometer for physics, chemistry and

materials science at the Institut Laue-Langevin
1,2 1 2
Garry McIntyre , Marie-Hélène Lemée-Cailleau , Clive Wilkinson
1 2
Institut Laue-Langevin, Grenoble, France, Department of Chemistry, University of Durham,
Durham, United Kingdom

The first eight years of operation of VIVALDI (Very-Intense Vertical-Axis Laue Diffractometer)
[1] at the ILL have been a resounding success, producing spectacular neutron Laue
diffraction patterns and exciting new science at a furious pace, with gains in data collection
rate over conventional neutron diffractometers of up to 100 fold. In just a few hours VIVALDI
has yielded detailed atomic structural information on large organic molecules [2], shown 2-D
magnetic ordering in astounding clarity [3], revealed new magnetic structures [4], allowed full
data collection from small crystals inside anvil pressure cells [5], followed variations of bond
lengths with temperature in fine detail [6], detected the multiple adsorption sites of hydrogen
in a metal-organic framework [7], and traced the kinetics of the continuous photo-induced
transition in a spin-crossover compound [8].

With an intense and broad incident thermal-neutron waveband, a large-solid-angle image-

plate detector, and fully automated readout and temperature variation, VIVALDI has proven to
3
be especially well suited to small crystals (0.1 mm is the norm!), rapid chemical
crystallography, reciprocal-space surveys, and studies of structural and magnetic phase
transitions. One copy of VIVALDI, called KOALA, is already in successful operation on the
OPAL reactor in Australia, and another, IMAGINE, is planned for HFIR at ORNL.

We briefly describe the unique technical aspects of VIVALDI and review the scientific
highlights of recent years to show why neutron Laue diffraction at a reactor may be the
technique of choice to solve your special crystallographic problem.

[1] G.J. McIntyre et al, Physica B 385-386 (2006) 1055-1058.

[2] M. Yousufuddin et al, J. Am. Chem. Soc. 130 (2008) 3888-3891.

[3] E.M.-L. Chung et al, J. Phys.: Condens. Matter 16 (2004) 7837-7852.

[4] P. Schobinger-Papamantellos et al, J. Phys.: Condens. Matter 16 (2004) 6569-6578.

[5] G.J. McIntyre et al, J. Phys.: Condens. Matter, 17 (2005) S3017-S3024.

[6] C. Dobe et al, J. Am. Chem. Soc. 126 (2004) 16639-16652.

[7] E.C. Spencer et al. Chem Comm. (2006) 278-280.

[8] F. Varret et al, Z. Krist. 223 (2008) 250-258.

07.16.7

MacCHESS: The Macromolecular Diffraction Facility at the Cornell High-Energy

Synchrotron Source

Richard Gillilan, Chae Un Kim, Ulrich Englich, Dave Schuller, Irina Kriksunov, Bill Miller, Scott
Smith, Mike Cook, Qingqui Huang, Søren Nielsen, Marian Szebenyi

Cornell University, Ithaca, NY, United States

MacCHESS is an NIH Research Resource at the Cornell High-Energy Synchrotron Source in

Upstate New York dedicated to providing researchers in the biological sciences with access
to state-of-the-art facilities for x-ray diffraction. As a synchrotron source located on an
academic campus, access to the facility is open to a very broad range of users by submitting
a simple one-page online express-mode proposal. During running periods, requests for time
are generally filled rapidly.

Of the five beamlines available to users, A1 and F1 support the majority of protein
crystallography. F1, with its large-area Q270 detector, finely-collimated beam, long sample-to-
detector distance, and BSL2 status is popular for large unit cell and virus work. A1 station is
comparable to F1 but is tuned just above the selenium edge and may be used for SAD data
collection. Automated sample mounting (based on ALS designs) is supported at F1 and is
currently being designed for A1.

Single-bounce monocapillary x-ray focusing optics developed at CHESS are routinely

available at all crystallography stations on request when small (20 micron), higher-flux beams
are desirable. F3 station provides a unique narrow band-pass (0.2%) multilayer system
compatible with low-energy SAD crystallography. F2 station, originally a tunable MAD
crystallography station, is in the process of being rebuilt as a high-flux multilayer-equipped
beamline for high-throughput biological small-angle x-ray scattering (BioSAXS). MacCHESS
also supports BioSAXS users part time on the CHESS G1 line, a high-flux (1.5% bandpass)
multilayer system.

MacCHESS currently provides the only high-pressure protein crystallography facility

available for routine use. Protein crystals flash cooled at high pressure sometimes show
significantly improved diffraction quality over those cooled at ambient pressure. High pressure
conditions can also shift binding equilibria of ligands, improving crystallographic occupancy
and may be valuable in trapping intermediates. MacCHESS engages in a wide range of
collaborative research and teaching activities and encourages proposals for unusual
experiments that would otherwise be difficult to do at dedicated crystallography beamlines
where equipment cannot easily be reconfigured.
 A

T-003

Five-Dimensional Crystallography
1 2 2
Marius Schmidt , Tim Graber , Robert Henning ,
2
Vukica Srajer
1
University of Wisconsin-Milwaukee, Milwaukee, WI,
2
United States, BioCARS, The University of Chicago,
B Chicago, IL, United States

A method for determining a comprehensive chemical

Right singular vectors from a
singular value decomposition of
two complete time series of time-
resolved crystallographic data at
o 0
(A) 20 C and (B) 30 C
exhibit measurable differences for the two temperatures, hence demonstrating that five-
dimensional crystallography is feasible.
kinetic mechanism for macromolecular reactions is
presented. The method is based on five-dimensional
crystallography [1], where in addition to space and
time, temperature is also taken into consideration and
an analysis based on singular value decomposition [2]
is applied. We present first results of such a time-
resolved crystallographic study. Temperature
dependent time-resolved X-ray diffraction
measurements were conducted on the newly
upgraded BioCARS 14-ID-B beamline at the
Advanced Photon Source. The measurements aimed
at elucidating a comprehensive kinetic mechanism of
the photocycle of the photoactive yellow protein.
Comprehensive time-series of crystallographic data
o o
were collected at two temperatures, 20 C and 30 C.
Relaxation times of extracted from these time-series

[1] Schmidt, Graber, Henning, Srajer (2010) Five-Dimensional Crystallography, Acta Cryst A,
66, 198-206.

[2] Schmidt, Rajagopal., Ren, Moffat (2003) Appli-cation of Singular Value Decomposition to
the Analysis of Time-Resolved Macromolecular X-ray Data. Biophys. J. 84 2112-2129
AW.02

Roles of Carbon-Oxygen Hydrogen Bonds in Methyl Group Recognition and Catalysis

in Protein Lysine Methyltransferases and Demethylases

Raymond Trievel

University of Michigan, Ann Arbor, MI, United States

Protein lysine methylation has emerged as a prevalent post-translational modification

associated with numerous biological functions. In eukaryotes, site-specific methylation of
lysine residues occurs in histones, transcription factors, ribosomal subunits, chromatin
modifying enzymes, and other protein substrates. Methylation of these targets has been
implicated in diverse functions, including gene regulation, DNA damage response, protein
turnover, and genome stability. The methylation status of protein lysine residues is
dynamically regulated through the concerted activities of lysine methyltransferases (KMTs)
and lysine demethylases (KDMs) and is biologically important because both the site and
degree of methylation are critical for intermolecular recognition in cellular signalling pathways.

Structural and functional studies of KMTs and KDMs have revealed key insights into their
respective substrate specificities and catalytic mechanisms. In the course of studying the
mechanisms of these enzymes, we have identified short-range interactions between the
methyllysine methyl groups and active site oxygen atoms that are indicative of carbon-oxygen
hydrogen bonds. This unusual type of hydrogen bonding can occur when a carbon atom and
its hydrogens are polarized by an adjacent covalently bonded heteroatom that enables
hydrogen bonding with a nearby oxygen atom. In KMTs, carbon-oxygen hydrogen bonds
facilitate the alignment of the methyllysine substrate for multiple methyl transfer reactions,
contributing to the product specificities of these enzymes. Similarly, hydrogen bonding to
methyllysine substrates within the active sites of KDMs promotes demethylation and defines
the methylation state specificities of these enzymes. These findings prompted us to examine
whether carbon-oxygen hydrogen bonding represents a general mechanism by which polar
methyl groups are recognized in biology. A survey of the PDB has revealed numerous
examples of these interactions in the structures of different classes of methyltransferases and
demethylases, highlighting the widespread nature of these hydrogen bonds in methyl group
coordination and catalysis.
02.02.1

Structural Characterization of Active Site Residues involved in Proton Transfer in

Catalysis by Carbonic Anhydrase II.

Dayne West, Rose Mikulski, Katherine Sippel, Balendu Avvaru, Seungjin Jang, Chingkuang
Tu, David Silverman, Robert McKenna

University of Florida, Gainesville, FL, 32610, United States

The reaction of physiological significance catalyzed by carbonic anhydrase (CA) is the

hydration of carbon dioxide. This catalysis involves an attack on CO2 by zinc-bound hydroxide
followed by a rate-limiting proton transfer from the active site to bulk solution to regenerate
the zinc-bound hydroxide. In human CA II, the intramolecular proton transfer residue H64
accepts protons from the zinc-bound water through a network of active-site hydrogen-bonded
6 -1
waters at a turnover rate as great as 10 s and transfers them to solution. The unifying goal
of this study is to expand our use of the CAs to understand such rate-limiting and long-range
proton transfer steps in a way that can be extended to other proteins. The current goal is to
understand the proton transfers in HCA II. We will present both structural and kinetic studies
on the site directed mutants of HCAII: N62Q/N67Q, N62L/N67L, H64A/N62L/N67L,
Y7N/N62L/N67L, and Y7F/N62Q. The structure-function studies of these mutations, when
taken together, provide information on the importance of specific residues for proton transfer.
These results provide a range of catalytic activities, geometries, and active-site environments.
18
Stopped-flow spectrophotometry, O exchange between CO2 and water measured by mass
spectrometry, and solvent H/D isotope effects will be used to investigate rate constants for
intramolecular proton transfer. Crystal structures of important mutants and the observed
structural alterations will be discussed and correlated to the influence of distances, location,
and environment on the rate of proton transfer and on CO2 binding at the active site.
02.02.2

Modeling Diffuse Scattering in a 1D Disordered Crystal Using a Probabilistic Layer

Stacking Model
1,2 3,4 4 1 1
Tara Michels-Clark , Hans-Beat Buergi , Jurg Hauser , Christina Hoffmann , Vickie Lynch
1 2
Oak Ridge National Laborator, Oak Ridge, TN, United States, University of Tennessee,
3 4
Knoxville, TN, United States, University of Zurich, Zurich, Switzerland, University of Bern,
Bern, Switzerland

We are reporting on a new approach for total scattering single crystal X-ray and neutron
pattern analysis. Highly sensitive area detectors are now able to measure complete diffraction
patterns from single crystal samples displaying local disorder. In many such cases interesting
functional responses are dependent on the disordered rather than the ordered part of the
structure. To elucidate the intricate interplay between local structure and overall structure
quantitatively, new computational methods are needed. We are using a combination of Monte
Carlo and Differential Evolution modeling techniques to simulate disorder and analyze diffuse
scattering. Modeling diffuse scattering from modulated disordered crystal structures
quantitatively proves to be a complex task with numerous optimization parameters.

We have studied tris(bicyclo[2.1.1]hexeno)benzene, which exhibits hexagonal symmetry and

1D streaks of diffuse scattering along the hexagonal axis. It is composed of ordered layers of
planar molecules stacked along the c-axis. The model takes into account layer-to-layer
interactions up to the fifth layer. Initially a four-layer model was developed to interpret the
diffuse intensity of fourteen of reciprocal lattice with four probabilistic stacking parameters [1].
The intensity distribution along c* (L) is given by
2
I ( L) 2 M F S ( L)

– — –W ˜ X–TcosU Lcos˜ Y cos

S L
L V cos 2 L —
2 L ˜ Z cos3 L —

F is the Fourier transform of a single P 6 2m layer of tris(bicyclo[2.1.1]hexeno)benzene

molecules in the unit mesh a,b with thickness c that corresponding to four layers. S(L) is the
interference function; its coefficients T, U, V, W, X, Y, and Z depend on the stacking
probabilities and M is the number of layers. The probabilites have been optimized utilizing a
differential evolutionary algorithm and parallel computing. Results of the four layer model of
tris(bicylco[2.1.1]hexeno)-benzene will be presented comparing calculated intensities I(L) to
experimental intensities. A five-layer model, which is expected to improve the model
intensities, will also be presented.

[1] H. B. Burgi, Marc Hostettler, H. Birkedal and D. Schwarzenbach, Z. Kristallography 2005,

220, 1066-1075.

This research is supported by UT Battelle, LLC under Contract No. DE-AC05-00OR22725 for
the U.S. Department of Energy, Office of Science and by a Swiss National Science
Foundation Sinergia Grant
02.02.3

Disrupting a Bacterial Enzyme Alleviates Cancer Drug Toxicity.

1 2 1 3 1 2
Bret Wallace , Hongwei Wang , Kimberly Lane , John Scott , Jillian Orans , Ja Seol Koo ,
1 3 2 1
Christian Jobin , Li-An Yeh , Sridhar Mani , Matthew Redinbo
1 2
University of North Carolina at Chapel Hill, Chapel Hill, NC, United States, Albert Einstein
3
College of Medicine, Bronx, NY, United States, North Carolina Central University, BRITE,
Durham, NY, United States

We show for the first time that a bacterial enzyme in human microbial symbiotes can be
selectively inhibited to improve chemotherapeutic tolerance. The dose-limiting side effect of
the colon anticancer chemotherapeutic CPT-11 is intense diarrhea caused by the GI
reactivation of a primary drug metabolite. The enzymes responsible are bacterial ™ -
glucuronidases present in the symbiotic gut microbiota. With the goal of reducing this side
effect, we sought to selectively eliminate GI-specific drug reactivation without killing the
commensal bacteria essential for human health. We identify potent bacterial ™-glucuronidase
inhibitors through high-throughput screening that have no effect on the mammalian
orthologue. Using crystal structures of E. coli ™-glucuronidase complexed with lead inhibitors,
we demonstrate that selectivity is based on a loop unique to the bacterial ™-glucuronidases.
We further establish that inhibitors are effective against the enzyme target in living bacterial
strains grown under aerobic or anaerobic conditions, but do not kill the host bacteria or harm
human colonic epithelial cells. Finally, we show that oral administration of an inhibitor lead
protects mice from CPT-11-induced toxicity. Thus, undesirable enzyme activities in essential
microbial symbiotes can be controlled to enhance chemotherapeutic efficacy.
02.02.4

Crystal structure of the engineered allosteric TEM-1 beta-lactamase fused to MBP

reveals insights into the mechanism of its intended and non-intended allosteric
regulation
1 2 2 1
Wei Ke , Jing Liang , Marc Ostermeier , Focco van den Akker
1 2
Case Western Reserve University, Cleveland, OH, United States, Johns Hopkins University,
Baltimore, Maryland, United States

RG13 is a molecular switch created by recombining the genes coding for the Escherichia coli
maltose binding protein (MBP) and the TEM-1 beta-lactamase (BLA). The beta-lactam
hydrolysis activity of RG13 is positively regulated by the binding of maltose. In addition,
RG13’s beta-lactam hydrolysis activity is switched off by zinc ion, which is serendipitously
found to be a non-competitive inhibitor against RG13. To answer the question how the activity
of TEM-1 beta-lactamase is heterotropically regulated by maltose and zinc ion, we
determined the crystal structure of RG13 in complex with zinc. Our structure reveals that
while the positions of catalytic S70, conserved active site residues Y105, S130, N132, E166
and N170 are relatively unchanged, the critical beta3-strand, one of the active site walls of
Class A beta-lactamase, is completely stripped away from BLA active site and instead
behaves as one of the loops connecting MBP domain and BLA domain. Consequently, new
residues move close to the active site especially an Arg residue which sticks into the active
site and makes hydrogen bonds with S130 and N132 and has the active site fully blocked. By
careful examination of the position occupied by Zn ion, we conclude that this inhibition
conformation is induced and stabilized by the zinc ion which is positioned near BLA active site
and by residues from both MBP domain and BLA domain. Together with previous NMR
insights of RG13 in complex with maltose, we propose that the maltose activation mechanism
for RG13 entails that, in the absence of zinc, maltose binding causes a subtle lengthening of
the linker region allowing the active site region to fully restore itself to render itself catalytically
active. Molecular dynamics data will be presented to complement these crystallographic
studies.
02.02.5

Resolution of pharmaceuticals via crystallization on chemically modified surfaces

Pranoti Navare, John MacDonald

Worcester Polytechnic Institute, Worcester, MA, United States

Chirality is important in development of pharmaceuticals because more than half of

commercial drugs are chiral, and the respective enantiomers often differ in their
pharmacological and toxicological effects. The FDA requires that enantiomers be separated
when racemic mixtures are produced during synthesis. Crystallization has been used to
separate enantiomers in select cases where the two molecules resolve spontaneously,
forming conglomerates. Unfortunately, crystallization energetically favors formation of racemic
crystals for 95% of chiral compounds. We are investigating resolution of chiral drugs via
crystallization on surfaces functionalized with chiral organic molecules as a means to bring
about enantioseparation. Two goals of this work are to determine (1) whether chiral surfaces
can act as templates that bias homochiral molecular aggregation at the surface, thereby
inducing nucleation of conglomerates over racemic crystals, and (2) whether chiral templating
can be used to control selective nucleation of one enantiomer leading to high enantiomeric
excess. To demonstrate chiral discrimination on surfaces, we are investigating crystallization
of the racemic 3-phenyllactic acid and N-acetylleucine on self-assembled monolayers (SAMs)
of D- and L-cysteine and their derivatives. Important findings show that one enantiomer can
be enriched over the other in up to 75% enantiomeric excess, and that the major enantiomer
can be selected by switching the chirality of the SAM. We also observed oriented crystal
growth and morphological changes indicating that diastereomeric interactions with chiral
groups on the surface promotes both homochiral aggregation and face-selective nucleation of
crystals of one enantiomer.
02.02.6

Evolution of Ligand Specificity in Steroid Hormone Nuclear Receptors

Jennifer Colucci, Eric Ortlund

Emory University, Atlanta, GA, United States

Steroid hormone nuclear receptors (SRs) are ligand-regulated transcription factors

that play critical roles in inflammation, development, and cancer progression. The SR family
of proteins include the estrogen, androgen, mineralocorticoid, glucocorticoid, and
progesterone receptors, which are activated by the endogenous hormones estrogen,
testosterone, aldosterone, cortisol, and progesterone, respectively. Each member of the SR
family is finely tuned to respond to its cognate hormone with high specificity. The SR family
arose from a single evolutionary precursor that was activated by the 3-hydroxy steroid
estrogen. Throughout a series of gene duplications, these proteins lost the ability to bind and
be activated by 3-hydroxy steroids, and gained the ability to bind and be activated by 3-keto
steroids. Currently, the molecular basis for hormone recognition is not well understood; this
is illuminated by the fact that most pharmaceuticals targeting these receptors are cross-
reactive.

In order to understand the molecular basis behind this functional switch, the ancestral
precursor to the modern-day androgen, progesterone, mineralocorticoid, and glucocorticoid
receptor, Ancestral Steroid Receptor 2 (AncSR2) was resurrected. This protein represents
the first protein to have lost 3-hydroxy sensitivity and gained 3-keto specificity. We took a
two-pronged approach to understanding ligand recognition and activation, using both
biochemical and structural techniques.

Using the crystal structure of AncSR2 in complex with progesterone, we made

mutations to alter the specificity of the receptor from 3-keto steroids (progesterone) to 3-
hydroxy steroids (estrogen). We showed that with three amino acid mutations (Q353E,
M384L, M387L) in the ligand binding pocket of AncSR2 that we can reverse steroid
specificity from progesterone to estrogen, a feat that remained elusive using modern
proteins.

We then wanted to investigate the mechanism behind this functional switch. We

hypothesized that the basis of the increased activation by estrogen in the Q353E M384L
M387L mutant was due to the increased receptor-ligand complex stability. Chemical
denaturation studies showed that the Q353E substitution conferred stability to the receptor.
The M384L and M387L substitutions decreased stability, but increased specificity. Thus, in
the forward evolutionary trajectory, this receptor gained specificity while losing stability.
02.02.7

Can Proper Selection of Data Reduction Program Optimize the Anomalous Signal for a
Particular Set of Diffraction Images?

James Swindell, John Rose, B.C. Wang

University of Georgia, Athens Ga, United States

To address the question as stated in the title, we have used two data sets from the same
crystal collected at SER-CAT and processed with HKL2000, d*TREK, XDS, MOSFLM and
PROTEUM2. We then compared the final results in terms of Rsym, I/σ I, heavy atoms
position(s), anomalous signal contribution, and phase comparison. The use of the data
processing programs and a comparison of results will be described and discussed.

Work supported by SER-CAT Member Institutions, University of Georgia Research

Foundation and Georgia Research Alliance.
02.02.8

Structural basis for nucleoside sugar discrimination in a thermostable DNA

polymerase I

Weina Wang, Eugene Wu, Lorena Beese

Duke University, Durham, NC, United States

DNA polymerases carry out accurate template-directed DNA synthesis by discriminating

hundreds to thousands fold in favor of 2’-deoxyribonucleotide triphosphate (dNTP) compared
to ribonucleotide triphosphate (rNTP). The two substrates differ only by the addition of 2’
hydroxyl group to the ribose sugar moiety, which makes it a larger substrate. Given the large
number of nucleotides synthesized during genome replication and greater concentration of
rNTP than dNTP in the cell, misincorporated ribonucleotides may compose the largest group
of DNA lesions in the genome. Thus, it is critical to understand how DNA polymerases
efficiently select the correct nucleoside sugar to incorporate. Site-directed mutagenesis and
kinetic characterization have led to the identification of several residues involved in sugar
discrimination. Here, we present five crystal structures of a high fidelity DNA polymerase I (pol
I) large fragment from a thermostable strain of Bacillus stearothermophilus (Bacillus fragment,
BF) complexed with DNA primer-template and dNTP, rNTP or ddNTP prior to chemistry. In
addition, BF complexed with enzymatically incorporated rNTP or ddNTP into the DNA primer
strand were captured after catalysis inside the crystals. The seven structures were
determined at resolutions between 1.5 Å and 1.9 Å. Taken together, these structures increase
our understanding of molecular mechanisms underlining sugar discrimination by DNA
polymerase.
01.02.1

Structure and Mechanism of a Protein Disaggregating Machine

Francis Tsai, Bernhard Sielaff, Jungsoon Lee, Sukyeong Lee

Baylor College of Medicine, Houston, Texas, United States

ClpB and Hsp104 are ring-forming AAA+ machines that recognize aggregated proteins as
substrates, and remodel them in an ATP-dependent manner. The ability to disaggregate
stress-damaged proteins is strictly dependent on the M-domain that is a hallmark of the
ClpB/Hsp104 family. While the three-dimensional structures of ClpB and Hsp104 have been
determined, the location of the M-domain is controversial and its exact function remains
unclear.

To provide an explanation for the observed structural differences, we determined the three-
dimensional structure of Hsp104 using a multi-pronged structural and biochemical approach.
The mechanistic implication of our new structural insight will be discussed at this meeting.
01.02.2

Structural Genomics Approach to Macromolecular Assemblies and Motors

Andrzej Joachimiak

Argonne National Laboratory, Argonne, IL, United States

Protein Structure Initiative (PSI) efforts are driven by genome sequence information and a
focus on protein families that have no structural information available. Initially, at the Midwest
Center for Structural Genomics (MCSG), as high-throughput methods were being adopted,
refined and optimized, the majority of targets included single chains of small and medium size
proteins. However, as technology matured and salvage pathways were implemented, MCSG
targets included proteins that are considered “challenging,” e.g. larger, multi-meric and, more
recently, multi-chain complexes as well as protein-NA complexes. Some of the selected
protein families represent very distant sequence relatives of macromolecular assemblies and
predicted macromolecular motors. These assemblies perform a variety of cellular roles,
although some may have still poorly understood function. These novel structures provide
new information and allow us to extract a common denominator, a function signature, or in
some cases, an alternative solution for a particular functional requirement. These structures
help to correlate function with structure and sequences, and generalize it for a set of proteins
in a large family that share the same or similar function. This information can also help to
transfer function from one protein to others, thereby, expanding functional coverage. The PSI
is a discovery-based program that contributes to studies of the co-evolution of protein
structure and function. It provides a wealth of ideas, concepts and understanding of
mechanisms for the acquisition of novel biological function and the evolution of biological
systems. Several examples will be provided, including tetrahedral protease, metal ion
transporter, and a portal protein.

This work was supported by NIH Grant GM074942 and by the U.S. DOE, OBER contract DE-
AC02-06CH11357.
01.02.3

Structure, mechanism, and regulation of a critical spliceosomal ATPase and helicase

Brr2
1 1 2 3 1 4
Lingdi Zhang , Tao Xu , Corina Maeder , Laura-Oana Bud , James Shanks , Jay Nix ,
2 3 1
Christine Guthrie , Jeffrey Pleiss , Rui Zhao
1 2
University of Colorado Denver, Aurora, CO, United States, University of California San
3
Francisco, San Francisco, CA, United States, Cornell University, Ithaca, NY, United States,
4
Lawrence Berkeley National Laboratory, Berkeley, CA, United States

Pre-mRNA splicing is an essential step in gene expression of all eukaryotes. Splicing of

introns is carried out through two transesterification reactions catalyzed by the spliceosome, a
huge RNA/protein complex composed of five snRNAs and over 100 protein factors.
Structural and functional analyses are essential for our understanding of the molecular
mechanism of pre-mRNA splicing. However, the structure and function of many spliceosomal
components remain elusive. Among these are several DExD/H-box RNA helicases that play
critical roles in the assembly and activation of the spliceosome. Brr2 is a large (>2,000 amino
acids) and essential helicase in the spliceosome. It is responsible for U4/U6 unwinding, a
critical step in spliceosomal activation. Brr2 has a unique domain structure, which contains
an N-terminal domain (NTD, ~500 amino acids) and two tandem sets of a helicase domain
followed by a Sec63 domain with unknown structure and function. Previous mutagenesis
studies have shown that the first helicase domain is responsible for the ATPase and helicase
activity of Brr2, but the function of all other domains is unclear. To understand the structure
and function of this critical spliceosomal helicase, we recently determined the crystal structure
of the second Sec63 domain, which unexpectedly resembles domains 4 and 5 of DNA
helicase Hel308. In addition, the helicase domain upstream of Sec63 has clear sequence
similarity with domains 1-3 of Hel308. We, therefore, hypothesize that Brr2 is composed of
an N-terminal domain and two consecutive Hel308-like modules, providing our first glimpse of
the overall structure of this unique spliceosomal ATPase and helicase. The structural
similarity between Brr2 and Hel308 suggests a helicase mechanism for Brr2 that is consistent
with our mutagenesis and biochemical studies. This mechanism is different from many
DEAD-box RNA helicases and is likely responsible for Brr2’s unique ability to unwind the
highly stable U4/U6 duplex. Furthermore, we demonstrated that the second Hel308 module
of Brr2 interacts with Prp8 and Snu114 in vitro and in vivo, potentially serving as a mediator
for the regulation of Brr2’s activity by Prp8 (an essential splicing factor known to stimulate
Brr2’s helicase activity). This is the first example of a helicase-like module serving as a major
protein-interacting domain. We further demonstrated that the C-terminal region of Prp8
(Prp8-CTR) facilitates the binding of the Brr2/Prp8-CTR complex to U4/U6, suggesting a
potential role of Prp8-CTR as an auxiliary substrate binding and specificity domain for Brr2.
In addition, we found that Brr2 NTD is required for yeast viability and have dissected the
specific regions of NTD that are essential. We are currently investigating the specific function
of this domain using a combination of genetic, biochemical, and structural approaches. Our
results in general have important implications for the mechanism and regulation of Brr2’s
activity.
01.02.4

Structure of a Membrane-associated AAA Machine

1 2 2 2 2
Sukyeong Lee , Steffen Augustin , Takashi Tatsuta , Florian Gerdes , Thomas Langer ,
1
Francis Tsai
1 2
Baylor College of Medicine, Houston, Texas, United States, University of Cologne, Cologne,
Germany

AAA proteases are membrane-associated AAA machines that mediate the processing and
turnover of soluble and membrane embedded proteins. We have determined the 12-Å
resolution cryoEM structure of a detergent solubilized, full-length, hetero-oligomeric m-AAA
protease hexamer, which reveals for the first time the structures of the twelve transmembrane
spanning segments and six intermembrane space domains. Our fitted structure offers an
explanation how m-AAA proteases dislocate and degrade membrane integral proteins, and
provides the stereo-chemical framework for further biochemical and mechanistic studies. The
structure and mechanism of m-AAA proteases will be discussed.
01.02.5

Crystal structure of the mammalian cytosolic chaperonin CCT in complex with tubulin
at 5.5 Å resolution
1 2 3 1 2
Ines G. Munoz , Hugo Yebenes , Min Zhou , Pablo Mesa , Marina Serna , Elisabeth
1 3 2 1
Bragado-Nilsson , Carol V. Robinson , Jose M. Valpuesta , Guillermo Montoya
1 2
Spanish National Cancer Research Centre (CNIO), Madrid, Spain, Centro Nacional de
3
Biotecnologia, Madrid, Spain, University of Cambridge, Cambridge, United Kingdom

Protein folding in the cell is assisted by a large group of proteins termed molecular
chaperones, one of the most important members being the chaperonins or Hsp60s (Heat
Shock Proteins of 60 kDa). The eukaryotic cytosolic chaperonin CCT (chaperonin containing
TCP-1, also know as TRiC) is the most complex of all chaperonins, an oligomeric structure
built by two identical rings, each composed of single copies of eight different 60kDa subunits
called α , β , γ , ζ , ε , δ , θ and η . This macromolecular complex of 1 MDa has crucial relevance in
several essential biological processes, emerging as a key molecule due to its role in the
folding of many important molecules including actin, α -, β - and γ -tubulins. Here we present
the crystal structure of this protein machine in complex with tubulin. The structure of CCT
trapping tubulin provides information about the molecular mechanism by which this
macromolecular complex aids the tubulin folding process. The structure reveals the presence
of one tubulin molecule in each CCT octamer showing the three-dimensional organisation of
the different components in the presence of a substrate and providing new important insights
into the function of this molecular chaperonin.
01.02.6

The structure of the phage T4 DNA packaging motor suggests a mechanism dependent
on electrostatic forces
1 2 2 2 1
Siyang Sun , Kiran Kondabagil , Bonnie Draper , Tanfis Alam , Valorie Bowman , Zhihong
2 2 1 1 2
Zhang , Shylaja Hegde , Andrei Fokine , Michael Rossmann , Venigalla Rao
1 2
Purdue University, West Lafayette, IN, United States, The Catholic University of America,
Washington, DC, United States

Viral genomes are packaged into "procapsids" by powerful molecular motors. The crystal
structure of the DNA packaging motor protein, gene product 17 (gp17), of bacteriophage T4
has now been determined. The structure consists of an N-terminal ATPase domain, which
provides energy for compacting DNA, and a C-terminal nuclease domain, which terminates
packaging. The structure showed that the function of the C-terminal domain also includes the
translocation of the genome into the procapsid. The two domains are in close contact in the
crystal structure, representing a "tensed state." A cryo-electron microscopy reconstruction of
the T4 procapsid complexed with gp17 showed that the packaging motor is a pentamer and
that the domains within each monomer are spatially separated, representing a "relaxed state."
These structures suggested a mechanism, supported by mutational and other data, in which
electrostatic forces drive the DNA packaging by alternating between tensed and relaxed
states. Similar mechanisms may occur in other molecular motors.
01.03.1

The Use of Longer X-ray Wavelengths in Macromolecular Crystallography

Manfred S. Weiss, B.-C. Wang

1 2
Helmholtz-Zentrum Berlin, Berlin, Germany, University of Georgia, Athens, United States

The use of longer X-ray wavelengths (š= 1.5-3.0 Å) in macromolecular crystallography has
over the past few years almost become a routine tool for phase determination using the
anomalous signal derived from the natively present sulfur and/or phosphorus atoms. Since
the obtainable signal is very small, the experiment has to be conducted with great care. The
challenges of the method are reviewed as well as some recent developments. Also, a survey
about successful experiments carried out a beamlines around the world will be given.
01.03.2

Development of a beamline for low energy (4 keV) SAD experiments

Soichi Wakatsuki, Naohiro Matsugaki, Yusuke Yamada, Leonard Chavas, Masato Kawasaki,
Ryuichi Kato, Noriyuki Igarashi, Masahiko Hiraki

Structural Biology Research Center, Photon Factory, Institute of Materials Structure Science,
High Energy Accelerator Research Organization (KEK), Tsukuba, Ibaraki, Japan

SAD (Single-wavelength Anomalous Dispersion) phasing with sulfur or phosphor atoms is

currently one of the most attractive methods to solve macromolecular crystal structures. The
method is particularly important for a range of macromolecules for which heavy atom or
selenomethionine derivative crystals are difficult to prepare. The Structure Biology Research
Center at the Photon Factory, Tsukuba, Japan, is developing a new beamline dedicated to
low energy (long wavelength) SAD experiments as part of the national project "Targeted
Proteins Research Program (TPRP)". It is a nationwide structural biology effort which includes
35 target-oriented structural biology projects and 10 R&D projects in protein production,
chemical library, structural analysis and bioinformatics. Each of the 35 target-oriented
structural proteomics projects aims to solve structures of challenging targets in close
collaboration with groups in cell biology, biochemistry, bioengineering, pharmacology, or
medicine. Two synchrotron radiation facilities, SPring-8 and Photon Factory undertook to
build two complementary micro-focus beamlines, and in collaboration with Hokkaido Univ.,
Osaka Univ., and Kyoto Univ. are developing techniques to facilitate user access and
experiments at both synchrotron sites. At SPring-8 a micro-beam beamline, BL32XU, has
been constructed to provide highly intense 1 micron by 1 micron beam while the Photon
Factory has built a micro-focus beamline, BL1A, optimized for low energy SAD experiments.
At PF BL1A beamline, an intense low energy beam at around 4 keV is provided using the first
harmonic of an in-vacuum mini-gap undulator source to enhance anomalous signals from light
atoms. A cryo freezing device designed by Isao Tanaka's group (Hokkaido Univ.) is available
for capillary-top mounting of cryo-cooled crystals on site. The beamline optics consists of a
cryo-cooled channel-cut monochromator and bimorph KB mirrors. The experimental hutch is
equipped with a diffractometer specifically designed to minimize the attenuation of the lower
energy beam and scattering by mother liquor and the air around the crystal. The beamline
has been commissioned in spring 2010, and the user operation due to start in May. Initial
results will be reported from small crystals and de novo structure determination using the 4
keV X-ray beam.
01.03.3

Solving structures with in-house long wavelength radiation

Cheng Yang, Jim W. Pflugrath, Joseph Ferrara

Rigaku American Corporation, Woodland, Texas, United States

Recent advances in technologies for phasing with in-house long wavelength radiation has
made it straightforward to solve protein structures by utilizing the intrinsic sulfur atoms and/or
added selenium atoms in those proteins. In-house chromium Kα radiation (2.29Å) currently is
the longest wavelength X-ray radiation which can be routinely used for data collection. This
wavelength is capable of doubling the anomalous signal of many intrinsic elements or oft-
used heavy atoms for derivatization compared to Cu radiation. For example, the contribution
to the anomalous terms of sulfur and selenium atom doubles to 1.14 and 2.28, electron
equivalents, respectively.

This report relates successful examples of protein structures solved by using chromium sulfur
or selenium-SAD phasing. Some of these proteins were either difficult to synthesize in
selenomethionine-substituted form or to produce more crystals. Furthermore, anomalous
scattering collected with an in-house Cr X-ray radiation source can be used to determine
protein bound ion positions, verify ligand orientation and help structure tracing. A properly
collected Cr data set can also be used refine the protein structure. These results demonstrate
that Cr radiation has become a routine phasing approach in the crystallographer’ s toolkit. Cr
Kα makes it possible to solve a crystal structure with native crystals only or before
synchrotron data collection when selenomethionine-substituted protein is available. This
method improves the productivity of macromolecular structure determination, usage of the
synchrotron beam time, and high throughput structure determination.
01.03.4

Crystallographic structure determination of an iron uptake regulatory protein (FeoA) by

sulfur SAD in C2 space group
1 1 2 2 3
Joseph Ng , Ronny Hughes , Yang Li , Zhi-Jie Liu , Bi-Cheng Wang
1 2
University of Alabama, Huntsville, Huntsville, AL, United States, Institue of Biophysics,
3
Beijing, China, University of Georgia, Athens, Athens, Georgia, United States

The recombinant iron uptake regulatory protein (FeoA) derived from Thermococcus
thioreducens was overexpressed in E. coli, purified and crystallized in the C2 monoclinic
space group with unit cell parameters of a=93.8Å, b=68.4Å, c=68.97Å and β =132.67o. The
asymmetric unit contained 4 molecules in which each monomer contained 6 sulfurs all
contributed by methionines within 76 amino acids. The overall number of sulfur atoms within
the asymmetric unit was 24. In the interest of obtaining phases by Sulfur Single-Wavelength
Anomalous Diffraction (SAD), synchrotron X-ray data collection was performed at the SER-
CAT ID22 (Argonne National Lab, Chicago, IL). A 360-frame data set was taken using 1.9Å
wavelength X-ray radiation with each frame consisting of a 1º wedge of data using a MAR300
CCD detector. Reflections extending out to 2.0 Å were recorded with an overall data
redundancy of 22.5. The positions of 24 sulfur anomalous scattering atoms were identified
and consequently phases were determined and refined. The final refined structure of FeOA
was determined to 2.0Å with Rwork and Rfree to be 17.6 and 22.8 respectively. We show
the anomalous signal of the sulfurs in FeOA crystals can be detected and used for phasing
with sufficient sulfur content in monoclinic space group.
01.03.5

Getting the most out of weak phase information

George Sheldrick

University of Goettingen, Goettingen, Germany

Longer wavelengths can achieve an increase in the ratio of anomalous to normal diffraction
but some systematic errors are also enhanced at longer wavelength so careful data scaling is
required and the attainable resolution is reduced. One approach to combat these problems is
to combine the long wavelength data with data collected to higher resolution at a short
wavelength, if possible from the same crystal. This also helps to reduce problems with the
1,2
location of heavy atoms using the program SHELXD when the low angle data are
incomplete or contain errors. In the case of SAD phasing using naturally occurring sulphur
3
atoms or the iodine atoms in a partially occupied sticky magic triangle as anomalous
scatterers, the known geometry of the disulfide units or the equilateral triangle of iodine atoms
4
should be useful in enhancing the heavy atom search. When the phase information derived
from the heavy atoms is very weak, iterative density modification and tracing of the peptide
2,5
backbone with the program SHELXE is proving particularly effective at bootstrapping onto
an interpretable structure; in borderline cases this procedure often requires several iterations
before it locks in and traces most of the peptide backbone.

1. Schneider, T. R. & Sheldrick, G. M. (2002). Acta Cryst. D58, 1772-1779.

2. Sheldrick, G. M. (2008). Acta Cryst. A64, 112-122.

3. Beck, T., da Cunha, C. E. & Sheldrick, G. M. (2009). Acta Cryst. F65, 1068-1070.

4. Debreczeni, J. É., Girmann, B., Zeeck, A., Krätzner, R. & Sheldrick, G. M. (2003). Acta
Cryst. D59, 2125-2132.

5. Sheldrick, G. M. (2010). Acta Cryst. D66, 479-485.

01.03.6

In-House SAD: experiences and structures.

Elspeth Garman, Edward Lowe

University of Oxford, Oxford, United Kingdom

Over the last four years we have used our in house copper high brilliance X-ray
source (Bruker Microstar generator) in conjunction with a kappa goniometer stage and a
SMART 6000 CCD detector to great advantage for SAD phasing. Key structure solution
determinations have been carried out using this method, utilising the high speed and
sensitivity of the detector. These include the determination of a number of novel structures
and complexes, such as domains CBEGF9-HYB2-CBEGF10 of fibrillin-1 in a calcium
saturated form using a combination of molecular replacement and SAD methods which made
use of anomalous signal from bound Ca ions and intrinsic sulphurs [1].
8 9
Other SAD structures solved have been the F1 F1 type 1 domains of human
fibronectin (S-SAD) [2], the sulphur oxidising protein, SoxB (SAD from a bound Mn ion) [3],
human HIF prolyl-hydroxylase, PHD2, in complex with an iodinated inhibitor (I-SAD) [4], the
vonWillebrand factor (vWF) domain of the proteasomal component Pus1 (S-SAD) [5] and
human PHYHD1 protein (SAD using iron and iodine derivatives).

Systematic experiments have been carried out to optimise our current S-SAD data
collection strategies, using crystals of insulin, lysozyme and glucose isomerase as test
systems. In the case of glucose isomerase (GI) this involved phasing the structure of the 388
residue protein using the signal from only 8 sulphur atoms. The absence of manganese in the
EDTA treated GI crystal was confirmed by microPIXE (proton induced X-ray emission) [6].

The results of these experiments and current protocols will be discussed.

References:

[1] Jensen, SA et al (2009) Structure, 17, 759-768

[2] Erat, M.C. et al (2009) PNAS, 106, 4195-4200

[3] Sauvé, V et al (2009) J Biol Chem, 284, 21707-21718

[4] McDonough, MA et al (2006) PNAS, 103, 9814-9819

[5] Riedinger, C. et al (in press)

[6] Garman, EF & Grime, GW Progress in Biophysics and Molecular Biology (2005) 89/2,
173-205.
01.03.7

The upgrade programme of the ESRF and a new structure.

daniele de sanctis, Christoph MUELLER-DIECKMANN

ESRF, Grenoble, France

Structural Biologists are tackling ever more ambitious projects, for example more complex
membrane proteins and larger macromolecular assemblies. Such systems often show
considerable inter- and intra- crystal variation in diffraction quality. Sample evaluation prior to
data collection, already widespread in Macromolecular Crystallography (MX), will thus
become more crucial as will data collection facilities optimised for the collection of diffraction
data at long wavelengths or from crystals that are very small and/or diffract to low resolution
(i.e. dmin > 5 Å).

As part of the Upgrade Programme of the European Synchrotron Radiation Facility (ESRF;
http://www.esrf.fr/AboutUs/Upgrade) its Structural Biology Group will develop a unique
resource, based on 2nd generation automation, designed to maximise the chances of a
successful conclusion to MX experiments. The hub of this resource will be a sample
evaluation and sorting facility, MASSIF. The most suitable crystals from which to collect data
will be distributed from this hub to upgraded MX data collection facilities that also form an
integral part of the ESRF’s upgrade plans.

The ESRF’s Structural Biology Group’s upgrade plans will be presented, with particular
emphasis on a refurbished ID29 which will be optimised to enable data collection from
crystals of macromolecules using X-rays of lower energies (E = 5 keV, › = 2.5 Å). Recent
successes in the solution of a macromolecular crystal structure exploiting only the small
anomalous signal from sulphur atoms innate to protein amino acid sequences will be also be
described.
01.03.8

The MDS Approach: A Data Collection Strategy for Routine Sulfur Phasing and Other
Applications that Require High-Quality and Higher Resolution Data

1 1 1 1 1 1 1 1
B.C. Wang , Z.-J. Liu , L. Chen , W. Zhou , H. Xu , H. Zhang , J.T. Swindell II , J.P. Rose ,
1 1 1 2
G. Rosenbaum , Z.-Q. Fu , J. Chrzas , M. Benning
1 2
University of Georgia, Athens, Georgia, United States, Bruker AXS, Madison, Wisconsin,
United States

A fundamental question facing the crystallographer is "For a given X-ray dose, what is the
best strategy for collecting a data set from a crystal that will increase structure solvability?" A
common answer to this question is to increase the exposure time for each diffraction image,
so that we may better “visualize” the recorded reflections to get better data.
Interestingly, we have found that a significantly better data set may be produced for a given
crystal and X-ray dose by using shorter exposures (i.e. reduced X-ray dose) and collecting
the complete data set multiple times such that the total X-ray dose the crystal receives
remains the same. This is the MDS (Multiple-Data-Set) approach.
In a recent case, a total of 8,600 images were collected using the MDS approach, from a
single protein crystal with an estimated (visual inspection) diffraction limit of around 2.5Å,
using a copper home X-ray source. The resulting merged and scaled data set gave good
statistics to better than 2.0Å resolution. Thus, this unlikely candidate crystal for sulfur phasing
has now produced a S-SAD structure from its MDS data collected with a highly sensitive
detector.
Both the theoretical and practical aspects of the MDS method as well as the future
perspectives of the MDS approach will be discussed
Work supported by NIH, SER-CAT, University of Georgia Research Foundation, Georgia
Research Alliance, and Bruker AXS
* Current Address: Institute of Biophycs, Chinese Academy of Sciences, Beijing, China
† Current Address: The College of Life Sciences, Nankai University, Tianjin, China.
02.03.1

Larry R. Falvello

University of Zaragoza - C.S.I.C., Zaragoza, Spain

Methods for obtaining undistorted views of the reciprocal lattice are described. The principle
of de Jong and Bouman for diffraction photography is presented along with a brief description
of a camera based on it. The Buerger precession method is described, including the role of
the de Jong - Bouman principle in the design of the "Mach 2" precession camera. The use of
undistorted reciprocal lattice views has been facilitated in recent years by the routine
availability of extensive digital data from contemporary diffractometers, together with the
advent of rapid pixel harvesting and layer reconstruction techniques. These are summarized,
and applications of these reciprocal lattice views to topical crystallographic problems are also
presented.
02.03.2

When the Structure is Difficult Look at the Whole Diffraction Pattern

Carolyn Brock

Department of Chemistry, University of Kentucky, Lexington, KY, United States

Synthetic “precession photos”, i.e., reciprocal-lattice (or, RL) slices, can be calculated easily
from the pixel values in the many frames typically collected with a CCD diffractometer. A
quick look at these images, which show the non-Bragg as well as the Bragg scattering, often
reveals the reason for a problem refinement.

If a second set of Bragg reflections is observed then the crystal is not single. Structured
diffuse scattering between the Bragg peaks indicates short- or medium-range order. If the
Bragg peaks are very elongated then the crystal either had a large mosaic spread or moved
during data collection. If the Bragg peaks seem a little large and seem to have surprisingly
similar intensities then there is reason to suspect pseudo-merohedral twinning. If large
numbers of Bragg peaks are very weak the structure may be modulated.

RL slices for a number of problem structures from our lab will be shown and discussed.
02.03.3

Electron-density maps. What insight can we gain from them?

Jenny Glusker

Fox Chase Cancer Center, Philadelphia, United States

Electron-density maps result from attempts to represent the material in a crystal that has
scattered X rays and given a diffraction pattern. In a similar way, neutron diffraction gives a
picture of nuclear density. However, since the first diffraction patterns were obtained in 1912,
it has been clear that experimentally measured intensities of diffracted beams will not give all
the information necessary for an electron-density map. The relative phases of the diffracted
beams have been lost because an appropriate X-ray lens is not currently available. This is
the “phase problem,” the most important part of a course in X ray crystallography. It is solved
in a variety of ways to give a picture of the scattering matter. Analyses of the preliminary
maps obtained, however, need care and attention to detail. Methods for finding good atomic
coordinates and identifying missing atoms, or movements or disorder of atoms, will be
described. Finally omit maps, deformation-density maps, nuclear-density maps and
anomalous dispersion effects will be discussed. The primary experimental data are the
intensities, directions, and orders of diffraction of the diffracted beams. Their relationship to
the required electron-density or nuclear-density maps is the subject of this talk.

I thank the National Institutes of Health (CA-10925 and CA-06927) for support through the
years.
02.03.4

Topotaxy HOWTO: Following Two-Phase Solid-State Reactions in the 1970s and Today
with a Modern Diffractometer

Aaron R. Gell, Shai R. Posner, Chun-Hsing Chen, Bruce M. Foxman

Brandeis University, Waltham, MA, United States

Topotactic reactions are single crystal-to-single crystal reactions (SCSCRs) where the
daughter phase(s) is(are) aligned in an explicit three-dimensional fashion with respect to the
1
mother phase. The largest class of SCSCRs are class T1, where the space group of the
original unit cell does not change, and the lattice parameters change smoothly over time. The
presentation will review a complex T1 example, followed by a number of T2 (two-phase)
cases. In the 1970’s, topotaxy was established using film techniques; today, the elegant
2
approach of Gougoutas and coworkers has been neglected, and it is time for a return to an
era where the alignment of mother and daughter crystals is firmly established in every
experiment. IT’S EASY! Using a modern diffractometer, a single crystal reaction may be
followed, and the alignment established in a relatively simple fashion: truly, only a few
3
minutes’ work. Examples from the 1970s will be compared with modern-day measurements,
4
and protocols for the future set out. Examples will be taken from the ancient and new work
on the topotactic transformations of coordination compounds as well as polymorphic phase
transformations.

1. Lotgering, F. K. J. Inorg. Nucl. Chem. 1959, 9, 113-23; Dent Glasser, L. S.; Glasser,
F. P.; Taylor, H. F. W. Quart. Rev. 1962, 16, 343-60; Shannon, R. D.; Rossi, R. C.
Nature 1964, 202, 1000-1001.

2. Gougoutas, J. Z. Isr. J. Chem. 1972, 10, 395-407.

3. http://www.nonius.com/cad4/manuals/user/chapter08.html

Cheng, K.; Foxman, B. M. J. Am. Chem. Soc. 1977, 99, 8102-8103.

02.03.5

Structure Analysis. Why do we always ..........?

David Watkin

Oxford University, Oxford, United Kingdom

In the last decade X-ray Crystal Structure Analysis has become increasingly automated, with
a growing number of crystallographic decisions being made automatically by the software. In
addition, a number of crystallographic Cook Books have been published which give
instructions for how to deal with more complicated tasks. Taken together, these aids enable
chemists with quite modest training to undertake routine analyses.

While this broadening of the crystallographic community is almost certainly a good thing
(because it makes X-ray crystallography more accessible), it carries the risk of misuse of the
technique, and potentially the failure of analyses which, with a little more experience, could
have been resolved. The very high levels of automation now available reduce the exposure
of novices to the actual processes of structure analysis. This leaves them ill-equipped to deal
with unusual situations.

This talk will aim to explain why we always do a selection of things, such as aim at high
completeness, aim at high redundancy, find a fancy weighting scheme for the refinement etc.
Once one knows why these things are done, one is in a position to judge when things might
be done differently, when the "rules" can be broken. A "hand-waving" (as opposed to a deep
mathematical) understanding of the physical background to structure analysis enables one to
carry out analyses more effectively.
02.03.6

The Structure of Fe3(CO)12 Revisited

1 2 2 2
Charles Campana , Ilia Guzei , Evgueni Mednikov , Lawrence Dahl
1 2
Bruker AXS Inc., Madison, Wisconsin, United States, Department of Chemistry, University
of Wisconsin - Madison, Madison, Wisconsin, United States

Triiron dodecacarbonyl was one of the first metal carbonyl clusters synthesized. The
molecular structure of the Fe3(CO)12 molecule has been a subject of interest since the 1950’s.
Dahl and Rundle proposed that Fe3(CO)12 consists of a triangle of iron atoms surrounded by
12 CO ligands. Ten of the CO ligands are terminal and two span an Fe---Fe edge. By
contrast, Ru3(CO)12 and Os3(CO)12 adopt D3h-symmetric structures, wherein all 12 CO ligands
are terminally bound to the metals. However, the elucidation of the detailed structure of
Fe3(CO)12 has proved to be a challenging crystallographic problem.

The original crystal structure by Wei and Dahl was done at room temperature, using data
collected on Weisenberg and precession cameras with visual estimation of intensities.
Subsequent datasets were collected with scintillation detectors on Syntex P-1 and Nonius
CAD-4 diffractometers. We report on the latest datasets collected with a Mo IœS micro-focus
source and a two-dimensional CCD detector at 100K.

As new instrumentation and software have become available, it is now possible to re-examine
this old structure to obtain a much more precise structure. The details of the modelling of this
disordered structure will be discussed.
03.01.1

New techniques in fiber diffraction illuminate the supramolecular organization of the

mammalian Extracelluar Matrix
1,3 1 3 4 1
Joseph Orgel , Olga Antipova , Irit Sagi , Yujia Xu , Sandra Bishnoi , Aruna
1 2
Kalyanasundaram , James San Antonio
1 2
Illinois Institute of Technology, Chicago, IL, United States, Orthovita, Inc, Malvern, PA,
3 4
United States, Weizmann Institute of Science, Rehovot, Israel, Hunter College, New York,
NY, United States

The fibrous collagens are the fundamental constituents of the Extracellular Matrix (ECM) of
animals, forming the structural basis of all known mammalian connective tissues and organ
systems (1). Yet, despite the fundamental biological importance of collagen, many of us are
perplexed by the complexity of the assemblies that the collagens form. This is particularly true
at what may be the most significant aspect of collagen structure from a cellular point of view,
at the intermediate sub-fibrillar and at fibril surface levels (i.e. collagens molecular packing)
where many important biological processes occur in growth, development and disease (1, 2).
These include but are not limited to: fibrillogenesis, tissue remolding and in forming the
scaffolding upon which organ systems, bones, cartilage, etc., i.e. the animal body, are built
upon. Clearly, obtaining an unambiguous and contextualized visualization of collagen
molecules would be of significant value to the scientific community. We have recently
determined the structure of the type I collagen microfibril (3) and fibril (2) at the molecular
level from whole intact rat-tail tendons and produced an initial one (and now two) dimensional
structure for type II collagen from lamprey notochord (4). Using these data, it is possible to
map the amino acid chemistry, ligand binding data and other observations onto the defining
shape modality of the fibrillar collagen ECM. In so doing, we have been able to propose the
first fibrillar based mechanism of collagenolysis and provide a number of illuminating
observations regarding other collagen fibril - ligand interactions involving cell adhesion,
hemostasis and matrix organization (1-5). Here we expand on these observations using new
technical developments in fiber diffraction and complimentary methods such as TEM and
AFM.

1. Sweeney S et al. (2008. J Biol Chem 283:21187-97; 2. Perumal S, Antipova O, Orgel J

(2008). Proc Natl Acad Sci U S A 105:2824-9; 3. Orgel J et al., (2006) Proc Natl Acad Sci U S
A 103:9001-5; 4. Antipova, A., and Orgel, J.P.R.O. (2010) In Press, Journal of Biological
Chemistry; 5. Joseph P.R.O. Orgel et al., (2009) Public Library of Science ONE. 4(9), e7028.
03.01.2

Using Micro- and Nano-beams for Scanning Diffraction Experiments on Fibrous

Materials

Manfred Burghammer, Sebastian Schoeder, Richard Davies, Aurelien Gourrier, Christian

Riekel

European Synchrotron Radiation Facility, Grenoble, France

X-ray diffraction on single fibres, instead of multi-fibre assemblies, has been enabled more
than a decade ago when highly focused X-rays became available at third generation
synchrotron radiation sources. Today the structure of fibrous materials can be probed with
unprecedented spatial resolution using x-ray beams in the range of one micron down to a few
hundred nanometres. Evidently, there is a high interest to combine such measurements with
in situ sample environments e.g. allowing to control temperature, humidity, and mechanical
deformation. However, in order to benefit from these possibilities challenging problems like
mechanical stability issues, high throughput data analysis, and, above all, radiation damage
issues have to be addressed.

The Instrumentation dedicated to high resolution scanning diffraction experiments at the

ESRF Microfocus Beamline (ID13) will be presented. Examples from the fields of life science
and polymer research will be discussed.
03.01.3

The Molecular Basis for Stretch Activation in Insect Flight Muscle

1 2 1
Thomas Irving , RJ Edwards , Michael Reedy
1 2
Illinois Institute of Technology, Chicago, IL, United States, Duke University Medical School,
Durham NC, United States

Deciphering the molecular mechanism of muscle contraction and its regulation using small-
angle fiber diffraction has been a driving force for the development of x-ray technology since
the beginning of this field in the 1950’s. In fact, the very first diffraction experiment using
synchrotron radiation (on anything) was done on the indirect flight muscle (IFM) of the giant
water bug Lethocerus indicus nearly 40 years ago in Hamburg by Rosenbaum, Witz and
Holmes. This muscle system is an attractive model system because of its high degree of
crystallinity which allows more detailed structural analysis than muscles from mammals.
When slightly calcium-activated glycerinated Lethocerus insect flight muscle (IFM) can be
2+
mechanically stretch-activated at constant [Ca ] to give a delayed active rise to peak force, a
response typical of asynchronous IFM that allows efficient flight. The structural mechanism
underlying stretch activation has long been elusive. We oscillated demembranated muscle at
2 HZ, and collected continuous 8ms time frame movies of x-ray fiber patterns, using a Pilatus
100K pixel array detector, reflecting structural changes within the muscle as it performs
oscillatory work, much as it would in the living insect. The results showed that stretch induces
movement of tropomyosin alternatively allowing attachment of crossbridges with every wing
beat. This movement appears to ce due to persistent mechanical linkages between the thick
and the thin filaments at the level of the troponins on the thin filament. In addition, we found
clear evidence for twisting and untwisting of the myosin containing thick filaments as the
muscle is stretched and released. During stretch, this would result in placing myosin heads
closer to their “target zones”, i.e. stereospecific binding sites on the actin-containing thin
filament. This, combined with stretch induced recruitment of myosin heads may finally provide
an explanation for stretch activation in these muscles. Since stretch activation is also a
feature of human cardiac muscle, this may have relevance to understanding this feature of
cardiac function. Supported by NIH 5R37AR014317 and RR08630.
03.01.4

Fibre diffraction flexes its muscles.

Carlo Knupp

Cardiff University, UK, United Kingdom

Muscle sarcomeres, the repeating structural units in muscle, contain two sets of fibrous
proteins namely actin and myosin filaments. In the sarcomere these filaments run parallel to
each other. The interaction of the actin filaments with globular domains projecting from the
myosin filament backbone (myosin heads) makes the two sets of filaments slide past each
other. This process ultimately result in muscle contraction, but the exact details of how force
and movement are produced at the molecular level in the sarcomere are still unknown.

X-ray fibre diffraction offers the possibility of probing the molecular events involved in muscle
contraction by recording and analysing time-resolved diffraction patterns from live contracting
muscles. Here we will review some of the computational techniques that can be used to
understand the changes in the diffraction patterns (including interference effects arising
between adjacent myosin head arrays) and to reconstruct, at different times within the
contracting cycle, the overall arrangement of interacting myosin and actin filaments in the
sarcomere.
ORMOPMT

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k\¢\„¡ ¡K?hmK?t‹ ¡ ?r \ ¡

Carrageenans represent a special class of biopolymers extracted from marine algae and are
utilized in food applications as thickeners, gelling agents, syneresis inhibitors and binders, to
name a few. They are recognized as GRAS materials and FDA approved the food usage.
Their anti-coagulant, anti-therapeutic, anti-tumor and anti-HIV activities resulted in the
pharmaceutical utilization as well. Further, their regular intake in human diet is proven to
reduce blood cholesterol and lipid levels. These hydrocolloids are made up of a disaccharide
galactan backbone with variable amounts of sulfation at different hydroxyl positions.
Depending on their source of extraction, presence or absence of a sulfate ester as well as
anhydro group fifteen carrageenans are known to date. However, only kappa-, iota- and
lambda-carrageenans have so far been exploited industrially and are subjected to extensive
x-ray and rheological studies for understanding their structure-function relationships. Insights
about structural organization and solution properties are very much needed for delineating
their functional behavior, especially in the presence of other biopolymers.

In this set, iota-carrageenan is well characterized with precise atomic details and insights
about the pertinent roles of metal ions in stabilizing packing structure. On the other hand, no
such detailed information exists for kappa-carrageenan. Among the two known hydrogen
bond disrupters urea and guanidine hydrochloride (GH), the later is reported to enhance
gelation in kappa-carrageenan. Our recent fiber diffraction results on guanidinium salt of iota-
and kappa-carrageenan yield interesting observations. The pattern of iota-carrageenan,
having one sulfate group on each sugar residue of its disaccharide repeat, contains three well
defined layer lines within 4.3 Å resolution. The polymer prefers a three fold, parallel, half-
staggered double helical structure. The stability of the helix is gained through two interchain
O-6H¨¨¨O-2 and O-2H¨¨¨O-5 hydrogen bonds, and the peripheral sulfate groups promote
interhelix association via cations and ordered water molecules. In contrast, kappa-
carrageenan, with only one sulfate group per disaccharide repeat, diffracts to generate six
layers lines within the same resolution period, suggesting a novel molecular structure.
Analysis reveals that non-half-staggered as well as anti-parallel double helical arrangements
are viable options. In the second case, the two chains are held together by three strong
interchain O-6H¨¨¨O-6, O-2H¨¨¨O-2 and O-2H¨¨¨O-2 hydrogen bonds two more than the former
indicating the preferred molecular structure for kappa-carrageenan might be an anti-parallel
double helix. Although both of them have similar backbones, absence of a sulfate group on
alternate residues seems to bestow additional freedom and a novel molecular assembly to
kappa-carrageen chains. These results unequivocally attest to the observed differences in
solution behavior between the two carrageenans.
03.01.6

Structural Insights into the Interactions of Amines with Cellulose

1 2 3 4,1
B. Leif Hanson , Yoshiharu Nishiyama , Masahisa Wada , Paul Langan
1 2
University of Toledo, Toledo, OH, United States, Joseph Fourier University of Grenoble,
3 4
Grenoble, France, University of Tokyo, Tokyo, Japan, Los Alamos National Laboratory, Los
Alamos, NM, United States

We have studied structural changes during the treatment of highly crystalline microfibers of
Cladophora cellulose with ethylenediamine (EDA) using time-resolved X-ray microprobe
diffraction methods. EDA molecules penetrate the cellulose crystals converting the naturally
occurring crystalline cellulose I phase to a crystalline complex of cellulose with EDA, called
EDA-cellulose I. The (200) direction of cellulose I is resistant to EDA penetration, with EDA
penetrating most effectively at the hydrophilic edges of the hydrogen bonded sheets of
cellulose chains. Most of the cellulose chains in the initial crystals of cellulose I are
incorporated into crystals of EDA-cellulose I, and there is no evidence of any gradual
structural transition from cellulose I to EDA-cellulose I involving a continuously changing
intermediate phase. Instead a rapid transition to EDA-cellulose I occurs in regions of the
microfibrils that have been penetrated by EDA. The size of the emerging EDA-cellulose I
crystals are limited to about half the size of the cellulose I crystals, due to strains introduced
by the penetrating EDA molecules.
03.01.7

Fiber diffraction, auto-florescence, and Raman microscopy studies of the time-

dependent solubilisation of lignocellulosic biomass with ionic liquids.
2,1 2 2 2 1 1
Paul Langan , Kirk Rector , Marcel Lucas , Greg Wagner , Indira Samayam , Lefi Hanson ,
1 3,1 4
Indira Samayam , Yoshiharu Nishiyama , Masahida Wada
1 2
University of Toledo, Toledo, OH, United States, Los Alamos National Laboratory, Los
3 4
Alamos, NM, United States, CNRS, Grenoble, France, University of Tokyo, Tokyo, Japan

Lignocellulosic biomass, the fibrous material derived from plant cell walls, is a potential clean
and renewable, non-food feedstock for future biorefineries. The components of lignocellulosic
biomass can serve as a source of carbon based feedstock for fuel and chemical production in
much the same way as crude oil serves as the carbon feedstock in petrochemical refineries.
In particular, the sugars derived from the cellulosic and hemicellulosic portions of biomass
can be converted to biofuels or other value added products using current technologies. The
deconstruction of lignocellulosic biomass into simple sugars constitutes a core barrier for
producing products from the sugar platform. Cellulose, a fibrous material biogenerated from
the linear polymer poly(1-4) b-D glucan, is difficult to break down into glucose monomers
because of its crystalline nature. Furthermore, cellulose fibers are encrusted in the branched
heterogeneous polymers of hemicellulose and lignin. Lignin adds structural rigidity to plant
cell walls, but it also protects the cellulosic component from hydrolyzing enzymes that can
release glucose. Recently, Ionic Liquids (ILs) have been investigated by several groups as a
promising new approach for the pretreatment of lignocellulosic biomass so that its complex
architecture can be disrupted, thereby making it accessible to water and enzymes for an
accelerated conversion to glucose. We have shown that pretreatment with ILs not only
disrupts the plant cell wall and separates its cellulosic, hemicellulosic, and lignin components,
but it also disrupts the crystallinity of cellulose making it more rapidly digestible by hydrolyzing
enzymes. Some advantages of this approach are that it is non-derivatizing, that it does not
produce fermentation inhibitors, and that it is amenable for "easy recovery" of the ILs
employed in the pretreatment. However, further optimization of the use of ILs as a
pretreatment will require a detailed chemical-level understanding of the composition and
structure of lignocellulosic biomass and the time dependant impact of pretreatment. Because
of the compositional and structural complexity of the cell wall and its interaction with ILs, no
one experimental technique can provide this understanding. Therefore we have taken the
approach of combining several experimental techniques including fiber diffraction, auto-
flourescence and Raman microscopy to provide a program that can be used to characterize
the intact plant cell wall at multiple length scales. The experimental techniques were chosen
not only for the complementarity of the information that they provide, but also because they
are non-invasive and non-destructive and therefore avoid interference from traditional
staining, embedding, and processing chemicals that can sometimes alter the material under
study. We will report on the application of our experimental program to investigate the time
dependent solubilization of lignocellulosic biomass from poplar trees by the IL 1-n-ethyl-3-
methylimidazolium acetate.
07.13.1

The Magic of Spin Ice and Emergence of Magnetic Monopoles

Jeffrey Lynn

National Institute of Standards and Technology, Gaithersburg, Md 20899, United States

Cubic pyrochlores such as Dy2Ti2O7 have a structure where the Dy sites form a corner-
sharing tetrahedral network, which is the prototype geometry to frustrate the magnetic
interactions that can lead to novel magnetic ground states that are fundamentally different
than conventional long range magnetic order. For this material the magnetic anisotropy of the
3+
Dy spins requires them to point along a local <111> axis, such that on each tetrahedron the
moment can only point into the center, or in the opposite direction. The ground state then
turns out to be where two spins on each tetrahedron point inward, and two point outward. But
you don’t know which two are in and which two are out, giving rise to six equivalent
configurations for any particular tetrahedron, and a macroscopic degeneracy and finite
entropy for the ground state. This “two-in two-out” description of the spin system is identical
to the proton configurational disorder for hexagonal ice discussed by Pauling [1], inspiring the
name “spin ice” [2]. Recently it was realized that the magnetic excitations of spin ice can then
be described as magnetic monopoles [3], and such excitations have now been observed
experimentally [4]. We will describe the nature of magnetic frustration, spin-ice, and how
magnetic monopoles emerge from the ground state of this very special magnetic material.

[1] L. Pauling, J. Am. Chem. Soc. 57, 2680 (1935).

[2] J. S. Gardner, M. J. P. Gingras, and J. E. Greedan, Rev. Mod. Phys. 82, 53 (2010).

[3] C. Castelnovo, R. Moessner, & S. L. Sondhi, Nature 451, 42 (2008).

[4] H. Kadowaki, N. Doi, Y. Aoki, Y. Tabata, T.J. Sato, J. W. Lynn, K. Matsuhira, and Z. Hiroi,
J. Phys. Soc. Japan 78, 103706 (2009).
07.13.2

Two ACuCl3(H2O) Compounds Containing Unusual Aggregation of the Copper(II)

Complexes.

Marcus Bond

Southeast Missouri State University, Cape Girardeau, MOI, United States

The structures of (N,N-dimethylpiperidinium)CuCl3(H2O (I)):monoclinic P21/c, a = 10.1958(3)

Å, b = 7.6080(3) Å, c = 16.3281(5) Å, β = 102.927(2)°, V = 1234.46(7) Å 3, Z=4; and of
(1,2,3-trimethylpyridinium)CuCl3(H2O) (II): monoclinic C2/m, a = 15.6948(6) Å, b =
9.2431(4) Å, c = 8.5103(3) Å, β = 103.488(2)°, V = 1200.53(8) Å 3, Z = 4; are reported. The
-
structures consist of distorted tetrahedral (CuCl3(H2O) complexes separated by organic
cations. The distorted geometry of the complexes precludes the stacking found for similar
compounds with quasi-planar complexes, in which copper(II) ions of one complex complete
...
their coordination spheres by forming long, semi-coordinate Cu Cl bonds to neighboring
complexes. In these compounds these complexes aggregate in more unusual ways. In (I)
the water molecule of a complex forms hydrogen bonds to chloride ions on neighboring
complexes to organize the complexes in stacks in which the water molecules are sequestered
into parallel channels within the crystal. In (II) the complexes form pairs in which Cu...Cl
semi-coordinate bond are shared to form weakly bound dicopper complexes which then
aggregate into sheets via O-H...Cl hydrogen bonding.
07.13.3

Unexpected and Incomplete Super-Triangles and Rectangles: When Designed Self-

Assembly Does Not Go According to Plan.
1 1 2 1
Louise Dawe , Konstantin Shuvaev , Santokh Tandon , Laurence Thompson
1 2
Memorial University, St. John's, Newfoundland, Canada, Kent State University at Salem,
Salem, Ohio, United States

The targeted synthesis of [4x4]M16 grids involves designing polytopic ligands with coordination
pocket composition that is complimentary to a metal ion’s coordination ‘ algorithm’. These
grids exhibit interesting, and well characterized magnetic properties, and have attracted
[1]
much attention for their possible nanotechnological applications. Incorporating large,
quinoline or bipyridine-type ligand end-pieces has been targeted, in an attempt to achieve
long-range ordering, and extended magnetic communication between grids. While several
[1-4]
synthetic successes with M = Mn, Cu, and Co have been achieved, subtle changes in
[5]
ligand design has also lead to unanticipated results. A Mn(II)12 rectangle, and a Cu(II)11
super-triangle, both with available coordination pockets will be presented, and their
intermolecular ©-© interactions highlighted.

1. Dawe, L.N., Shuvaev, K.S., Thompson, L.K., Inorg. Chem., 2009, 48, 3323-3341.

2. Dey, S.K., Abedin, T.S.M., Dawe, L.N., et al., Inorg. Chem., 2007, 46, 7767-7781.

3. Dey, S.K., Thompson, L.K., Dawe, L.N., Chem. Commun., 2006, 4967-4969.

4. Dawe, L.N., Thompson, L.K. Angew. Chem., 2007, 46, 7440-7444.

5. Shuvaev, K.V., Tandon, S.S., Dawe, L.N., Thompson, L.K., Chem. Comm., 2010,
Submitted.
07.13.4

Crystal Structures of BaSrR4Zn2O10, R = La, Nd, Sm, Eu

1 2
James Kaduk , Winnie Wong-Ng
1 2
Poly Crystallography Inc, Naperville IL, United States, NIST, Gaithersburg MD, United
States

The crystal structures of BaSrR4Zn2O10 have been determined using synchrotron powder
diffraction data collected at 11-BM at the Advanced Photon Source at Argonne National
Laboratory. BaR2ZnO5 crystallize in the tetragonal space group I4/mcm for R = La and Nd,
and in orthorhombic Pbnm for smaller lanthanides.

All four structures are tetragonal. The Nd, Sm, and Eu compounds crystallize in I4/mcm,
and the trends in lattice parameters follow those of the BaR2ZnO5. Four weak peaks in the
BaSrLa4Zn2O10 pattern could not be attributed to any impurity phase, but corresponded to the
102, 122, 124, and 128 peaks of the tetragonal unit cell. The body centering condition was
thus violated, and the true space group is P4/ncc.

The 10-coordinate sites in all four compounds are occupied by Ba and Sr. The 8-
coordinate sites are occupied primarily by the lanthanide cations, but small concentrations of
Sr are apparently present at this site for R = La and Nd. The tetrahedral Zn sites are similar
in all four compounds.

The larger size of the R = La cell apparently results in movement of the Ba/Sr off the
center of the 10-coordinate cage. The largest errors in the Rietveld plots are in the tails of the
peaks, and the errors are not random. Density functional quantum chemical geometry
optimizations for Ba-only and Sr-only structures help understand local microstructural
distortions and the potential effects of local compositional variations.
07.13.5

Charge density study of NaI3O8: Origin of the non linear optics properties
1,2 1,2 1,2
CLAUDE LECOMTE , CHRISTIAN JELSCH , EMMANUEL WENGER , ISABELLE
3 3 4,1
GAUTIER LUNEAU , YAN SUFFREN , PIERRE FERTEY
1 2 3
NANCY UNIVERSITE, NANCY, France, CNRS UHP UMR7036, NANCY, France, CNRS
4
UPR 2940, GRENOBLE, France, SOLEILSYNCHROTRON, SACLAY, France

NaI3O8 is a promising material for infrared parametric generation [1]. It crystallises in

an acentric space group P-4 , prerequisite for quadratic nonlinear properties. NaI3O8 is among
the rare materials having a large domain of transparency from visible to the beginning of the
far IR (12.5 μ m) allowing applications in the atmospheric transparency windows. It is not
hygroscopic, has a good thermal stability (up to 350°C) and shows high optical damage
-2
thresholds on powder (4.2 GW.cm ). Moreover, NaI3O8 could be grown as millimetric single
crystal which is a key point to study its NLO properties and further for the development of
-
device systems. The crystal structure reveals a novel oxo anion, [I3O8] which is the first
polynuclear anion of pentavalent iodine. This anion is formed from the condensation of three
iodate anions in concentrated acidic solution, and contains three polarisable lone electron
pairs (LEP) favouring high nonlinear susceptibilities. In previous calculations of NLO
polarisabilities coefficients in iodate compounds, the contribution of LEP of iodine atom was
neglected, or has been considered to be equal to the I-O bond influence.

In order to establish relations between the crystal structure of NaI3O8, and its optical
properties, as done for KTiO(PO4) [2],a charge density study must be performed. Modeling
the electron density of inorganic materials containing heavy elements is very far from a
routine work: accurate absorption and extinction corrections are the key for a realistic
electron density modeling. Low temperature multiple wavelength diffraction data have been
collected on the CRISTAL beamline at SOLEIL from 30KeV to 8 KeV in order to extrapolate
the extinction correction to zero wavelength before collecting the 30KeV ultra high resolution
data set used for the electron density model [1] with the program suite MOPRO [1] This
communication will describe the preliminary electron density results concentrating on both
methodological aspects and NLO properties .

[1] D. Phanon & I. Gautier-Luneau, Angew. Chem. Int. Ed., 46, 8488-8491, 2007.
"Promising Material for Infrared Nonlinear Optics: NaI3O8 salt containing a New
-
Octaoxotriiodate(V) Anion formed from Condensation of [IO3] Iodate”[2] Hansen N K, Protas J
P and Marnier G; C. R. Acad. Sci., Paris 307, 475, 1988 “Structure atomique, densité
électronique et propriétés optiques non linéaires de KTiOPO4.” ; Hansen N K, Protas J P and
Marnier G; Acta Cryst., B47, 660-672, 1991. “The Electron Density Distribution in
KTiOPO4.”[3 C.Jelsch, B.Guillot, A.Lagoutte & C.Lecomte, J. Appl. Cryst., 38 , 38-54,2005. "
Advances in proteins and small molecules charge density refinement methods using software
MoPro"
07.13.6

A Non-Isostructural Series of Lanthanide complexes that puts the “Fun” in

Dysfunctional.

Christine Beavers, Guoxin Tian, Linfeng Rao

Lawrence Berkeley National Lab, Berkeley, CA, United States

In an attempt to observe the lanthanide contraction, a series of lanthanide complexes

(and the occasional actinide) was synthesized and crystallized. To the eager
crystallographer, these compounds seemed destined for the esteemed realm of “isostructural
series.” However this series has proved to be much more difficult than imagined, but also
much more complex and intriguing. Identical stoichiometry did not lead to identical structures.
Phase changes, twinning and a possible modulated structure all found their way into this
collection of complexes.

Asymmetric unit of Pr-TMDA with hydrogen sites omitted for clarity

07.13.7

Weird Structures A Moving Framework Structure, a P1 Compound with Unusual

Bonding Correlations and a Disappearing Molecule.
Abraham Clearfield, Aaron Celestian, Houston Perry, Paul Zhang

CUNY Queens College, New York, United States

A. Clearfield, Aaron Celestian, Houston Perry, and Paul Zhang Chemistry Department, Texas
A&M University College Station, TX 77842
The compound Na2Ti3O3(SiO4)•2H2O has a tunnel structure and is highly selective for Cs+.
It is able to remove cesium ions from highly alkaline solutions in the presence of 6M Na+ and
therefore is under consideration to remove the cesium from nuclear waste solutions. The
structure of this titanium silicate and to its Cs+ form have been determined from x-ray powder
data. However, treatment of the Cs+ phase with HCl fails to remove the cesium. In situ time
resolved x-ray and neutron diffraction revealed the mechanism of exchange which showed
that the framework can rotate to narrow the tunnel and change the shape of the tunnel to trap
the Cs+. A video of the movement of the framework will be displayed.
A second structure of a strange nature is the copper complex of 1,3,5-benzene
tris(methylphosphonic)acid and 4,4'-bipyridyl. This compound was refined in space group and
found to be a dimer of copper square pyramids linked into a supermolecular array. However,
there were indications that violations of centrosymmetric symmetry occurred in the placement
of the protons. Refinement in P1 resolved the symmetry problems. However it revealed an
unusual correlation between the positioning of oxygen atoms in a sixth (or octahedral)
position of the square pyramids and the Cu-O bond distances of the water molecules in the
apex position of the pyramids.
If time permits we will include the structure of a Zr PMIDA derivative that self exfoliates and
reconfigures as a function of pH.
T-006

EVIDENCE FOR CONFORMATIONAL CHANGES UPON

INHIBITOR BINDING IN SERINE RACEMASE

Myron Smith, Volker Mack, Andreas Ebneth, Isabel Moraes, Brunella Felicetti, Michael Wood,
Dorian Schonfeld, Owen Mather, Andrea Cesura, John Barker

Evotec UK Ltd, Oxfordshire, United Kingdom

Serine racemase is responsible for the synthesis of D-serine, an endogenous co-agonist for
N-methyl-D-aspartate receptor-type glutamate receptors (NMDARs). This pyridoxal 5’-
phosphate-dependent enzyme is involved both in the reversible conversion of L- to D-serine
and serine catabolism by α ,β -elimination of water, thereby regulating D-serine levels. Since
D-serine affects NMDAR signalling throughout the brain, serine racemase is a promising
target for the treatment of disorders related to NMDAR dysfunction. To elucidate the reaction
mechanism and provide a molecular basis for rational drug design the X-ray crystal structures
of human and rat serine racemase were determined at 1.5 Å and 2.1 Å resolution
respectively, and in the presence and absence of the orthosteric inhibitor malonate. The
structures revealed a fold typical of β -family PLP-enzymes, with both a large domain and a
flexible small domain associated into a symmetric dimer, and indicated a ligand-induced
rearrangement of the small domain that organises the active site for specific turnover of the
substrate.
T-009

Trapping and structurally characterizing true catalytic intermediates in a target enzyme

Ronald Viola

University of Toledo, Toledo, Ohio, United States

The biosynthetic pathway derived from aspartic acid is unique to plants and microorganisms,
producing four of the essential amino acids required for protein synthesis. In addition,
intermediates in this pathway are involved in bacterial cell wall cross-linking, sporulation in
Gram-positive bacteria, and quorum sensing in Gram-negative bacteria. ASA dehydrogenase,
a core enzyme in this pathway, has been targeted for inhibitor design and drug development.
Two proposed enzyme-bound intermediates in the catalytic cycle have been trapped and
structurally characterized. These structures have helped delineate the mechanism of this key
metabolic enzyme and provided guidance for selective inhibitor design. Structures have also
been determined for this target enzyme isolated from Gram-negative and Gram-positive
infectious bacteria and from a fungal species. The structural differences between these
mechanistically identical enzymes are being exploited for the development of species-specific
antibiotics.
T-012

A Joint X-ray and Neutron Diffraction Study on a Copper Protein Reveal the Role of
Protein Dynamics in Electron Transfer
1 2 3 4
Narayanasami Sukumar , Scott Mathews , Paul Langan , Victor Davidson
1
NE-CAT and Dept. of Chemistry and Chemical Biology, Cornell University, , Argonne
2
National Laboratory, Argonne, IL 60439, Dept. of Biochemistry and Molecular Biophysics,
3
Washington Univ. School of Medicine, St. Louis, MO 63110, , Bioscience Div., Los Alamos
4
National Laboratory, Los Alamos, NM 87545, Dept. of Biochemistry, Univ. of Mississippi
Medical Center, Jackson,MS 39216.

Amicyanin from Paracoccus denitrificans contains a single copper site and mediates the
electron transfer (ET) from methylamine dehydrogenase (MADH) to cytochrome C551i. The
protein has a molecular mass of about 12.5 kDa and folds as a β - sandwich, with nine β -
strands forming two mixed β -sheets. Though several X-ray crystallographic studies in
combination with spectroscopic and kinetic studies have been carried out on amicyanin, a
detailed understanding of its electronic properties has been hampered by lack of information
on the precise positions of hydrogens. In order to determine precisely the position of
hydrogen atoms, the extent of hydrogen/deuterium exchange (HDX), and the flexibility and
dynamic nature of the protein, a joint X-ray/neutron (XN) diffraction study was performed with
amicyanin.
The amide hydrogen atoms of ~86% residues including the residues that provide the copper
ligands are either partially or fully exchanged, indicating that the structure of amicyanin is
highly dynamic. An analysis on the crystal structure of the tertiary complex of MADH,
amicyanin, and cytochrome c-551i indicated that the likely point of inter-protein ET from
amicyanin to cytochrome c-551i is Glu31. Further ET analysis in solution yielded a value for
-1
electronic coupling, HAB of 0.3 cm which was unusually large, given the ET distance of ~23
Å. Subsequently a study of this same ET reaction in crystals of the protein complex yielded a
-4 -1
much slower rate and a much smaller value for HAB of 7.3 ´ 10 cm . The experimentally
determined reorganization energies for the reactions in solution and in the crystal state were
identical; only the HAB was affected. The dynamic motion of the residues during the ET was
not considered when the calculations were performed using X-ray crystal structures. In the
present XN structure, the dynamic nature of individual residues could be ascertained based
on HDX. The difference in the ET studies between solution and crystal state may be
explained by the finding in this present study that the amicyanin molecule, particularly the
portion of the protein through which ET occurs, is highly dynamic.
Despite this dynamic nature, all of the conventional H-bonds around ~8Å of copper site that
are predicted to occur in the X-ray structure are directly observed in the XN structure. A
careful analysis of N-H...X and C-H...X types of hydrogen bonds reveals that five of the C-
H....X bonds involve the copper ligand-His95 are absent in the reduced structure, which
undergoes a pH-induced conformation change in the reduced state. The results reveal
previously unknown role of these weaker C-H...X bonds as they, like conventional H-bonds,
collectively influence the structure, redox and electron transfer properties of amicyanin.
T-015

Crystallization and structure determination of a transient electron transfer complex

1 2 3 4
Miki Senda , Shigenobu Kimura , Tetsuo Ishida , Toshiya Senda
1 2
Japan Biological Informatics Consortium, Tokyo, Japan, Ibaraki University, Ibaraki, Japan,
3 4
Shiga University of Medical Science, Shiga, Japan, National Institute of Advanced Industrial
Science and Technology, Tokyo, Japan

Electron transfer complexes of redox proteins are characterized by weak affinity, transient
interaction, and redox-dependent affinity regulation. These characteristics are critical to fast
and efficient electron transfer. We have studied an electron-transfer system for a multi-
component dioxygenase, BphA, derived from Acidovorax sp. strain KKS102. BphA3 and
BphA4 are a Rieske-type [2Fe-2S] ferredoxin and an FAD-containing NADH-dependent
ferredoxin reductase, respectively (1, 2). To reveal the electron-transfer mechanism from
BphA4 to BphA3, crystal structure analysis of the BphA3-BphA4 complex is indispensable.
However, oxidized BphA3 and oxidized BphA4 hardly form a stable complex in solution,
because the Kd value for their interaction was too large, 294 ªM, to form a stable complex in
solution. The Kd value, however, changed significantly upon their reduction (Kd = 14.6 ªM);
BphA3 and BphA4 can form a stable complex under the reduced conditions. This complex
seems to resemble a productive complex for the electron transfer reaction. We have
developed an anaerobic chamber to crystallize the BphA3-BphA4 complex under anaerobic
conditions and have succeeded in crystallizing the BphA3-BphA4 complex (3). This system
has also been used to crystallize free BphA3 and free BphA4 in their reduced form. We have
so far determined the crystal structures of the BphA3-BphA4 complex (productive electron
transfer complex); free BphA4 in oxidized, hydroquinone, and semiquinone forms; and free
BphA3 in oxidized and reduced forms. Comparison of these crystal structures revealed that (i)
BphA4 undergoes conformational changes upon reduction and (ii) BphA4 shows
conformational changes while forming a complex with BphA3. Since these conformational
changes found in BphA4 are similar to one another, they seem to be essential to the
formation of an electron-transfer complex; they are likely to contribute to the formation of a
high-affinity binding site for BphA3 on BphA4 (1, 2). References: 1. Senda, T. et al., (2009).
Antioxid. Redox. Signal., 11, 1741-1766;

2. Senda, M. et al., (2007). J. Mol. Biol. 373, 382-400; 3. Senda, M. et al., (2007). Acta Cryst.
F63, 520-523.
T-018

Redox-dependent conformational changes of NADH-dependent ferredoxin reductase

under various pH conditions
1 2 3 4
Toshiya Senda , Miki Senda , Shigenobu Kimura , Tetsuo Ishida
1 2
National Institute of Advanced Industrial Science and Technology, Tokyo, Japan, Japan
3 4
Biological Informatics Consortium, Tokyo, Japan, Ibaraki University, Ibaraki, Japan, Shiga
University of Medical Science, Shiga, Japan

BphA4, which is an FAD-containing NADH-dependent ferredoxin reductase, receives two

electrons from NADH and delivers one electron each to ferredoxins (BphA3). To elucidate the
molecular mechanism underlying the electron-transfer reaction and the redox-dependent
interaction between BphA3 and BphA4, we determined the crystal structures of the productive
BphA3-BphA4 complex as well as of free BphA3 and BphA4 in all redox states occurring in
the catalytic cycle. Our results revealed that (i) FAD in BphA4 changes its conformation
depending on the redox state, (ii) BphA4 also changes its conformation depending on the
redox state of FAD, and (iii) the conformational changes of BphA4 are required to form a high-
affinity binding site for BphA3. These results suggested that the conformational change of
FAD could be one of the main reasons for the conformational changes in the overall structure
of BphA4 and affinity regulation between BphA3 and BphA4. However, it has been known that
FAD changes its conformation and chemical properties depending on pH and environment.
Since BphA4 was crystallized at pH 5.4, there might be some artifacts in the FAD
conformations found in the crystal structures. We therefore analyzed the effects of pH on the
structures of FAD and the overall structure of BphA4 in oxidized and semiquinone (BphA4-
NAD complex) forms. BphA4-NAD crystals were prepared by the soaking method as reported
earlier. The crystal structures of the BphA4-NAD complex and oxidized BphA4 have been
determined at higher than 1.5 Å resolution under various pH conditions (pH 5.4–8.5). These
crystal structures showed that semiquinone FAD in BphA4 adopts different conformations in a
pH-dependent manner. However, in the pH range from 5.4 to 8.5, no significant differences
were found in the bound NAD conformation and in the overall structures of the BphA4-NAD
complex. In addition, BphA4 formed a high-affinity binding site for BphA3 throughout the pH
range 5.4-8.5. Our biochemical analysis demonstrated that reduced BphA4 has an
approximately 100-fold higher affinity for NAD than the oxidized BphA4 at pH 5.4-8.5. These
results suggested that the binding of the nicotinamide moiety of NAD to reduced BphA4
facilitates the formation of the high-affinity site to BphA3. This mechanism seems to contribute
to the efficient electron transfer from BphA4 to BphA3 under various pH conditions.
T-021

Structural Basis for Allosteric Activation of Ubiquitylation Mediated by Ube2g2 and

gp78 RING Finger
1 2 2 3 3 2
Yuhe Liang , Ranabir Das , Jess Li , Jennifer Mariano , Allan Weissman , Andrew Byrd ,
1
Xinhua Ji
1
Macromolecular Crystallography Laboratory, National Cancer Institute, Frederick, MD 21702,
2
United States, Structural Biophysics Laboratory, National Cancer Institute, Frederick, MD
3
21702, United States, Laboratory of Protein Dynamics and Signaling, National Cancer
Institute, Frederick, MD 21702, United States

Ube2g2 is an E2 ubiquitin-conjugating enzyme and gp78 is an endoplasmic reticulum-

associated E3 ubiquitin ligase with the RING finger. Distinct from the RING finger, gp78
recruits Ube2g2 with its G2BR domain. The Ube2g2-G2BR interaction is specific with high
affinity, induces significant conformational changes near the active site of Ube2g2, causes a
50-fold increase in the affinity between Ube2g2 and the RING finger, and results in markedly
increased ubiquitylation by Ube2g2 and the gp78 RING finger (Molecular Cell 34, 674-685,
2009). Here, we report the crystal structure of the ternary Ube2g2-G2BR-RING complex at
2.3-Å resolution. The crystal belongs to space group P41212, with unit cell parameters a = b =
58.25 and c = 158.43 Å. The structure shows that the G2BR and RING finger of gp78 bind to
the opposite sides of the Ube2g2 molecule. Comparative analysis of the ligand-free Ube2g2
(PDB entry 2CYX), Ube2g2-G2BR (3H8K), and Ube2g2-G2BR-RING (this work) structures
reveals structural basis for the allosteric activation of ubiquitylation mediated by Ube2g2 and
the gp78 RING finger. The Ube2g2-G2BR-RING structure presented here is the first of its
kind, shedding lights on the mechanism of other E3 ubiquitin ligases with the RING finger.

This research was supported by the Intramural Research Program of the NIH, National
Cancer Institute, Center for Cancer Research. X-ray diffraction data were collected at
beamline 22-ID of SER-CAT, Advanced Photon Source, Argonne National Laboratory.
T-024

Crystal Structures of Human Exonuclease I/DNA Provide Insight Into Catalytic

Mechanism

Jillian Orans, Elizabeth McSweeney, Paul Modrich, Lorena Beese

Duke University Medical Center, Durham, NC, United States

Human exonuclease I (hEXOI) plays a significant role in human mismatch repair through the
5’ to 3’ excision of the non-template DNA strand, as well as possessing additional roles in
DNA replication and recombination. Here we present crystal structures of the hEXOI catalytic
domain as both the wild-type protein and an inactive mutant (D173A) in complex with a 5’
recessed DNA substrate. The initial structure was determined using selenomethionine SIRAS
phasing and refined to a resolution of 2.5 Å. The model shows a common core of a seven-
stranded β -sheet surrounded by flanking helices which is similar to those seen in a variety of
homologous flap endonuclease (FEN1) structures; however, certain commonly conserved
nucleotide binding motifs have altered secondary structural elements and are significantly
repositioned in our structure. We have also identified three metal ion-binding sites, two in the
active site surrounded by conserved acidic residues, and another in close proximity to the
DNA phosphate backbone. The active site metals are appropriately positioned relative to
each other and the scissile phosphate such that we can propose a two-metal ion catalytic
mechanism analogous to that observed in the 3’ to 5’ exonuclease sites of DNA Polymerase I
Klenow fragment. This structure of hEXOI is the first known structure of a Class III member of
the RAD2 nucleases, a family with highly divergent sequences and DNA structure
specificities. Structural knowledge of hEXOI will greatly assist in our understanding of
disease-associated mutations, as well as aid in the modeling and rationalization of similar
mutations in additional RAD2 nucleases such as XPG. Furthermore, analysis of this structure
in conjunction with previously determined DNA-complexed homologs and recently acquired
SAXS data will allow identification of possible protein binding surfaces and aid in the
elucidation of a mechanism for substrate specificity.
T-027

Structural Analysis of Glucokinase Activators and Inhibitors

1 1 1 1
Christine Lukacs , Joseph Grimsby , Nancy-Ellen Haynes , R. Ursula Kammlott , Robert
1 2 1 1
Kester , Paige Mahaney , Frank Podlaski , Ramakanth Sarabu
1 2
Roche, Nutley, NJ, United States, Boehringer Ingelheim Pharmaceuticals, Ridgefield, CT,
United States

Glucokinase is an enzyme of the glycolytic pathway which converts glucose to glucose-6-

phosphate. It plays an essential role in the maintenance of glucose levels in the liver and in
controls glucose stimulated insulin release in pancreatic beta cells, making it an attractive
target for the development of small molecules to treat type II diabetes.

Roche has previously published the discovery of small molecule glucokinase activators
(GKAs). These compounds do not bind at the enzyme active site (glucose binding site), but
rather at a distant allosteric site 20Å away. Modulating the kinetic binding properties of GKAs
in turn affect the kinetics of the glucokinase enzyme. We have been studying these
properties via biochemical and cellular assays, kinetic assays, and biophysical methods,
including X-ray crystallography.

We have identified compounds within our phenylacetamide series which have either
activating, inhibitory, or no effect on the glucokinase activity. We have solved the crystal
structure of GK in its closed conformation with several of these compounds bound. This
poster will show details of these structures and correlate the binding features of these small
molecules with their effect on GK activity.
T-031

Communication Between Thiamin Cofactors in the E. coli Pyruvate Dehydrogenase

Multienzyme Complex E1 Component Active Centers
1,2 1 3 3
Palaniappa Arjunan , Krishnamoorthy Chandrasekhar , Natalia Nemeria , Frank Jordan ,
1,2
William Furey
1 2
University of Pittsburgh, Pittsburgh, PA 15261, United States, VA Healthcare System,
3
Pittsburgh, PA 15240, United States, Rutgers University, Newark, NJ 07102, United States

Structural, spectroscopic and kinetic analyses tested the hypothesis that a chain of residues
connecting the 4’-aminopyrimidine N1’-atoms of thiamin diphosphates (ThDPs) in the two
active centers of the E. coli pyruvate dehydrogenase complex (PDHc) E1 component
provides a signal transduction pathway. Substitution of the three acidic residues (E571, E235,
E237) and R606 resulted in impaired binding of the second ThDP, once the first active center
was bound to the cofactor suggesting a pathway for communication between the two ThDPs.
Titration of the E235A and E237A variants with methyl acetylphosphonate (MAP, an analog
for the substrate pyruvate), monitored by circular dichroism suggested that only half of the
active sites were filled with the related, covalently bound, predecarboxylation intermediate
analog. We have determined the crystal structures of the active site variants E571A in
complex with cofactor ThDP, and E235A in complex with ThDP as well as with MAP, and
compared the structural changes with biochemical activity data. Crystal structures of E235A
and E571A in complex with ThDP revealed the structural basis for the spectroscopic and
kinetic observations and showed that either substitution affects cofactor binding, despite the
fact that E235 makes no direct contact with the cofactor nor does it block entrance to the
active site. The structural results also support the idea of non-equivalence of active sites
within the E1 dimer. While there are general similarities between the native and these two
mutant structures, there are significant differences in the active sites, and the role of the
conserved E571 residue in both catalysis and in defining cofactor orientation was revealed by
the structural results. Details and implications of ThDP binding on catalytic activity for the
mutant structures will be presented.
T-034

Crystal Structure of Human Prethrombin I at 1.66 ä Resolution

Zhiwei Chen, Leslie Bush-Pelc, Enrico Di Cera

Department of Biochemistry and Molecular Biology, Saint Louis University School of

Medicine, St. Louis, Mo 63104, United States

Prethrombin I is an inactive precursor of thrombin along the prothrombin activation pathway.

Prethrombin I consists of a single chain composed of fragment 2 (F2), A and B chains. A
single peptide bond cleavage between R15 and I16 results in generation of the active
intermediate meizothrombin and further cleavage between F2 and the A chain produces
thrombin. Here we report the first structure of prethrombin I carrying the double mutation
W215A/E217K. Comparison with the recent structure of meizothrombin reveals that F2 has
shifted by at least 10 Å and the rms deviation is 3.65 ä. The region around residues 215-223
is partially collapsed as seen in the inactive E* form of thrombin, presumably as a result of the
double substitution W215A/E217K. Interestingly, the region around residues 186-193 is also
collapsed and assumes a conformation never before observed in thrombin structures. The
side chain of R15 from a second molecule in the lattice is bound to the active site. The side
chain of residue D194 that in the active enzyme H-bonds to the amino terminus of the B
chain, I16, interacts with the backbone N atoms of residues 141 and 142. The structure
suggests a pathway of activation from prethrombin 1 to thrombin that takes into account the
allosteric equilibria between the E* and E forms of the enzyme.
T-037

Structural studies of enduracididine biosynthetic enzyme MppR

Nicholas Silvaggi, Tyler Voegtline

University of Wisconsin-Milwaukee, Milwaukee, WI, United States

Antibiotic-resistant pathogens are a serious and persistent threat to public health. The recent
isolation of methicillin-resistant Staphylococcus aureus (MRSA) with significant resistance to
vancomycin, the current last line of defense against this and other antibiotic-resistant
pathogens, is particularly ominous and underscores the need for new, effective antibiotics.

The nonribosomal peptide antibiotics mannopeptimycin and enduracidin are promising

candidates for development into chemotherapeutic agents for the treatment of MRSA,
vancomycin-resistant enterococci (VRE), and penicillin-resistant Streptococcus pneumoniae.
Mannopeptimycin is a lipoglycopeptide with a cyclic hexapeptide core of alternating D- and L-
amino acids. Enduracidin is a 17-residue cyclic lipopeptide. Both compounds contain the rare
nonproteinogenic amino acids beta-hydroxy L-enduracididine or L-enduracididine,
respectively. In mannopeptimycin biosynthesis, L-enduracididine is produced from L-arginine
through the action of three enzymes, MppP, MppQ, and MppR. The details of this
transformation are currently unknown. The success of a semi-synthetic mannopeptimycin
analog, AC98-6446, has prompted much interest in producing additional derivatives of these
promising natural products. These efforts will be facilitated by a ready supply of
enduracididine and its analogs for use in combinatorial biosynthesis or semi-synthetic
approaches.

Herein we present the X-ray crystal structure of MppR (32.2 kDa) from Streptomyces
hygroscopicus at 1.6Å resolution (Rcryst=0.152, Rfree=0.179). The structure was determined by
SAD from a single data set collected from a SeMet-derivitized crystal at LS-CAT beamline
21ID-D at the Advanced Photon Source. The overall fold of the protein is nearly identical to
acetoacetate decarboxylase (PDB ID 3BH2; SSM RMSD over 218 Cα atoms = 1.78Å). In
addition, despite low sequence identity (<10%), the active site residues in these two enzymes
are very similar, suggesting that MppR is also a decarboxylase.
T-040

Mechanism of catalysis and structure of pharmaceutically relevant enzymes in the

biosynthetic pathways of NAD and pyrrolobenzodiazepines (PBDs)
1 1 1 2 1
Watchalee Chuenchor , Melissa Resto , Kaiti Chang , Tzanko Doukov , Barbara Gerratana
1 2
University of Maryland, College Park, United States, Stanford Synchrotron Radiation
Lightsource, Menlo Park, United States

We are studying the mechanisms and structures of biosynthetic pharmaceutically relevant

enzymes. The first project aims at elucidating the mechanism and structure of NAD
synthetase, an essential NAD biosynthetic enzyme in M. tuberculosis for the design of
TB
structure- and mechanism-based inhibitors. The structure of wild type NAD synthetase in
complex with DON and NaAD, previously solved by the Gerratana’s laboratory, is
homooctameric and features a 40 angstrom long inter-subunit ammonia tunnel for transport of
ammonia from the glutaminase active site to the synthetase active site. An 180-fold activation
of the glutaminase active site was measured when the synthetase intermediate complex is
formed. We hypothesize that ordering of loop P2 at the synthetase active site induces a
conformational change that activates the glutaminase active site and we reasoned that
inhibitors that will trap the enzyme in this closed activated conformation will have high affinity.
TB
To obtain the NAD synthetase bound to ligands that stabilize the disordered loop P2 at the
synthetase active site, we solved several structures of different ligands complexes in the
resolution range of 2.4 to 3.0 angstroms. The analysis of these structures is reported in this
poster. We have also expressed the human NAD synthetase in insect cell and purified for
kinetic and structural characterization. With the characterization of this ortholog, we will be
able to clarify the basis of substrate specificity and design high affinity inhibitors selective for
TB
NAD synthetase .

The second project is focused on characterizing novel enzymes of the biosynthesis of

pyrrolo[1,4]benzodiazepines (PBDs). A glycosylated member of this class, sibiromycin, has
remarkable potency against cancer cells (pM range). We have solved the apo structure of
SIbS from Se-Met and native crystals at 2.3 and 2.1 angstrom resolutions, respectively. SibS
is hypothesize to catalyze an unusual C-C hydrolase reaction and shows very little sequence
homology to the phenazine biosynthesis-like proteins. This is the initial step in the
characterization of the enzymes involved in the unique transformation of L-tyrosine to
hydropyrrole identified in PBD biosynthesis.
T-043

Structural and Functional Characterization of Outer-Sphere Mutations of Toluene 4-

Monooxygenase

Lucas Bailey, Justin Acheson, Brian Fox

Department of Biochemistry, University of Wisconsin-Madison, Madison, WI, United States

Diiron hydroxylases are multicomponent enzyme complexes that catalyze the oxidation of a
wide range of hydrocarbons. Toluene 4-monooxygenase (T4moH) is a four-component
hydroxylase consisting of two electron transfer components, T4moF and T4moC, responsible
for the reduction of the diiron hydroxylase (T4moH). Additionally, a small cofactorless effector
protein, T4moD, forms a high affinity complex with T4moH and is responsible for the
modulation of a number of catalytic phenomena associated with enzymatic function. A series
of crystal structures of the T4moH-T4moD complex have revealed how residues outside of
the first coordination sphere rearrange to create an active site pocket poised for turnover.
These include Asn202 and Gln228, which form a hydrogen-bonding network with conserved
Thr201 and a newly ordered HOH5. T4moH variants N202A, Q228A and Q228E all had lower
enzyme activity as determined by a combination of steady state and transient kinetic
approaches. High-resolution structures of the T4moH variant in complex with T4moD provide
new insight into the role of these residues in catalysis.

Funded by NSF MCB 0843239 to BGF.

T-046

Hydroxymethylglutaryl-CoA lyase (HMGCL): Insights into the Reaction Mechanism

++ ++
from Structures of HMGCL-Mg -hydroxyglutaryl-CoA and HMGCL R41M-Mg -HMG-
CoA Ternary Complexes
1 1 2 1
Jennifer Runquist , Zhuji Fu , Henry Miziorko , Jung-Ja Kim
1 2
Medical College of Wisconsin, Milwaukee, WI, United States, University of Missouri-Kansas
City, Kansas City, MO, United States

HMGCL is crucial to ketogenesis and inherited human mutations result in potentially fatal
disease. Detailed understanding of the HMGCL reaction mechanism as well as the molecular
basis for correlating recently reported human mutations with enzyme deficiency have been
limited by the lack of structural information for enzyme bound to an acyl-CoA substrate or
inhibitor. Soaking crystals of wild-type HMGCL with the competitive inhibitor 3-
hydroxyglutaryl-CoA (HG-CoA) or of the R41M HMGCL mutant with substrate HMG-CoA has
supported determination of X-ray structures for complexes of wild type with inhibitor (2.4 Å)
and R41M with substrate (2.2 Å). Comparison of these β /α barrel structures with those of
unliganded HMGCL and R41M reveal substantial differences for positioning of the flexible
loop containing the conserved “signature” sequence of HMGCL. In the ternary complex
++
formed with substrate, Mg , and R41M, loop residue C266 (implicated in active site function
by mechanistic and mutagenesis observations) is more closely juxtaposed to the catalytic site
++
than in the case of either unliganded enzyme or the complex of wild-type enzyme with Mg
and inhibitor HG-CoA. In both ternary complexes, the S-stereoisomer of substrate or inhibitor
is specifically bound, in accord with the observation that oxygens from both the C3 hydroxyl
++
and the C5 carboxyl groups are Mg ligands. In addition to H233 and H235 imidazoles, other
++
Mg ligands are the D42 carboxyl oxygen and an ordered water molecule. This water is
positioned between D42 and the C3 hydroxyl of bound acyl-CoA substrate/inhibitor and may
function as a proton shuttle. Results also display the interaction of R41 with the acyl-CoA’ s C1
carbonyl oxygen, in agreement with the observed effects of R41 mutation on reaction product
enolization. This substrate enzyme interaction offers an explanation for the drastic enzyme
5
deficiency (10 –fold) seen in human R41 mutation.
T-049

Structural insights into allosteric activation of pyruvate carboxylase: the asymmetric

tetramer organization of Rhizobium etli PC is independent of acetyl coenzyme A.

Martin St. Maurice, Adam Lietzan, Sudhanshu Kumar

Marquette University, Milwaukee, WI, United States

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T-052

Domain Movement in NADPH Cytochrome P450 Oxidoreductase: Effect of an

Engineered Disulfide Bond between the FAD- and FMN Domains
1 2 3 3 2
Chuanwu Xia , Djemel Hamdane , Anna Shen , Vivian Choi , Sang-Choul Im , Haoming
2 3 2 1
Zhang , Charles Kasper , Lucy Waskell , Jung-Ja Kim
1
Department of Biochemistry, Medical college of Wisconsin, Milwaukee, WI, United States,
2
The University of Michigan and Veterans Affairs Medical Research Center, Ann Arbor, MI,
3
United States, McArdle Laboratory for Cancer Research, University of Wisconsin, Madison,
WI, United States

NADPH-cytochrome P450 oxidoreductase (CYPOR) is a multidomain microsomal diflavin

protein that transfers electrons from NADPH to cytochromes P450 via FAD and FMN. It has
been suggested that interflavin domain movement must occur, resulting in a more open
conformation than that seen in the crystal structure, to form a productive electron transfer
complex with the redox partners. In order to directly evaluate whether this hypothesis is
structurally relevant in solution, we constructed a mutant CYPOR (D147C-R514C) in which a
disulfide bond has been engineered between residue 147 of the FMN domain and 514 of the
FAD domain. The crystal structure of the mutant determined at 2.2 Å resolution reveals that
the two flavin domains are indeed joined by the disulfide linkage and that the distance
between the two flavins is ~5 Å (wild type, ~4 Å) and the two flavin rings are twisted ~20º from
that of wild type. The steady state kinetic characterization of this mutant showed that the
affinity of NADPH (and NADP+) is slightly decreased by 2-4 fold. Although the hydride
transfer from NADPH to FAD proceeds normally, electron transfer from FADH2 to FMN is
significantly impaired (~2% of wild type). This slow interflavin electron transfer does not,
however, explain the dramatically decreased ability of the crosslinked D147C- R514C mutant
-3
to reduce ferric cytochrome P450 (10 of wild type). The reduction of cytochrome P450 by the
mutant is slow and incomplete, indicating that the FMN domain is no longer competent to
transfer electrons to cytochrome P450 when the FMN and FAD domains are locked in a
closed conformation. These results, together with our previous studies showing that a
CYPOR variant with an open conformation is capable of reducing cytochrome P450
(Hamdane et al., J Biol Chem. 2009,284(17):11374-84), directly demonstrate that CYPOR
adopts large conformational changes during its catalytic cycle. The enzyme adopts a closed
conformation for electron transfer from NADPH to FAD to FMN and an open conformation
when it interacts with cytochrome P450, its electron transfer partner.
T-055

Product specificity and the role of active site water molecules in the methyltransfer
reaction of SET7/9.
1 1,2 3 1 1
Paul Del Rizzo , Jean-Francois Couture , Lynnette Dirk , Bethany Strunk , Marijo Roiko ,
4 3 1
Joseph Brunzelle , Robert Houtz , Raymond Trievel
1 2
University of Michigan, Ann Arbor, MI, United States, University of Ottawa, Ottawa, ON,
3 4
Canada, University of Kentucky, Lexington, KY, United States, Northwestern University
Center for Synchrotron Research, Argonne, IL, United States

SET domain lysine methyltransferases (KMTs) are responsible for the methylation of specific
lysine residues in histone proteins as well as other substrates found in cellular signaling
pathways. One such enzyme is human SET7/9, a monomethyltransferase that transfers a
methyl group from S-adenosylmethionine (AdoMet) onto target lysine residues in histone and
non-histone substrates. In the work presented here, we have characterized the mechanism
underlying the product specificity of SET7/9, and two active site mutants, Y305F and Y245A,
that convert the enzyme into a di- and trimethyltransferase, respectively. High resolution
structures of the enzymes in complex with the product S-adenosyl-homocysteine (AdoHcy)
and a series of unmodified and methylated peptide substrates (mono-, di- and tri-methylated)
were obtained. Comparisons between these complexes reveal the role of active site
residues, as well as the coordinating water molecules, that help to position the lysine epsilon-
amine group for successive rounds of methylation. These results provide the first molecular
snapshots of the mono-, di- and trimethyltransfer reactions catalyzed by SET domain
enzymes.
T-058

Crystal Structure of Chicken Muscle Lactate Dehydrogenase in the Presence of

Oxalate, NAD and Pyruvate.

Milan Draganov, L Grant, E Greiner, J Warfel, N Polder, G Watanabe, C Smith, B Rupp, X

Ouyang, S Herron, C Meyer, C Srinivasan, K Kantardjieff

CSU Fullerton, Fullerton, CA, United States

Lactate dehydrogenase (LDH) is an essential enzyme in carbohydrate metabolism. The

structure of chicken muscle LDH-A has not been reported previously, although coordinate
data for orthologs are available. Protein crystallography has been used for a comprehensive
structure determination and analysis of LDH-A in the upper division biochemistry laboratory
and subsequent student research at Cal State Fullerton, providing insights into structural
features that regulate LDH kinetic and stability properties. LDH-A was isolated and purified
using established protocols. Enzymatic activity was assayed spectrophotometrically. LDH-A
was co-crystallized with substrate, cofactor and inhibitor by vapor diffusion methods using
commercially available screens. Diffraction data were collected from flash cooled crystals at
SSRL BL9-2. Model building and refinement was achieved using the CCP4 program suite.
The 1.9Å native structure has been determined by molecular replacement, using porcine
LDH-A as probe, in space group P212121, a = 84.04, b = 126.78, c = 252.74, two tetramers
with 222 pseudo-symmetry in the asymmetric unit, R = 0.22, freeR = 0.27. The 1.9Å structure
of LDH-A complexed with pyruvate has been solved by molecular replacement, using the
native chicken structure as probe, in space group C2, a = 90, b = 92.77, c = 90, β = 93.5, with
one tetramer in the asymmetric unit, R= 0.19, freeR =0.24. A 1.9Å structure of the LDH-A
+
complexed with oxalate and NAD has been determined by molecular replacement using the
native structure as probe, in space group P1 21 1, a= 72.665, b= 147.836, c= 139.642, two
tetramers each with 222 pseudo-symmetry in the asymmetric unit, R = 0.21 and freeR = 0.26.
A 3Å structure of the LDH-A complexed with pyruvate has been determined by molecular
replacement using the native structure as a probe, in space group P1 21 1, a = 72.019, b =
146.518, c = 139.469, two tetramers each with 222 pseudo-symmetry in the asymmetric unit.
Model building and refinement are in progress. Overall topology in each of the structures is
consistent with crystal structures of orthologs, although secondary structure active site flexible
loop rearrangements (residues 96-112) result from ligand binding and crystal contacts. The
flexibility in the active site entails an “extended unit” involving much of the structure.
T-067

Correlated Single-Crystal Spectroscopy and X-ray Crystallography at Beamline X26-C

of the NSLS and Plans for Spectroscopy + MX Beamlines at the NSLS-II

Allen Orville, Deborah Stoner-Ma, John Skinner, Dieter Schneider, Robert Sweet

Brookhaven National Laboratory, Upton, NY, United States

Understanding the complex relationships among atomic structure, electronic structure, and
chemistry is crucial for obtaining fundamental insights into biological processes. Inspiration
and breakthroughs will come from new tools developed to probe biological processes with
several complementary techniques. To achieve these goals, we are accelerating the
construction of an integrated beamline to enable the measurement of spectroscopic and high
resolution crystallographic data. Beamline X26-C of the National Synchrotron Light Source
(NSLS) is the only user facility in the US to support correlated measurements of up to three
types of complementary data -- X-ray diffraction to high resolution, optical absorption
spectroscopy, and Raman spectroscopy -- from the same sample and under nearly identical
experimental conditions. Single-crystal electronic absorption spectra correlated with X-ray
diffraction data are routinely collected from a 25µm diameter region of the crystal that
intersects the X-ray beam during the readout time of each X-ray detector image. Raman
spectra are also collected with either 785nm or 532nm laser excitation from the same 25µm
region of the crystal that intersects the X-ray beam and the electronic absorption optical path.
Integration of the controls for these spectroscopic techniques into the X-ray beamline
operations software yields fully correlated atomic and electronic structures for deposition to
the PDB. A complementary off-line laser spectroscopy laboratory immediately adjacent to the
beamline will support additional spectroscopic techniques (e.g. fluorescence, time-resolved
fluorescence). Some recent findings will be presented including correlated studies of heme-
based and flavin-based macromolecules.

NSLS-II is a new 3 GeV, 500 mA storage ring currently under construction at BNL. The 1nm
spatial resolution, 0.1meV energy resolution, flux density (« 10 ph/s/0.1%BW) and brightness
15

(2 keV - 10 keV; « 10 ph/mm /mrad /s/0.1%BW) will be state-of-the-art. Several MX and

21 2 2

Spectroscopy+MX beamlines are being designed now, with construction to commence in

2011, and full operations by 2015. Some of these plans will be discussed.

Supported by the NIH National Center for Research Resources and the US Department of
Energy, Office of Biological and Environmental Research.
T-070

Human UDP-Glucose Dehydrogenase Reveals a Bifunctional Active Site: The Pin in

Fischer’ s Lock
1 2 1 1 1
Renuka Kadirvelraj , Stephen Weitzel , Nicholas Sennett , Samuel Polizzi , Zachary Wood
1 2
University of Georgia, Athens, GA, United States, University of Oregon, Eugene, OR, United
States

UDP-glucose dehydrogenase (UGDH) oxidizes UDP-glucose to UDP-glucuronic acid using

+
two molecules of NAD . UGDH is regulated in vivo by another nucleotide sugar, UDP-xylose,
which acts as a cooperative feedback inhibitor. Using sedimentation velocity studies, we show
that the substrate UDP-glucose or the inhibitor UDP-xylose induce human UGDH (hUGDH) to
form a hexameric complex, while the unliganded enzyme favors the dimeric state. The
available crystal structures of active hUGDH reveal a hexameric complex with 32 symmetry,
best described as a ‘trimer of dimers’. We have solved four different crystal structures of the
inhibited UDP-xylose:hUGDH complex, revealing a horseshoe-shaped ‘broken hexamer’
conformation. These crystal structures show that UDP-xylose binds in the active site, causing
a buried loop to repack. The restructured loop alters the oligomerization interface to favor the
+
broken hexamer conformation. The NAD -binding site is occluded in the broken hexamer
structure, suggesting a link between the oligomeric state of the enzyme and the observed
cooperative inhibition kinetics. Our results show that hUGDH has evolved a bifunctional active
site that can either facilitate catalysis or respond to a heterotropic effector to promote the
formation of an inactive enzyme complex.
T-073

Structure Determination and Characterization of Klebsiella pneumoniae HpxO, a FAD-

Dependent Urate Oxidase
1 2 2 1
Katherine Hicks , Sean O’Leary , Tadhg Begley , Steven Ealick
1 2
Cornell University, Ithaca, NY, United States, Texas A&M University, College Station, TX,
United States

HpxO is involved in a novel purine catabolism pathway in Klebsiella pneumoniae, a multi-drug

resistant pathogen. This pathway consists of five enzymes that convert hypoxanthine to
allantoic acid. HpxO is responsible for the third step in the pathway, the hydroxylation of uric
acid to 5-hydroxyisourate. Here we report the structural and biochemical characterization of
HpxO.

Our detailed biochemical studies have illustrated that, in contrast to most urate oxidases,
HpxO requires a FAD cofactor for catalysis and is catalytically similar to the well-
characterized enzyme p-hydroxybenzoate hydroxylase. Here we present the crystal structure
of HpxO at 2.3 Å resolution which was solved by SeMet SAD phasing and a 2.0 Å resolution
structure in complex with its substrate, uric acid. To gain further mechanistic detail, we have
biochemically characterized a number of active site mutants including R204Q HpxO, which
appears to uncouple the uric acid hydroxylation and FAD reduction reactions. Recently, a 2.7
Å dataset of this mutant has been collected. Based on these structures, we are able to
propose a mechanism for the HpxO hydroxylation reaction.
T-076

Toward understanding an unusual His-Tyr ligated c-heme: X-ray crystal structures of

MauG point mutants.
1 2 2 1
Lyndal Jensen , Nafez Abu Tarboush , Victor Davidson , Carrie Wilmot
1 2
University of Minnesota, Minneapolis, MN, United States, The University of Mississippi
Medical Center, Jackson, MS, United States

MauG is a di-heme enzyme responsible for the post-translational modification of two

tryptophan residues to form the tryptophan tryptophylquinone cofactor (TTQ) of methylamine
dehydrogenase (MADH). MauG converts preMADH, containing monohydroxylated-β Trp57, to
mature MADH by catalyzing the insertion of a second oxygen atom into the indole ring and
covalently linking β Trp57 to β Trp108. We recently reported the X-ray crystal structure of
MauG from Paracoccus denitrificans in complex with its substrate, preMADH (PDB code
3L4M). The structure revealed that the two c-hemes of MauG and the nascent TTQ site of
preMADH are separated by long distances over which electron transfer must occur to achieve
catalysis. One of the c-hemes has an atypical His-Tyr axial ligation involving residues His205
and Tyr294. While cytochrome c peroxidases (CCPs) have the highest sequence similarities
with MauG (up to ~30% homology), they exhibit His or Met axial ligands at the MauG Tyr294
position. A key distinction for MauG is that its 6-coordinate c-heme iron attains the Fe(IV)
state during enzyme turnover (i.e. TTQ catalysis), with concomitant formation of an Fe(IV)=O
species at its other c-heme. As such, we are interested in understanding the coordination and
stabilization of this new member of the c-heme family. We present here the X-ray crystal
structures of MauG-Tyr294X mutants in their diferric resting states, complexed with
preMADH.
T-079

Twisting of the DNA Binding Surface By A Beta-Strand-Bearing Proline Modulates DNA

gyrase Activity
1 1,2 1 1,2 2
Nei-Li Chan , Tung-Ju Hsieh , Tien-Jui Yen , Te-Sheng Lin , Hsun-Tang Chang , Shu-Yun
2 1 3
Huang , Chun-Hua Hsu , Lynn Farh
1 2
National Taiwan Univ., Taiwan, Taiwan, National Chung Hsing Univ., Taiwan, Taiwan,
3
National Pingtung Univ. of Education, Taiwan, Taiwan

DNA gyrase is the only topoisomerase capable of introducing (-) supercoils into relaxed DNA.
The C-terminal domain of the gyrase A subunit (GyrA-CTD) and the presence of a gyrase-
specific “GyrA-box” motif within this domain are essential for this unique (-) supercoiling
activity by allowing gyrase to wrap DNA around itself. Here we report the crystal structure of
Xanthomonas campestris GyrA-CTD and provide the first view of a canonical GyrA-box motif.
This structure resembles the GyrA-box-disordered Escherichia coli GyrA-CTD, both adopting
a non-planar ¬-pinwheel fold composed of 6 seemingly spirally arranged ¬ -sheet blades.
Interestingly, structural analysis revealed that the non-planar architecture mainly stems from
the tilted packing seen between blades 1 and 2, with the packing geometry likely being
defined by a conserved and unusual β -strand-bearing proline. Consequently, the GyrA-box-
containing blade 1 is placed at an angled spatial position relative to the other DNA-binding
blades, and an abrupt bend is introduced into the otherwise flat DNA-binding surface.
Mutagenesis studies support that the proline-induced structural twist contributes directly to
gyrase’ s (-) supercoiling activity. To our knowledge, this is the first demonstration that a β -
strand-bearing proline may impact protein function. Potential relevance of β -strand-bearing
proline to disease phenylketonuria is also noted.
T-082

Structural Basis of the Nucleotidase Activity for HAD Super Family Member P4 from
Haemophilus influenzae.

Harkewal Singh, Thomas J. Reilly, John J. Tanner

Univ. of Missouri, Columbia, MO, United States

Class C non-specific acid phosphatases catalyze the transfer of phosphoryl group from
phosphomonoesters to water at acidic pH using an active-site aspartate residue and
represent a major subgroup of haloacid dehalogenase (HAD) superfamily. The acid
phosphatase e (P4) from Haemophilus influenzae is a major component of outer membrane
of the organism, and it is highly conserved among H. influenzae strains, making it an
attractive vaccine candidate. The main biological role of e (P4) is to catalyze the conversion of
+
nicotinamide mononucleotide (NMN) to nicotinamide riboside (NR) as part of vestigial NAD
utilization pathway. We show that e (P4) is promiscuous enough to catalyze the hydrolysis of
various 2’, 3’ and 5’ mononucleotide phosphates. Furthermore, we used high resolution X-ray
crystallography to understand the basis of substrate promiscuity. An active site mutant (Asp to
Asn) of e (P4) was engineered using site directed mutagenesis and structure of NMN, 5’AMP,
2’AMP and 3’AMP enzyme-substrate complexes were obtained at 1.35 Å, 1.55 Å, 1.90 Å and
1.85 Å resolution respectively. We also obtained a high-resolution crystal structure of e (P4)
complexed with its very potent inhibitor adenosine 5′ -O-thiomonophosphate (AMPS) at 1.35
Å. The e (P4) –AMPS complex structure shows a different conformation of AMPS in the active
site as compared to the binding of its natural substrate NMN. The difference is due to the
larger size and lower electro negativity of sulfur (S) compared to oxygen (O) Finally, steady
state kinetics parameters of P4 with its substrates NMN, 5’AMP, 2’AMP, 3’AMP and inhibitor
AMPS, were determined using discontinuous colorimetric assay.
T-085

Structural basis for molecular recognition in the mouse AKR1C13-NAD complex

Debanu Das, Ashley Deacon

JCSG-SSRL, SLAC National Accelerator Laboratory, Menlo Park, CA, United States

Proteins in the Aldo-Keto Reductase (AKR) superfamily are involved in metabolism

and human disease, including cancer. Subfamily C of AKR family 1 (AKR1C) has 25
members that are all mammalian proteins (AKR1C1-C25) and they share more than 60%
sequence identity. Of these, only 7 members C1-C5, C9 and C21 have been structurally
characterized. AKR1C13 is believed to play an important role in detoxification in the mouse
stomach. Biochemical characterization by others has shown that AKR1C13 has some key
differentiating features compared to its subfamily members: a broader substrate specificity
and preference for non-steroidal alcohols; and the use of NAD as cofactor instead of NADP.

We have determined an ultra-high resolution 1.18 Å crystal structure of the mouse AKR1C13-
NAD complex in the JCSG that now provides a 3D basis for explaining some of the
biochemical differences. The structure reveals that Glu276 is the determinant for selecting
NAD as a cofactor, compared to Arg276 in family members that use NADP, which requires
additional interactions with the phosphate moiety. Tyr55 and His117 interact with the
nicotinamide ring and are conserved in family members. Residue 54, a determinant of
substrate specificity, is present as Ala (Leu, Val or Phe in C1-C5 and C9) and nearby an
MPD-like molecule in the active site. Tyr24, Asp128 and Phe129 that are known to be
important in substrate interactions are poised around the MPD. Lys27 and Ser137, located
near the substrate entry point, may play a role in initial substrate recognition. The JCSG is
funded by NIGMS/PSI, U54 GM074898. SSRL operations are funded by DOE BES, and the
SSRL SMB program by DOE BER, NIH NCRR BTP and NIH NIGMS.
T-091

Kinetic and Structural Charaterization of the GTP-Dependent Enterococcal

Aminoglycoside Phosphotransferases from the APH(2”) family
1 2 2 3 2
Clyde Smith , Marta Toth , Hilary Frase , Laura Byrnes , Sergei Vakulenko
1 2
SSRL, Menlo Park, CA, United States, University of Notre Dame, Notre Dame, IN, United
3
States, Cornell University, Ithaca, NY, United States

The aminoglycoside (2”) phosphotransferases (APH(2”)s) are a family of four protein kinase-
like enzymes (APH(2”)-Ia, APH(2”)-IIa, APH(2”)-IIIa, and APH(2”)-IVa) which are a major
cause of acquired resistance to the aminoglycoside antibiotics in enterococci and
staphylococci. The APH(2”)-Ia enzyme is the best studied and is capable of phosphorylating
virtually all known aminoglycosides including 4,5-disubstituted drugs such as neomycin, and
4,6-disubstituted drugs such as kanamycin and gentamicin. The enzyme exists as the C-
terminal half of a bifunctional molecule, the N-terminal domain comprising an aminoglycoside
acetyltransferase, AAC(6’)-Ie. The other three APH(2”) enzymes are all monofunctional,
single domain enzymes which react with a smaller subset of the drugs, and appear to be
selective for the 4,6-disubstituted aminoglycosides. The APH(2”)-IIa, APH(2”)-IIIa and
APH(2”)-IVa enzymes phosphorylate only the 4, 6-disubstituted aminoglycosides at the 2”
3 6 -1 -1
position with kcat/Km values in the range 10 - 10 M s . The nucleotide substrate specificity
for each enzyme has been analyzed and we have found that GTP is a substrate for all four
enzymes and that furthermore, two of the enzymes have a preference for GTP over ATP
(APH(2”)-Ia has a 250-fold preference and APH(2”)-IIIa has a 400-fold preference in terms of
Km). This is unprecedented amongst the phosphotransferase enzymes, and in the protein
kinases in general. The crystal structures of all four APH(2”) enzymes have been solves in
order to ascertain the structural determinants of substrate binding, specificity and selectivity.
Analysis of the nucleotide binding site shows the presence of two overlapping hydrogen
bonding templates responsible for the selective binding of GTP and ATP. We feel that the
understanding we now have of the ways in which these enzymes interact with nucleotides
and aminoglycoside antibiotics may lead to novel ways of inhibiting these enzymes.
T-094

Atomic resolution structure of the cytoplasmic domain of Yersinia pestis YscU, a

regulatory switch involved in type III secretion

George Lountos, Brian Austin, David Waugh

Macromolecular Crystallography Laboratory, National Cancer Institute at Frederick, Frederick,

MD 21702, United States

Yersinia pestis and many other gram-negative bacterial pathogens use a type III secretion
system (T3SS) as a protein transport apparatus to inject a number of effector proteins through
a hollow needle that penetrates the cytosol of eukaryotic cells, thereby enabling the pathogen
to defeat the immune response of the host. The export apparatus consists in part of
cytoplasmic and inner-membrane proteins that identify T3SS substrates and control the
switching of substrate specificity during morphogenesis and host-cell contact. YscU, an
essential component of the secretion apparatus, is composed of a transmembrane N-terminal
domain and a C-terminal cytoplasmic domain that are separated by a long, conserved linker
region. The cytoplasmic domain undergoes auto-cleavage of the N263-P264 peptide bond at
a conserved NPTH motif, resulting in N- and C-terminal fragments that remain tightly bound.
The N263A mutant inhibits auto-cleavage and blocks the export of pore-forming translocators
but not effector proteins. Therefore, it has been proposed that cleavage results in a
conformational change that triggers the recognition and export of translocators during
assembly of the T3SS. Crystal structures of the noncleaved and cleaved YscU cytoplasmic
domain were solved at 1.5 and 1.1 Å resolution, respectively, by SAD phasing. The structure
of the noncleaved YscU-N263A mutant consists of a four-stranded, mixed ­ -sheet that is
surrounded by five ®-helices. The NPTH motif is located on a ­ -turn between strands 1 and 2.
The structure of cleaved YscU reveals that the core protein structure remains essentially the
same, however, cleavage of the N263-P264 scissile bond results in a rearrangement of the
NPTH loop that exposes residues that were buried by the ­-turn, thus forming a new surface
that may be a recognition site for other T3SS proteins. Additionally, there is a conformational
change that also occurs in the N-terminal linker region in which the N-terminal helix switches
into a mostly disordered conformation that likely has a significant impact on the orientation of
the cytoplasmic domain with respect to the membrane-bound domain. This conformational
change may imply a regulatory role for this region which influences substrate specificity.
T-097

The crystal structure of AroE from Pseudomonas putida and insights into substrate
binding in the shikimate dehydrogenase superfamily
1 2 1 1
James Peek , Sasha Singh , Kay Chen , Dinesh Christendat
1 2
University of Toronto, Toronto, Ontario, Canada, Harvard Medical School, Boston,
Massachusetts, United States

AroE catalyzes the reversible NADPH-dependent reduction of dehydroshikimate to shikimate.

This reaction represents the fourth step in the shikimate pathway, the common route for the
biosynthesis of the aromatic amino acids in plants, fungi, bacteria and apicomplexan
parasites. The absence of this pathway in metazoans makes the enzymes in the pathway
attractive targets for herbicides and antimicrobials. AroE belongs to the shikimate
dehydrogenase (SDH) superfamily which consists of at least five members that share similar
overall structures, but bind a range of substrates. Through comparative analysis, we are
exploiting the limited structural variability among SDH homologs to understand modes of
substrate discrimination used by the enzymes. The results of this study may be relevant to the
rational design of drugs targeting the SDH homologs. We have recently solved the crystal
structure of one member of the SDH superfamily, AroE-Like1 (Ael1) from Pseudomonas
putida. Here, we present the structure of AroE from the same species. Interestingly, both
enzymes are capable of accepting shikimate, but Ael1 binds the substrate with a dramatic
reduction in Km. Comparison of the AroE and Ael1 structures reveals a high level of
conservation among substrate binding residues. However, subtle variations in active site
architecture appear to result from differences in residues in a secondary active site layer.
These variations may contribute to the observed difference in the binding affinity of the two
enzymes.
T-100

A pair-based approach to structural homology using quaternion SLERP averaging

1 2
Herbert J. Bernstein , Lawrence C. Andrews
1 2
Dowling College, Oakdale, NY, United States, Micro Encoder, Kirkland, WA, United States

We present an atom-pair-based alternative to the Kabsch algorithm [Kabsch 1978] for

measuring structural homology between commensurate molecular fragments that is able to
reproduce the results of the Kabsch algorithm for fragments that can be mapped into each
other by a single rigid body motion. In addition, the pair-based algorithm is able to illuminate
cases in which different rigid body motions are required to bring various substructures into the
alignment. The Kabsch algorithm is the gold standard for measuring structural homology
between two molecular fragments with the same numbers of atoms in each of two fragments
with the same connectivity. The Kabsch algorithm is based on first centering each of the two
fragments on their centroids and then computing the covariance matrix of the first fragment
against the other. Our pair-based approach takes pairs of atoms, one atom from the first
fragment and the matching atom from the second fragment, and computes the plane dividing
the line between them. Then, taking an appropriate sampling of pairs of pairs yields an axis,
and, most importantly, an angle of rotation from the intersections of the planes separating the
pairs of atoms. Each axis plus an angle determines a quaternion. If the quaternions are
averaged using Spherical Linear Interpolation (SLERP), the result agrees with the results of
the Kabsch algorithm. As noted by Ye and Godzik [Ye Godzik 2003], “When flexible
molecules in different conformations are compared to each other as rigid bodies, even strong
structural similarities can be missed and significant errors in alignments can occur because
such algorithm compensate global rearrangements with local alignment shifts.” Their
“FATCAT” algorithm works in terms of rigid body movements of fragments. Our pair-based
algorithm can be used in a similar manner, but is not dependent on the identification of
fragments, nor on the identification of translations, going directly to the identification of the
rotations needed.

[Kabsch 1978] Kabsch, W. (1978), “A discussion of the solution for the best rotation to relate
two sets of vectors”, Acta Cryst. A34:5, 827 – 828.

[Ye Godzik 2003] Ye, Y., Godzik, A. (2003), “Flexible structure alignment by chaining aligned
fragment pairs allowing twists,” Bioinformatics 19, suppl. 2., ii246 -- ii255.
T-103

The Relevance of H-Bonding to the Relative Thermodynamic Stability of Polymorphs

Alicia Ng, Chiajen Lai, Feng Qian, Baoqing Ma, Qi Gao

Bristol-Myers Squibb, New Brunswick, NJ, United States

Relative thermodynamic stability between crystalline polymorphs can be established in most

cases, however, the phenomenon is not usually well understood. Nevertheless, insight into
this important property can be obtained by examining the crystal structure, which offers a
wealth of information about the intrinsic nature of a crystalline solid. In this presentation, the
contribution of H-bonding to relative thermodynamic stability of true polymorphs is evaluated.
The compounds showcased in this discussion offer a good system for comparing structures
containing strong H-bonding to their polymorphic counterparts which have only weak or no H-
bonding. In essence, the effects of H-bonding to 3-dimensional molecular packing is
compared to molecular packing that relies mainly on van der Waals forces and/or stacking
interactions, and how these differences translate into thermodynamic stability of true
polymorphs. The correlation between molecular conformation and H-bonding characteristic is
also henceforth discussed.
T-106

PSI:BIOLOGY: High-Throughput-Enabled Structural Biology

Ward Smith, Ravi Basavappa, Jean Chin, Charles Edmonds, Paula Flicker, Peter Preusch,
Janna Wehrle, Catherine Lewis, Jeremy Berg

National Institute of General Medical Science, National Institutes of Health, Bethesda, MD,
United States

The National Institute of General Medical Sciences has announced PSI:Biology (Protein
Structure Initiative:Biology) to continue the development of high-throughput structural biology
methods and apply them to important biological problems. This will be accomplished by
establishing a network of collaborations between centers for structure determination and
biologists with interests in problems involving particular proteins or collections of proteins that
would benefit from structural information. The collaborations will be established through
separate awards to investigators outside the structure determination centers. These awards
are named Consortia for High-Throughput-Enabled Structural Biology Partnerships and
successful applicants will help to define targets for structure determination by the centers and
will receive funds for functional studies in the applicants’ laboratories. This mechanism
provides an on-going opportunity for the wider biomedical research community to obtain
funding to participate in the PSI through collaboration with the high-throughput structure
determination centers and with the centers for membrane structure determination.

The PSI:Biology Network will include Centers for High-Throughput Structure Determination,
Centers for Membrane Protein Structure Determination, The PSI:Materials Repository and
The PSI:Biology Knowledgebase and the Consortia investigators.

In addition, R01 and P01 applications are solicited from individual investigators or groups of
investigators to participate in the PSI:Biology Network. The applicants may propose to further
develop high-throughput structural biology methods, novel methods for comparative
molecular modelling or additional biological partnerships as member of the PSI:Biology
Network

Individual researchers may also suggest proteins for structure determination by the PSI
Structure Determination Centers on the PSI Knowledgebase Community Nomination site.
T-109

Crystallographic studies of human Kynurenic Acid Transaminase II (hKAT II), a novel

schizophrenia target.
1 2 1 1 1
Marie Anderson , Artem Evdokimov , Jay Pandit , Kim Fennell , Patrick Verhoest , Brian
1 1
Campbell , Jim Valentine
1 2
Pfizer, Groton, CT, United States, Monsanto, St.Louis, United States

KATII has emerged as a potentially attractive target for the treatment of Schizophrenia.
KATII, a pyridoxal phosphate dependant enzyme catalyzes the conversion of L-kynurenine to
kynurenic acid (KYNA), and is part of the tryptophan metabolism pathway. KYNA can act as
a competitive antagonist at the glycine site of NMDA receptors. Reduced NMDA receptor
function is predicted to contribute to symptoms of Schizophrenia. It is hypothesised that
reducing KYNA blockade of the glycine co-agonist site should increase NMDA receptor
function, which could lead to a novel antipsychotic agent.

A high-throughput screen of our corporate sample collection led to the discovery of several
potent inhibitors of KAT II activity. In this report we describe the x-ray structure of one such
inhibitor co-crystallized with KAT II, which can serve as the starting point for structure-based
optimization.
T-113

Use of the ScreenMachine as a tool to augment graduate-level Biophysics education

Bryan Sutton

Texas Tech University Health Sciences Center, Lubbock, TX, United States

For biologically-oriented students, X-ray crystallography is a daunting aspect of their graduate

education. They are typically ill-prepared for a comprehensive mathematical treatment of the
subject, and they are usually not familiar with the basic principles of optical physics; however,
it is crucial that these students gain a working knowledge of the strengths and weaknesses of
the technique. At Texas Tech University, we are experimenting with an innovative concept in
graduate education to bridge between the familiar discipline of biochemistry and the less
familiar physics of X-ray crystallography.

Our Deep Saturation Mutagenesis (DSM) course seeks to study all possible mutations at all
possible sites of one enzyme. Each student will be assigned a single point mutation to
introduce into the Synechocystis glutaredoxin gene. Analysis of each mutation will range from
the enzymatic consequences of the mutagenesis to the X-ray crystal structure of the soluble,
mutated enzyme and management of the data through LIMS systems. Throughout the
course, documentation of all results will be maintained with the intention of publication once
the entire DSM space has been covered (~8000 mutations). Our aim is to make this a viral
course by pre-packaging lecture material as YouTube-style lectures and tasks for the course
to accommodate a wide variety of potential students.

The X-ray diffraction aspects of the DSM course will center on the ScreenMachine. This is a
non-threatening, robust instrument that does not require specialized personnel or extensive
instruction. Further, it does not present as great of a radiation hazard for students relative to a
full-sized x-ray instrument, so it is well-suited for this task.

Our objective is to provide these students with a workable knowledge of x-ray crystallography,
with the hope that they will pursue a more in depth study of the discipline afterward. Further,
this concept links real-world biophysical research with graduate education to accomplish
useful science for the community at large.
T-116

Study of Serine Dehydrogenase from Mycobacterium tuberculosis

1 1,2
Dana Hogan , William L. Duax
1 2
Hauptman Woodward Medical Research Inst., Buffalo, NY, United States, State Univ. of New
York at Buffalo, Buffalo, NY, United States

Mycobacterium tuberculosis, the etiologic agent of tuberculosis in humans, is responsible for the
global resurgence of the disease affecting more than 30 million people worldwide. Current
preventative drugs have been effective at inhibiting the expression of the bacterium; however
several antibiotic-resistant strains are starting to emerge. This study was undertaken to identify
and characterize the structure of a putative L-Serine Dehydrogenase (SerDh), an important
enzyme involved in the metabolic pathway of at least 8 other amino acids. SerDh belongs to the
superfamily of proteins known as the Short Chain Oxidoreductase Enzymes (SCORs), which are
important in growth and development of all organisms. The cofactor predicting sequence XR,
*
occurs 19 residues from the Gly-rich N-terminal motif, indicting NADP binding . Substrate binding
in SCOR proteins occurs within 3 loops. There are 5 quasi-conserved amino acids in 2 of those
loops that can predict a substrate for the enzyme. SerDh contains 4 of the 5 identical positions
with the known serine dehydrogenase fingerprint (AG-YGG represents 164 proteins). The 4
positions are the amino acids [G]G-YGG with the bracketed G replacing the A in the fingerprint.
A deeper understanding of the proteins that are critical to the function of Mycobacterium
tuberculosis could lead to the design more effective inhibitors that will interrupt the expression of
the disease. Support in part by: Mr Roy Carver, Stafford Graduate Fellowship, Caerus Forum
Fund and The East Hill Foundation.

*
Proteins. 2003 Dec 1;53(4):931-43.
T-119

A Fast And Fully Automated Solution For Lipidic Cubic Screening (LCP) using
Mosquito LCP

Joby Jenkins, Patricia Edwards, Rob Lewis, Joanne Franklin

TTP LabTech Ltd., Melbourn, United Kingdom

Membrane proteins such, as G-protein coupled receptors, are known to be much more
difficult to purify and crystallise than soluble proteins due to their native environment within
the lipid bilayer of the cell membrane. As a result aqueous solutions are unsuitable for their
reconstitution as they require lipids or detergents to retain their structural integrity.

The in meso crystallisation technique revolutionised the process of crystallising membrane

proteins. This method utilises highly viscous lipid mesophases to contain the membrane
proteins for crystallisation. However, there are a number of technical difficulties associated
with the LCP method which makes this process difficult to perform and challenging to
automate.

One problem is the viscous nature of the lipids which can be almost solid at room
temperature. As a result the addition of protein to the lipid and subsequent reconstitution can
be hard to achieve. In addition, the accurate dispensing of LCP, required for efficient
miniaturisation, and the precise positioning of drops required for efficient imaging of
membrane crystals present two other challenges.
®
TTP LabTech have solved this problem by developing mosquito LCP, a dedicated
instrument that offers a fully automated solution to LCP screening. This instrument offers fast
throughput, high precision and unrivalled reproducibility. Here we describe the benefits of the
instrument and how the renowned and reliable positive displacement tip technology ensures
that the LCP screening preparation is performed to the highest standard with the minimum
amount of effort.
sLPQQ

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c¡« \‹?q ¦¦\‒ K?p \‹£?b· K?f¡›‒£¡?mM?o⁄ fi K?i‒

¯°±² ³´ µ±¶·³°¶±°K?¸¹º±¶³°K?µhK?t°±»¼ ?r»¹»¼¶

In this study, the variance-covariance matrix of protein motions is used to compare several
elastic network models within the theoretical framework of X-ray scattering from crystals. A
set of 33 ultra-high resolution structures is used to characterize the average scaling behavior
of the vibrational density of states and make comparisons between experimental and
theoretical temperature factors. Detailed investigations of the vibrational density of states,
correlations, and predicted diffuse X-ray scatter are carried out for crystalline Staphylococcal
nuclease; correlations and diffuse X-ray scatter are also compared to predictions from the
TLS (translation, libration, screw) model and a liquid-like dynamics model. We show that
elastic network models developed to best predict temperature factors without regard for the
crystal environment have relatively strong long-range interactions that yield very short-ranged
atom-atom correlations. From atom-atom correlations, which are found to require more
modes to converge than is typically assumed, the diffuse X-ray scatter is computed to explore
practical implications for such models; for this we use a novel approach that decomposes the
intensity into contributions from different types of correlated motions.
T-125

Fiber diffraction, electron microscopy, and molecular modeling in studies of complex

assemblies

Wen Bian, Amy Kendall, Michele McDonald, William Wan, Gerald Stubbs

Vanderbilt University, Nashville, TN 37235, United States

Fiber diffraction has been used to determine the structures of filamentous macromolecular
assemblies that are not amenable to conventional crystallography or nuclear magnetic
resonance methods. However, many such assemblies are highly disordered and diffract
poorly even with the best available sample preparation and diffraction techniques, so that
their structures cannot be solved using fiber diffraction alone. We have obtained fiber
diffraction data for two important types of assembly, amyloids (including prions) and flexible
filamentous plant viruses, and have used a combination of fiber diffraction, electron
microscopy, and molecular modeling to derive important structural features and produce low
to medium resolution models for these systems. We are continuing to develop computational
methods to make use of the data provided by fiber diffraction and electron microscopy,
providing important constraints for helical reconstruction and molecular modeling.

Supported by NIH grant P01 AG010770 and NSF grant MCB-0743931

T-128

The Absolute Intensity Calibration of a Small-Angle X-ray Scattering Instrument with a Laboratory
X-ray Source
1 1 1 1 2
Lixin Fan , Mike Degen , Scott Bendle , Nick Grupido , Jan Ilavsky
1 2
Rigaku Innovative Technologies Inc., Auburn Hills, MI, United States, Advanced Photon Source,
Argonne National Laboratory, Argonne, IL, United States

Absolute calibration of small-angle scattering data (in units of differential cross-section per unit sample
volume per unit solid angle) is necessary for the determination of molecular weights, the number density
of particles, the scattering-length densities of phases in multiphased systems, volume fraction, the
specific surface area of the scatters and to restrict the parameters of a given model to the set which
reproduces the observed intensity. It is also a useful means for the detection of artifacts in SAS
experiments. Absolute intensities from the same sample also allows intercalibration among different
instruments. This work details the absolute calibration procedure of a small-angle X-ray scattering
instrument, the Rigaku S-Max3000. Absolute calibration was achieved by using two standards:
homogeneous and stable glassy carbon and water. The scattering intensity of glassy carbon is calibrated
by comparison with the absolute-calibrated measurements taken on the USAXS instrument located at the
32ID beamline of the Advanced Photon Source in Argonne National Laboratory. This instrument has
primary calibration capability. The scattering from water is angle-independent and only depends on the
physical property of isothermal compressibility. The absolute calibrations using two standards were
compared. The agreement of scale factors obtained using two standards suggests that precalibrated
glassy carbon can serve as a convenient standard for all type materials under study.
T-131

High-Efficiency SAXS/GISAXS/WAXS instrument for the Laboratory: Rigaku S-Max3000

Nick Grupido, Lixin Fan, Michael Degen, Scott Bendle

Rigaku Innovative Technologies, Inc., Auburn Hills, MI, United States

The Rigaku S-MAX-3000 instrument provides excellent SAXS, GISAXS and simultaneous
WAXS capabilities while maintaining maximum flexibility in controlling sample environment
[1]. This instrument utilizes a high brilliance X-ray microfocus source running at 40W of
power. Its state-of-the art design concentrates the applied power into a tiny spot which, when
coupled with a confocal graded multilayer focusing optic, yields a high intensity x-ray beam
comparable to conventional laboratory sources operating at kilowatt power. Combining this
intense beam with three-pinhole collimation, a fully evacuated beam path and a photon-
counting MWPC detector, this instrument is capable of making highly sensitive measurements
from both isotropic and anisotropic materials without neeeding desmearing corrections. A
photodiode embedded inside the beamstop allows continuous monitoring of the beam
intensity yielding a direct measurement of the sample transmission. A second optional sample
chamber allows exploration of a middle Q range without moving the detector or realigning the
beam. High throughput SAXS measurements can be performed by running a user friendly
script which automatically controls sample movement and environment. Simultaneous WAXS
measurements can be collected on image plates at scattering angles up to 68°. An automated
high weight capacity stage for GISAXS provides better than 5 arc second angular precision
and motion ranges of ½8 in plane, ½10 out of plane and ½12.5mm vertical. The high weight
o o

capacity can be utilized to support in-situ vessels or samples of all types. An alternative high
precision stage is available with sub-arc second motion but lower weight capacity. Both
stages are fully automated with automatic determination of the zero incident angle as well as
automatic collection of the scattering data. The Rigaku S-MAX-3000 is capable of
characterizing a large variety of materials, ranging from colloids of all types, cements,
nanoparticles, oils, polymers, plastics, proteins, surfactants, foods and pharmaceuticals. In
this presentation, we demonstrate Rigaku GISAXS capability by determining structural
morphology from polymer thin films.

http://www.rigaku.com/saxs/index.html
T-134

Case studies from the structural genomics of infectious disease

1,4 1,4 1,4 1,4
Anna Gardberg , Thomas Edwards , Jan Abendroth , Michelle Dietrich , Becky
1,4 1,4 1,4 1,4 1,4
Poplawski , Jameson Bullen , Jeff Christensen , Eric Smith , Nathan Ng , Taryn
1,4 1,4 1,4 1,4 3,4
Haffner , Amy Raymond , Don Lorimer , Bart Staker , Alberto Napuli , Wes
3,4 2,4 2,4 1,4
VanVoorhis , Robin Stacy , Peter Myler , Lance Stewart
1 2
Emerald BioStructures, Bainbridge Island, WA, United States, Seattle Biomedical Research
3
Institute, Seattle, WA, United States, University of Washington, Dept of Medicine, Seattle,
4
WA, United States, Seattle Structural Genomics Center for Infectious Disease, Seattle, WA,
United States

The Seattle Structural Genomics Center for Infectious Disease (SSGCID) is one of two
consortia funded by NIAID to apply genome-scale approaches in solving protein structures
from biodefense organisms, as well as those causing emerging and re-emerging disease. In
its first two years, the SSGCID has submitted ~170 protein structures to the Protein Data
Bank (PDB) and is on track to solve a further 100 per year going forward. For several
organisms, this represents the majority of PDB submissions during this time, including 100%
of the structures for Ehrlichia, Anaplasma, and Burkholderia. SSGCID’s target selection
strategy has focused on drug targets, essential enzymes, virulence factors and vaccine
candidates from a number of bacterial (Bartonella, Brucella, Ehrlichia, Anaplasma, Rickettsia,
Burkholderia, Borrelia and Mycobacterium) and eukaryotic (Babesia, Cryptosporidium,
Toxoplasma, Giardia, Entamoeba, Coccidioides and Encephalitozoon) pathogens, as well as
ssDNA and negative-strand ssRNA viruses. More than 3000 targets have been selected to
date, with >700 proteins being purified for crystallization trials. Crystallization screening and
analysis of X-ray diffraction datasets for structure solution are performed at Emerald
BioStructures. We present a selection of protein crystal structures solved at Emerald as part
of its work with the SSGCID. Individuals or groups of investigators interested in proposing a
target for structure determination at the SSGCID are requested to submit a “Target Selection
Proposal”.
T-137
The c-AMP Receptor-Like Protein CLP is a Novel c-di-GMP Receptor Linking Cell-Cell
Signaling to Virulence Gene Expression in Xanthomonas campestris
1,2 1
Shan-Ho Chou , Ko-Hsin Chin
1 2
National Chung-Hsing U., Taichung, Taiwan, National Chung Hsing University
Biotechnology Center, Taichung, Taiwan
C-di-GMP controls a wide range of functions in eubacteria, yet little is known about the
underlying regulatory mechanisms. In the plant pathogen Xanthomonas campestris,
expression of sub-set of virulence genes is regulated by c-di-GMP and also by the CAP-like
protein XcCLP, a global regulator in the CRP/FNR superfamily. Here, we report structural and
functional insights into the interplay between XcCLP and c-di-GMP in regulation of gene
expression. XcCLP bound target promoter DNA with sub-¾M affinity in the absence of any
ligand. This DNA-binding capability was abrogated by c-di-GMP, which bound to XcCLP with
¾M affinity. The crystal structure of XcCLP showed that the protein adopted an intrinsically
active conformation for DNA binding. Alteration of residues of XcCLP implicated in c-di-GMP
binding through modeling studies caused a substantial reduction in binding affinity for the
nucleotide and rendered DNA binding by these variant proteins insensitive to inhibition by c-
di-GMP. Taken together, the current study reveals the structural mechanism behind a novel
class of c-di-GMP effector protein in the CRP/FNR superfamily and indicates that XcCLP
regulates bacterial virulence gene expression in a manner negatively controlled by the c-di-
GMP concentrations.
T-143

Structure Determination and Biochemical Analysis of TR4, a Retinoid-activated Nuclear

Receptor
1,2 2 2 2 2 2
Ross Reynolds , X. Edward Zhou , Kelly Suiino-Powell , Yong Xu , Schoen Kruse , Eric Xu
1 2
Grand Valley State Univ., Allendale, Michigan, United States, Van Andel Institute, Grand
Rapids, Michigan, United States

The atomic structure of the apo form of the LBD of the orphan nuclear receptor TR4 has been
determined. Similar to the structure of COUP II TF the protein crystallizes in the
autorepressed inactive apo form. Evidence is presented for the active form of the receptor to
be a homodimer as that present in the crystal. Biochemical assays and mutagenesis studies
have been performed which show that the ligand is likely to be a retinoic Acid derivative and
that a cofactor similar to SRC-1 is necessary for ligand binding and activation. Molecular
3
modeling indicates an active site pocket in the 600 – 700 Å range, consistent with a retinoid
ligand and results of binding experiments of over 60 retinoid related ligands are presented.
Based on this lab’ s studies one can describe TR2/TR4, the COUP-TF’ s, and the RXR’ s as a
group of retinoid-activated nuclear receptors.
T-146

Structural Proteomics Effort with Protein Tyrosine Phosphatases : Experience &

Perspective
1,2 1,2
Dae Gwin JEONG , Tae-Sung YOON
1 2
KRIBB, Daejeon, Korea, Republic of, University of Science & Technology, Daejeon, Korea,
Republic of

Protein tyrosine phosphatases (PTPs) consisting of 103 human genes encoding the conserved catalytic
domains with CXXGXXR motifs [Alonso A et al, (2004). Protein Tyrosine Phosphatases in the
Human Genome, Cell 117:699-711] are an important family of signal transduction proteins, together
with other protein phosphatases and protein kinases, controlling cellular protein phosphorylation which
plays an important role in human disease conditions. We have participated in an ‘in-house’ proteomics
project, of which resources are now commercially available as more than 80 purified proteins
(www.bioneer.com) and several monoclonal antibodies (www.younginfrontier.com). Along with this
line of effort, we have contributed in the structural elucidation of more than sixteen catalytic domains
of PTPs. Among them, we have deposited eight structures of dual-specifity protein phosphatises
(DUSPs) at PDB (3EZZ, 2NT2, 2GWO, 2G6Z, 1ZZW, 2ESB, 1YZ4, 1XM2). Complementary with the
world-wide efforts of structural genomics consortium, we have focused on the structural elucidation of
catalytic domains of DUSPs, which consist of more than half of PTPs and ‘relatively’ less understood
compared to ‘classical’ PTPs. We believe that the development of specific ‘modulators’ for DUSPs is
of significant importance to further elucidate their physiological roles in molecular contexts. Together
with the progress of structural elucidation of DUSPs, we hope that the proteomics research better to
understand the physiological role of DUSPs in molecular context will be leveraged with the ‘better’
development of specific modulators among DUSPs.
T-149

1 1 2 3 1
Franck Borel , Isma Hachi , Andres Palencia , Marie-Claude Gaillard , Jean-Luc Ferrer
1 2
Intitut de Biologie Structurale, Grenoble, France, Europeen Molecular Biology Laboratory,
3
Grenoble, France, Institut de Biologie et de Technologies de Saclay, Gif-sur-Yvette, France

Mu-Crystallin (or CRYM) was first described as a major structural component of the
eye lens in Australian marsupials. This cytoplasmic protein was identified, although in much
lower quantities, in other mammals where it has been found in eye, ear, heart, kidney, brain,
muscle, skin.

CRYM is structuraly close to bacterial ornithine cyclodeaminase (OCD) and alanine

dehydrogenase (alaDH) but do not display any of their enzymatic activities. To date there is
no enzymatic activity identified for CRYM. However CRYM has been characterized as an
NADPH-dependent cytosolic T3 thyroid hormone binding protein. Thyroid hormones are
produced by the thyroid gland and play important regulatory roles in process such as growth,
metabolism, homeostasis or development. They are secreted under two forms T4 (thyroxine
or 3,5,3',5'-tetraiodo-L-thyronine) and T3 (triiodothyronine or 3,5,3'-triiodo-L-thyronine); the
most abundant T4 is later converted, by a selenium deiodinase, into the less abundant but
more active T3 form. Thyroid hormones exert their action by interacting with their cognate
nuclear receptor to regulate the transcription of target genes.

Currently the mechanism of CRYM action involves its dimerisation in the cytoplasm
followed by the binding of NADPH. NADPH activated CRYM binds T3 and induces an
increase of hormone concentration in the cytoplasm. Whereas the action of CRYM-bound T3
is suppressed, dissociation of NADPH enables the release of free T3 that can transactivate
genes expression. Abnormal CRYM expression have been linked to syndromes as diverse as
hyperglycemia, muscular dystrophy, deafness or prostate cancer.

In the our study we compare three crystal structures of mouse CRYM. We solved the
structure of the apo form, the structure of the complex with NADPH and also the structure of
the ternary complex with NADPH and T3; the first one of a NADPH-dependent cytosolyc T3
binding protein containing the two ligands. This structural analysis coupled to in silico docking
experiments, thermodynamic and kinetic parameters determination provide new insight into
the sheltering of T3 hormone by CRYM protein.
T-152

Structural Characterization of Inositol Catabolic Enzymes

Karin van Straaten, Ryan Stubbing, David Palmer, David Sanders

University of Saskatchewan, Saskatoon, SK, Canada

Our research is focused on inositol catabolic enzymes. Inositol dehydrogenase from Bacillus
subtilis (BsIDH) is the first enzyme in the myo-inositol catabolic pathway, a primary carbon
+
source for soil bacteria. BsIDH catalyses the NAD -dependent oxidation of myo-inositol to
scyllo-inosose. BsIDH is able to oxidize other substrates, including the mono-saccharides α -
D-glucose and α -D-xylose but does not oxidize β -D-glucose, D-mannose or D-galactose. IDH
also oxidizes the α -(1,6)-linked disaccharides melibiose and isomaltose. Scyllo-inositol, the
equatorial stereoisomer of myo-inositol is neither a substrate nor an inhibitor for BsIDH.
These observations indicate that an axial hydroxyl group is required for the substrate and that
the active site of BsIDH can selectively discriminate between structural variations in substrate.
IolG1 from L. plantarum is annotated as a putative inositol dehydrogenase (structural
genomics consortia). It shows 24% sequence identity to BsIDH. However, our primary
biochemical data on IolG1 indicates that this enzyme is not an inositol dehydrogenase but
shows activity towards inosose.
Understanding the structural basis of inositol dehydrogenase substrate selectivity and the
residues involved in catalysis form the basis for our structural studies of BsIDH. We are also
using X-ray crystallography to probe potential substrates for IolG1 to understand its role in
inositol metabolism.
So far BsIDH crystal structures have been solved for apo, holo and ternary complex with
inositol and inosose. These results allowed us to identify key residues involved in cofactor
and substrate binding. Recently, we have solved the crystal structures of the ternary complex
of IolG1 with an inosose product, scyllo-inositol and myo-inositol. Although both enzymes
share the same tetramer arrangement, their substrate recognition site is different.
T-155

Biological Implications of Trimer Self-assembly in Solution State and in Crystal

Structure of Mtb UreA
1 1,2
Jeff Habel , Li-Wei Hung
1 2
Lawrence Berkeley National Laboratory, Berkeley, CA, United States, Los Alamos National
Lablratory, Los Alamos, NM, United States

Crystal Structure of the Urease γ subunit, UreA, from Mycobacterium tuberculosis (Mtb) was
determined previously (RCSB: 2FVH), and revealed a homotrimetric arrangement akin to the
UreA trimers in the Urease structures from Klebsiella aerogenes (RCSB: 2KAU), and from
Bacillus pasteurii (RCSB: 1UBP). Analysis of the inter-molecular contacts strongly suggests
that the Mtb UreA self-organizes into such trimeric assembly. The oligomeric state in solution
together with a low-resolution envelop of Mtb UreA calculated from Small Angle X-ray
Scattering data further support this hypothesis. The homotrimer formation in addition to the
3
gene organization within the Mtb genome support the Mtb Urease composition of (α β γ ) .
While not having any known associated catalytic activity, the Mtb UreA has the potential to be
the driving force of the trimer of trimers formation seen with known bacterial Urease
structures. The need of oligomerization beyond catalytic efficiency might play a role in Mtb
Urease’ s extreme tolerance to environmental challenges.
T-158

High Throughput Screening Identifies Ligands that Disrupt Exonuclease-SSB

Interactions
1,2 1 1
Kenneth Satyshur , Duo Lu , James Keck
1
University of Wisconsin, Department of Biomolecular Chemistry, Madison, Wisconsin, United
2
States, University of Wisconsin, Small Molecule Screening Facility, Madison, Wisconsin,
United States

Exonuclease I (Exo1) is a DNA repair enzyme whose action is mediated by the interaction
of single strand DNA binding proteins (SSB). The far C-terminal end of SSB contains the
evolutionary conserved MDFDDDIPF sequence that binds in two separate hydrophobic sites
on Exo1 stimulating Exo1 activity. A process of chemical High Throughput Screening (HTS)
has identified several compounds that

disrupt this interaction and compete for the SSB binding site on Exo1. Crystal structures of 2
of these compounds bound to the secondary (B) site of Exo1 reveal the mechanism of this
binding. (Lu, et.al.). The hydrophobic modified phenyl groups of the ligands bind in a deep
pocket while the charges on the core of the ligands bind to the electropositive surface near
the pocket.

Thru an in-silico HTS drug docking process, we have identified more potential ligands for
these binding sites. Phase I of the in-silico HTS enriches the 340,000 compound Life
Sciences chemical database using the docking program Surflex Dock (Prof. Ajay N. Jain,
UCSF) as implemented in the Sybyl (Tripos Crop) molecular modeling program. In Phase II,
the top 1% of Surflex Dock results were further docked with Autodock4 (Garrett A. Morris,
David Goodsell, Scripps Inst.) and re-scored. The best scoring compound was further docked
using receptor flexibility for 5 charged groups in the binding pocket, and resulted in
substantially increased binding energy and a docking similar to that of the crystal structure.
Efforts are ongoing to identify more ligands that disrupt this vital interaction of DNA repair
proteins.

Lu, D, Bernstein, D.A., Satyshur, K.A., and Keck,J.L., (2010), Proc.Nat.Acad.Sci., 107, 2,
633–638.
T-162

Structural and Biochemical Characterization of Seasonal Influenza Virus Hemaggluinin

1 1 1 1 1 2
Ki Joon Cho , Ji-Hye Lee , Seokha Kang , Yi Ho Park , Jun Young Lee , Joo Yeon Lee ,
2 1
Chun Kang , Kyung Hyun Kim
1 2
Korea University, Seoul, Korea, Republic of, Korea National Institute of Health, Seoul, Korea,
Republic of

Influenza is one of the most important respiratory infectious diseases causing seasonal
epidemics or pandemics. It was reported that structural feature of antigenic region of 2009
pandemic influenza hemagglutinin (HA) is different from that of seasonal influenza HA, but
similar to that of 1918 pandemic influenza HA. We determined the crystal structure of
seasonal H1N1 HA protein and compared with that of 1918 pandemic influenza HA.
Biochemical properties of both seasonal and 2009 pandemic HA proteins were also
investigated. In order to find a universal antiviral drug for influenza virus, various plant
extracts and compounds were screened, and KC2002, SC2740A, GA007, GA1002, and TY10
were screened to show affinities to seasonal HA, but not to 2009 pandemic HA.
T-165

Structure of the catalytic domain of glucuronoyl esterase from Hypocrea jecorina

1 1 1 2 2 3
P. Raj Pokkuluri , Stephen Wood , Norma Duke , Michael Cotta , Xin-Liang Li , Peter Biely ,
1
Marianne Schiffer
1 2
Argonne National Laboratory, Lemont, IL, United States, USDA-ARS, Peoria, IL, United
3
States, Slovak Academy of Sciences, Bratislava, Slovakia

We have determined the structure of the catalytic domain (Cip2S) of glucuronoyl esterase,
Cip2 from Hypocrea jecorina (formerly known as Trichoderma reesei). This is the first
structure of the recently established carbohydrate esterase family 15
(http://www.cazy.org/fam/CE15.html). The catalytic activity of the enzyme is important in
degradation of lignocellulosic material.

The crystals of Cip2S have three independent molecules and diffracted X-rays to 1.9 Å
resolution. The structure was determined by the conventional “heavy-atom soaking” method
followed by a SAD experiment at the Structural Biology beam line, 19BM (APS). The structure
was refined to a crystallographic R-factor of 19.5% and R-free of 23.4%. The catalytic domain
Cip2S has 375 amino acids and its structure has an α /β hydrolase fold. Inspection of the
structure revealed a triad arrangement of Ser – His – Glu residues on the surface of the
protein suggesting a putative active site. To confirm the active site and to understand the
substrate binding site we have obtained crystals of Cip2S in presence of a serine inhibitor,
phenyl methyl sulfonyl fluoride (PMSF) and a synthetic substrate, methyl ester of 4-O-methyl-
D-glucuronic acid. The structure of the native enzyme and the results obtained from co-
crystallization of the enzyme with the above compounds will be presented and discussed.
T-167

Small-angle neutron scattering study of Sindbis virus produced from vertebrate and
invertebrate hosts
1 2 2,3 1,3 2
Lilin He , Amanda Piper , Flora Meilleur , Dean Myles , Raquel Hernandez , Dennis
2 1
Brown , William Heller
1
Oak Ridge National Laboratory, Center for Structural Molecular Biology, Oak Ridge, TN,
2
United States, North Carolina State University, Department of Molecular & Structural
3
Biochemistry, Raleigh, NC, United States, Oak Ridge National Laboratory, Neutron
Scattering Sciences Division, Oak Ridge, TN, United States

Understanding the life cycle of viruses that are vectored between in nature different hosts,
such as insects and mammals, presents many challenges, yet this knowledge is crucial for
addressing some of the most devastating mosquito-transmitted infectious diseases. The
Sindbis virus, an Arbovirus and prototypic alphavirus, transitions between insect and
vertebrate hosts. It has inner protein and outer glycoprotein shells separated by a lipid
membrane. Host-specific differences in the composition of Sindbis virus have been observed,
but not structurally characterized. Here, we present the results of a small-angle neutron
scattering (SANS) investigation of mammalian- and insect-grown Sindbis virus that provide
the first evidence of host-derived differences in virus structure. The non-destructive nature of
SANS allowed for the characterization without decreasing the infectivity of the Sindbis virus
particles studied. The results demonstrate that the radial position of the lipid membrane does
not change significantly, but the lipid membrane of the mammalian-grown virus contains
significantly more cholesterol. Additionally, the outer glycoprotein coat of the mammalian
Sindbis virus is more extended. The SANS data also indicate that the inner nucleocapsid
protein and the RNA of Sindbis virus interact more closely in the mammalian-grown virus than
in Sindbis virus grown in insect cells.

Research sponsored by the Laboratory Directed Research and Development Program of

Oak Ridge National Laboratory (F.M.). Dennis Brown and Raquel Hernandez are supported
by The Foundation for Research, Carson City, Nevada. This research at Oak Ridge National
Laboratory's Center for Structural Molecular Biology was supported by the Office of Biological
and Environmental Research, using facilities supported by the U. S. Department of Energy,
managed by UT-Battelle, LLC under contract No.DE-AC05-00OR22725. This manuscript has
been authored by UT-Battelle, LLC, under Contract No. DE-AC05-00OR22725 with the U.S.
Department of Energy. The United States Government retains and the publisher by accepting
the article for publication, acknowledges that the United States Government retains a non-
exclusive, paid-up, irrevocable, world-wide license to publish or reproduce the published form
of this manuscript, or allow others to do so, for United States Government purposes.
T-171

Crystal structure of the human IQGAP1 calponin homology domain.

Vinodh Kurella, David Worthylake

LSU Health Sciences Center, New Orleans, Louisiana, United States

The 190 kDa scaffold protein IQGAP1 reversibly binds to a variety of cellular proteins
including active forms of Cdc42 and Rac1, calmodulin, and actin; the latter activity requiring
the IQGAP1 amino terminus which contains a calponin homology domain. In cells, IQGAP1
forms homodimers that bind to and cross-link filamentous actin and cross-linking activity is
2+
reduced in the presence of Ca -calmodulin. Recently it has been shown that amino terminal
fragments of IQGAP1 bind to both actin and calmodulin and that calmodulin competes with
actin for binding to these fragments. In this study, we have determined the crystal structure of
the human IQGAP1 calponin homology domain (CHD). The overall structure of the protein is
very similar to other type -3 calponin homology domains such as those found in calponin,
Vav-3 and the yeast IQGAP ortholog Rng2. In the crystal, the CHD has associated into a
parallel homodimer that displays a high degree of surface-shape complementarity at an
interface that is predominantly hydrophobic in nature. Gel filtration experiments verify the
presence of a dimer in solution. Isothermal titration calorimetry indicates that the CH domain
2+
binds to Ca -calmodulin but not apo-calmodulin, via a two-site-sequential mode of binding
and that mutation of two adjacent lysine residues within the CHD significantly reduces this
interaction. Using an actin-pelleting assay and SDS-PAGE, we find that both wild-type and
2+
mutant CHD bind to filamentous actin and that pre-incubation with Ca -calmodulin does not
reduce actin binding. Interestingly, we do not detect even small amounts of calmodulin in the
pellet fraction. These results suggest that full-length IQGAP1 may associate as a parallel
homodimer and that the binding sites for calmodulin and actin on the CHD overlap to a large
2+
degree with actin able to out-compete Ca -calmodulin for binding.
T-176

The Tautomerase Superfamily and its Structural Relatives - New Insights into Possible
Functions

Marvin Hackert, Youzhong Guo, Hector Serrano, William Johnson, Jr., Christian Whitman

The University of Texas at Austin, Austin, Tx, United States

The tautomerase superfamily has been divided into five families represented by 4-
oxalocrotonate tautomerase (4-OT), 5-(carboxymethyl)-2-hydroxymuconate isomerase
(CHMI), cis-3-chloroacrylic acid dehalogenase (cis-CaaD), malonate semialdehyde
decarboxylase (MSAD), and macrophage migration inhibitory factor (MIF). 4-OT and many of
its homologues are homo- or heterohexamers composed of small (60-75 a.a. residue)
subunits while CHMI, MSAD, cis-CaaD and MIF are nearly twice that size and form trimers.
The subunits of this family share two distinguishing features – one or two beta-alpha-
beta structural motifs and a catalytically important N-terminal Pro residue. Several different
catalytic activities are known to utilize this same structural motif - tautomerase, isomerase,
decarboxylase, dehalogenase, etc.

The smaller members of the 4-OT family had been previously categorized into five
subfamilies and a representative member from each group has been crystallized and its X-ray
structure determined. In spite of knowing the X-ray structures, several members of this family
have defied attempts to identify their biological / catalytic activities. However, it is observed
that several other proteins with similar folds, but some lacking an N-terminal proline, have
now been implicated in binding and regulation. While MIF has an N-terminal proline and can
function as a phenylenolpyruvate tautomerase, its major role is immunosuppression and MIF
is known to bind to the receptors CD74, CXCR2 and CXCR4. Thus many members of the
tautomerase superfamily may play roles in receptor-based regulation instead catalysis. A
summary of these findings and a comparison of the representative structures will be
presented.

This work is supported in part by The Welch Foundation (F 1219, F1334).

T-182

Structural basis for neuropilin ligand binding.

Craig Vander Kooi, Matthew Parker, Hou-Fu Guo, Ping Xu

University of Kentucky, Lexington, KY, United States

Neuropilin is an essential cell surface receptor that function in VEGF dependent angiogenesis
and semaphorin dependent axon guidance. Accumulating evidence indicates that neuropilin
may mediate cross-talk between the two pathways but the mechanism is unclear. We
demonstrate that both VEGF and semaphorin binding to neuropilin requires a C-terminal
arginine residue. The crystal structure of the core ligand binding domains of neuropilin bound
to ligand derived peptides reveals the structural basis for this interaction. The C-terminal
residue is tightly bound in a pocket on the b1 domain of neuropilin. Further, proteolytic
processing of semaphorin is found to regulate competition of the two classes of ligands for
binding to neuropilin.
T-185

Solution Studies of DNA bound Gyrase

1 2 1 1
Nicole Baker , Steven Weigand , Sarah Maar-Mathias , Alfonso Mondragón
1 2
Northwestern University, Evanston, IL, United States, DND-CAT Synchrotron Research
Center, Argonne, IL, United States

DNA gyrase, a type II topoisomerase, is unique amongst topoisomerases due to its ability to
introduce negative supercoils into DNA. While many details of its mechanism are still not
completely understood, it is known to involve the assembly of a large gyrase/DNA complex
and coordinated combination of DNA strand movements and protein rearrangements
modulated by ATP hydrolysis. Although structures of gyrase domains have been elucidated,
structures of the intact GyrA2-GyrB2 heterotetramer or complexes with DNA are still unknown.
To establish the arrangement of its domains during the binding of a DNA substrate that
directs the reaction towards negative supercoiling, gyrase complexes with large DNA
fragments representing the starting conformational state of the catalytic cycle were
characterized. Purified Escherichia coli and Deinococcus radiodurans gyrase bound to 137 or
217 base pair DNA fragments were characterized by sedimentation velocity and small angle
x-ray scattering (SAXS) experiments and revealed elongated complexes with hydrodynamic
radii of ~70Å. Molecular envelopes calculated from these SAXS data show elongated, two-
fold symmetric molecules with the carboxy-terminal domain (CTD) of the A subunit and the
ATPase domain of the B subunit at opposite ends of the complexes. This domain placement
is supported by experiments using a mutant gyrase lacking the CTD, by DNA footprinting
analysis, as well as SAXS and AUC simulations. All SAXS models suggest an initial
arrangement where the CTDs are found near the exit gate of the protein and with the DNA
wrapping along the sides of the molecule and around the CTDs. Overall, this arrangement is
consistent with mechanisms previously proposed for gyrase, but with a different arrangement
of the CTDs.
T-188

Structure-based Antibody Engineering: Mechanism of Action and Binding Affinity

Determinants for anti-IL13 Antibodies Derived from Co-structures with Antigen

Thomas Malia, Alexey Teplyakov, Galina Obmolova, Raymond Sweet, Gary Gilliland

Centocor R&D, Inc., Radnor, PA, United States

The three-dimensional structures of antibodies and antibody/antigen complexes provide

detailed insight into epitope and mechanism of action and are valuable for guiding
engineering. Therapeutic antibodies derived from mouse sources require humanization to
minimize their immunogenicity and often subsequent affinity maturation to restore or improve
binding. We determined the structures of a set of antibody Fab fragments from various
stages of engineering in complex with their target IL-13 in order to understand their
mechanism of action and to evaluate the process of engineering that was employed (1). IL-
13 is a pro-inflammatory cytokine produced in Th2 immune cells and has been implicated in
asthma and allergy pathogenesis. IL-13 mediates its action by engaging with receptors IL-
4Rα and IL-13Rα 1 to form a heterotrimeric signaling complex. A high affinity neutralizing
antibody against human IL-13 was isolated from mouse hybridoma (m836), which was then
human framework adapted into h826 and affinity matured into am836. Using a combination
of high-throughput screening and manual optimization with microseed-matrix screening
(MMS), we crystallized the anti-IL-13 Fabs from these three stages of antibody engineering in
complex with IL-13 and determined their structures. The structures of the complexes indicate
that the epitope and paratope were preserved throughout humanization and affinity
maturation. We describe the crystallization process, how the antibodies block binding of IL-13
to its receptors, and the structural features of the engineered antibodies that contribute to
their changes in binding affinity.

Fransson, J., et al. Human Framework Adaptation of a Mouse Anti-human IL-13 Antibody. J.
Mol. Biol.(2010), doi:10.1016/j.jmb.2010.03.004
T-191

De novo structure of a putative GAF-domain protein from S. aureus by SeMet

SAD.

Kevin Battaile1, Rob Lam2, Kathy Johns2, Jean Brawn2, Vlad Romanov2, Emil Pai2,3,
Nickolay Chirgadze2,4
1
IMCA-CAT/Hauptman Woodward Medical Research Institute, Argonne, IL, United
States, 2Division of Cancer Genomics and Proteomics, Ontario Cancer Institute,
University Health Network, Toronto, ON, Canada, 3Departments of Biochemistry,
Molecular Genetics and Medical Biophysics, University of Toronto, Toronto, ON,
Canada, 4Department of Pharmacology and Toxicology, University of Toronto,
Toronto, ON, Canada

It is imperative to the survival of a cell, be it a unicellular organism or a part of a

multicellular organism to be able to sense the chemicals in it surroundings and
respond to them. This could be through chemotaxis for a motile microorganism, or as
part of the metabolic control of an individual cell in a larger organism. GAF domains
(named for cGMP-phosphodiesterases, adenylate cyclases and FlhA) represent one
of a class of chemical sensors. Here we describe the structure of a hypothetical GAF-
domain protein (SA1058) from Staphylococcus aureus. SA1058 is a 150 amino acid
protein that whose structure was determined by SeMet SAD. SAS1058 crystallized in
space group P21212 (a=109, b=36, c=38) and diffracted to 2.1Å. Three methionines
are present in the sequence but only two were identified in density with the positions
of both confirmed by anomalous difference maps, of which the position of one was
well defined while the other appeared to assume two conformations. The asymmetric
unit contains one peptide, of which residues 8-133 were sequenced in the map. The
final model has an R=0.209 and Rfree=0.288. The general structure of SA1058 is of a
5-stranded β sheet with the N-terminal α helix and C-terminal strand on one face and
a grouping of several smaller helical regions on the other face of the sheet. The
secondary structure is similar to domains found in other proteins in the PDB including
phosphodiesterase 5, profiling 1 and bacteriophytochrome. The wide role in chemical
sensing of the GAF domain in various organisms could make it an interesting target
for antimicrobial therapy.
T-194

Towards the crystal structure of a lectin purified from the marine sponge Cinachyrella
1 1 2 1 1 2
Pamela Focia , Caleb Smith , Yuka Nakamura , Bryan Copits , Martin Gill , Ryuichi Sakai ,
1
Geoffrey Swanson
1 2
Northwestern University, Chicaog, IL, United States, Hokkaido University, Hakodate, Japan

Marine sponges represent a rich source of natural products, some of which have already
been characterized and found to be medically interesting. Analysis of lectins from marine
sponges can illuminate a deeper understanding of their biologically relevant role, as well as
how they might be used as valuable tools in biomedical research. Lectins are proteins that
bind carbohydrates and agglutinate cells. The sponge in this study was collected in the waters
off of Iriomote Island in Japan. It is a yellow ball sponge of the genus Cinachyrella, however
the species is not yet known. Its purified galectin, BaL, was determined to be an allosteric
modulator of glutamate-gated ion channels of the mammalian AMPA and kainate receptor
families in physiology studies. The functional protein appears to be a ~49 kDa trimer
comprised of two ~18 kDa subunits and one ~16kDa subunit, each having a unique but
conserved N-terminal sequence.

As this protein was purified from the marine sponge, rather than expressed, very limited
amounts were available. We have thus set up two commercial crystallization screens (192
conditions) using a drop size of 200nL:200nL with 10 mg/mL protein, and obtained 6
conditions with protein crystals, and 4 crystals that allowed datasets to be measured and
processed with good statistics. Among those crystals we have identified 3 different space
groups; the crystal which diffracts to the highest resolution, 2.1Å, takes the space group P21,
with one trimer expected in the asymmetric unit.

There are two structures in the PDB that are likely to have a similar fold as BaL, and after
superimposing them, removing the parts of the structures that are different, and creating a
polyalanine search model, we have initiated molecular replacement to solve the phase
problem. However, all crystals are held in LN2 storage in case the need for derivatives arises.

That we have only the N-terminal sequence of 20 amino acids for each monomer makes this
an exciting project of sequencing by X-ray crystallography!
T-197

Diffuse scattering in scanning x-ray nanodiffraction as a probe for high-resolution

mapping of strain distribution around epitaxial nanostructures
1 2 2 1 1 2
Tao Sun , Zixiao Pan , Xujing Xie , Zhonghou Cai , Jin Wang , Vinayak Dravid
1 2
Argonne National Laboratory, Argonne, IL, United States, Northwestern University,
Evanston, IL, United States

In the recent decade, the unexpected behavior and enhanced properties of functional
materials, induced by the spatial confinement and dimensional constraints, have been
extensively reported. Such constrained nanostructures are believed to be one of the central
themes in materials science, because of their significance in potential applications and
fundamental scientific underpinning. Although considerable efforts have been spending on the
fabrication/synthesis and function assessments of nanopatterned systems, quantitative
structural characterization has remained elusive, despite its importance for elucidating the
microstructure-property relationship. Strain issue in electronic and magnetic materials is a
good manifestation of confinement effects, and possessing powerful tools for measuring
strain is prerequisite for strain engineering and property controlling.
Scanning x-ray nanodiffraction (SXND) is one of the very few techniques that can be
used to investigate the local strain distribution of nanostructures. However, in the traditional
SXND experiment, strain information is obtained by evaluating Bragg diffraction signal from
either substrate or nanostructures. It faces great challenges when applied for small-strain
systems, because the strong Bragg diffraction from the un-strained lattices of single-
crystalline substrates can easily shadow the small perturbation induced by the structural
imperfection. In order to solve this problem, we developed a novel approach based on SXND
technique for probing spatially varying and small values of strain at/around individual epitaxial
nanostructures. By presenting an example of CoFe2O4/MgO system, i.e. single-crystalline
epitaxial CFO nanolines on (100) MgO substrate, we demonstrate that diffuse scattering
obtained by setting the incident angle slightly off the Bragg angle can be used to quantitatively
reveal the nanostructure-induced lattice imperfection in the system. The results indicate an
edge-induced strain distribution, which is consistent with the strain simulation based on the
edge-force model. Moreover, the shape parameter in the scattering intensity line-fit can
differentiate the contribution of mosaic structure and elastic residual strain in a quantitative
manner. We show the strain map around an oxide-on-oxide nanostructure, which has
extremely small strain values and cannot be effectively characterized by other techniques. We
believe that a thorough understanding of strain issues in such spatially and dimensionally
confined (oxide) nanostructures will not only facilitate the elucidation of their size-dependent
behaviors, but also guide for development of novel devices based on strained functional
oxides for advanced applications.
T-203

Flurbiprofen Tris polymorph I: the slog to R = 11%

Carl Schwalbe, Miren Ramirez

Aston University, Birmingham, United Kingdom

We have systematically studied a series of salts of the anti-inflammatory drug flurbiprofen,

starting with the t-butylammonium salt and progressively changing methyl to hydroxymethyl
groups, ending up with the Tris salt.

Me + CH 3 + CH 2 OH
H3N CH 3 to H3N CH 2 OH
COO - CH 3 CH 2 OH
F

This salt was prepared by mixing equimolar amounts of flurbiprofen and Tris in acetonitrile
solution and harvesting the precipitate that formed. Single crystals grew from different
solvents as different polymorphs. Recrystallization from methanol gave polymorph I, but
methanol:acetonitrile 40:60 yielded polymorph II. Although crystals of polymorph II diffracted
well, polymorph I gave small poorly diffracting crystals with Z’=2. Data collected by the
National Crystallography Service on a powerful conventional small molecule diffractometer
revealed the molecular connectivity, but the R factor never went below 20%. Next, we raised
the stakes by requesting re-collection of data on beamline I19 of the Diamond synchrotron.
The specimen crystal measured 0.1 x 0.01 x 0.01 mm. These data gave a stable refinement,
3
but unfortunately it converged at R>15%, and numerous peaks around 1 e/Å remained in the
difference map. Some disorder was apparent: the fluorine atom could be either side of its
benzene ring, and enantiomer discrimination was imperfect with some switching of H and Me.
Other peaks were uninterpretable. Eventually, inspection of the coordinates revealed that the
-
two independent cations and COO were related by a pseudo-glide plane while the biphenyl
units were related by a pseudo-translation! The extra peaks on the difference map arose
from applying the “opposite” pseudosymmetry operation, i.e. the translation, to the cations.
3
Now R=11% and the largest difference peak is 0.40 e/Å . We thank Drs. S. Callear and R. W.
Harrington and Prof. W. Clegg for data collection and Bristol-Myers Squibb for support.
T-206
II
Seeing is Believing? The first structurally characterized [4×4] Ni 16 Grid by Designed
Self-Assembly.

Louise Dawe, Konstantin Shuvaev, Laurence Thompson

Memorial University, St. John's, Newfoundland, Canada

[1,2]
Tritopic and tetratopic bis-hydrazone ligands have been prepared, and produce [3x3]M9
[1-4]
and [4x4]M16 grids respectively, by self-assembly reactions with metal salts (M = Mn(II),
Cu(II), Co(II)). These molecules have attracted much attention for their possible
nanotechnological applications, and with molecular footprints of ~ 3 nm x 3 nm, they could
eventually lead to the production of magnetic nanoparticles. Until now, however, there has
been no report any [nxn] Ni(II) grid with nuclearity exceeding n = 3, and the one report where
[5]
n = 3, did not yield a satisfactory single crystal X-ray structure . The structural and magnetic
characterization of a novel [4x4]Ni(II)16 grid (Fig. 1) will be presented. While this is a very
exciting result, details have yet to be published, and, as will be discussed, beg the question,
should they be published?

1. Dawe, L.N., Shuvaev, K.S., Thompson, L.K., Inorg. Chem., 2009, 48, 3323-3341.

2. Dey, S.K., Abedin, T.S.M., Dawe, L.N., et al. Inorg. Chem., 2007, (46), 7767-7781.

3. Dey, S.K., Thompson, L.K., Dawe, L.N. Chem. Commun. 2006, 4967-4969.

4. Dawe, L.N., Thompson, L.K. Angew. Chem., 2007 (46), 7440-7444.

5. Niel, V., Milway, V., Dawe, L.N., Grove, H., et al. Inorg. Chem., 2008, 47, 176-189.

II
Fig.1: [4x4] Ni 16 grid.
T-209

The HB2A High Resolution Powder Diffractometer at the High Flux Isotope Reactor

Ovidu Garlea, Clarina dela Cruz

Neutron Scattering Science Division, Oak Ridge National Laboratory, Oak Ridge, TN, United
States

Neutron powder diffraction is increasingly recognized as one of the most powerful techniques
for studying the structural and magnetic properties of advanced materials. We are presenting
an overview of the HB2a diffractometer that has recently been installed at the High Flux
Isotope Reactor in Oak Ridge. The instrument has been designed to provide an optimum
balance between high neutron flux and high resolution. Due to its versatility the diffractometer
can be employed for a large variety of experiments, but it is particularly adapted for
refinements of structures with large interplanar spacings as well as of complex magnetic
structures. Instrument capabilities will be illustrated by recent studies undertaken on various
materials ranging from rare-earth iron oxyarsenides to zeolites.
T-212

An Advanced Small-Angle X-ray Scattering Station for Structural Biology: Automated

High Throughput Solution Scattering, Time-Resolved Studies and Beyond.

Thomas Weiss, Ping Liu, Anne Martel, Marc Niebuhr, Hiro Tsuruta

Stanford University, Menlo Park, CA, United States

Beamline 4-2 at the Stanford Synchrotron Radiation Lightsource (SSRL) is a small angle x-
ray scattering/diffraction facility dedicated to structural studies on mostly non-crystalline
biological systems. The facility recently received an extensive array of optics and in-hutch
instrumentation upgrades to take full advantage of the high brightness beam produced by the
third generation storage ring SPEAR3. The instrument features a pin-hole geometry camera
configurable in one of seven sample-to-detector distances ranging from 0.3m to 3.5m,
-1
providing access to the Q-range 0.003-4.2 Å (at 11keV). It is equiped with a high-
sensitivity/stability CCD detector as well as a silicon pixel array detector, both of the latest
generation. The latter achieves high frame rates up to 300Hz, suitable for time-resolved
studies. The instrument and all experiments are controlled by a version of Blu-Ice software
customized for non-crystalline diffraction experiments. We have developed several sample
handling devices specific to each distinctive class of experiments such as a stopped-flow
rapid mixer for time-resolved solution x-ray scattering and a humidity controlled sample
chamber for lipid/fiber studies. Our high-throughput solution x-ray scattering data collection
system integrates an automatic sample changer in the 96-well microplate format with the Blu-
Ice data collection tab SolSAXS. The system achieves high throughtput without compromizing
high reliability in data collection. Our data processing software SasTool keeps up with the
high throughtput of data collection, generating fully processed data in real time. Remote data
collection is currently in trial. The high level of beam and detector stabilities enables routine
use of dilute protein solutions in the sub-mg/ml range. The multilayer monochromator option
14
provides an extremely high beam flux level of approx. 10 photons/s for time-resolved
studies, achieving sub-millisecond time resolution in single trigger events. This presentation
will discuss relevant characteristics of the instrumentation on BL4-2 and a few recent scientific
applications in structural molecular biology, complementing crystallographic studies.
T-215

Capabilities at GM/CA CAT Beam Lines at the APS

1 1 1 1
Nagarajan Venugopalan , Michael Becker , Stephen Corcoran , Mark Hilgart , Oleg
1 1 1 1 1
Makarov , Craig Ogata , Sudhirbabu Pothineni , Ruslan Sanishvili , Sergey Stepanov ,
1 1 1,2 1
Shenglan Xu , Derek Yoder , Janet Smith , Robert Fischetti
1 2
Argonne National Laboratory, Argonne, IL, United States, University of Michigan, Ann Arbor,
MI, United States

GM/CA CAT operates two independent undulator beamlines, 23ID-B and 23ID-D, at the APS.
Both beamlines are rapidly tunable for MAD and are equipped with ALS-style sample
automounters. Several features of the beamlines have been developed or improved over the
past year, all controlled within the Bluice-EPICS interface.
Over the past three years we have provided Micro-diffraction capabilities at both beamlines
through versatile collimator systems. Recently we have developed a robust quad-collimator
monolith containing user-selectable beam-sizes of 5 μ m, 10 μ m, 20μ m or "full beam" (~25
mm (V) x ~75 mm (H) FWHM). Several groups have used these mini-beams to solve
structures that otherwise would not have been possible.
For challenging samples, such as, invisibly small membrane protein crystals in lipidic cubic
phases, larger inhomogeneous crystals or multiple crystals, we developed a semi-automated
rastering procedure that records diffraction images over a user-defined region of the sample
on a 2-D grid. Automated analysis, or visual inspection, of each diffraction pattern indicates
the best position to collect data. A similar semi-automated rastering feature based on
fluorescent signal is also available for locating metallo-proteins, Se-Met derivatives or back
soaked heavy atom derivatives. Also, to facilitate data collection on radiation sensitive
crystals, we recently developed an automated procedure to collect data along a user-defined
3D vector.
The automounter is used by nearly two-thirds of GM/CA CAT users to screen and collect
data. The Web-ice package has been implemented to facilitate strategy calculations and
scoring of samples. Remote access is available to experienced GM/CA CAT users.
GM/CA CAT is supported by NIGMS and NCI within the NIH.
T-218

The Gulf Coast Protein Crystallography Consortium Beamline at the Center for
Advanced Microstructures and Devices.
1 2
Henry D. Bellamy , Robert O. Fox
1 2
Louisiana State University, Baton Rouge LA, United States, University of Houston, Houston
TX, United States

The Gulf Coast Protein Crystallography (GCPCC) beamline is a fully equipped

tunable MAD experimental station at the CAMD synchrotron in Baton Rouge LA. The
beamline has been in operation since 2002. Construction was jointly funded by the NIH and
NSF through a Major Research Instrumentation grant to a consortium of Universities in LA,
TX and OK. The beamline is open to general users as well as GCPCC consortium members.
Access is free for non-proprietary users.

The detector is a MAR (Rayonix) 165 mm CCD mounted on a MAR dtb goniostat.
The beamline is equipped with a fluorescence counter and all standard equipment and
software required for MAD data collection. Data collection uses the marccd program supplied
by the detector vendor and the beamline is controlled through the MX software package,
which has been customized and extended to meet our requirements. Users have access to a
small wet lab next to the beamline with an anaerobic chamber, a cold room, and incubators
for sample storage. The beamline has an active “FedEx” data collection program.

Because of the relatively low energy of the CAMD ring (1.3 GeV) the beamline uses a
7 T single-pole superconducting wavelength shifter as its source. We have recently received
a NSH MRI grant to replace the wavelength shifter with a 7.5 T 11-pole wiggler which will
increase the flux about 8 fold. The new wiggler will become operational in the fall of 2011.

We will describe the beamline and the associated equipment and software. We will
also briefly describe CAMD and its ring and the other beamlines at CAMD. Finally we will
discuss our future plans for the beamline and for structural biology at CAMD.
M-219

The PSI Structural Biology Knowledgebase – Search Online for Protein Seqeunces,
Structures, Models, Methods, and More
1 1 1 1 1
John Westbrook , Margaret Gabanyi , Wendy Tao , Raship Shah , Andrei Kouranov , Torsten
2 2 2 3 3
Schwede , Konstantin Arnold , Lorenza Bordoli , Paul Adams , Lester Carter , Wladek
4 1
Minor , Helen Berman
1 2
Rutgers, the State University of New Jersey, Piscataway, NJ, United States, Swiss Institute
3
of Bioinfomatics & Biozentrum, Basel, Switzerland, Lawrence Berkeley National Laboratory,
4
Berkeley, CA, United States, University of Virginia, Charlottesville, VA, United States

¿ÀÁ? ÂÃÄÅÁÆÇ? ÈÅÃÉÊÅÉÃÁ? ËÇÆÅÆÌÅÆÍÁ? ÈÅÃÉÊÅÉÃÌÎ ÏÆÄÎÄÐÑ? j‹›• ¡ £¡ \ ¡? Gorh? rajaK? ‹¡•? tqkY
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s⁄¡?orh?raja? ?¢·‹ ¡ ? „? ⁄¡?mhflrM

T-221

The Structural Biology Center User Program at the Advanced Photon Source, Argonne
National Laboratory

Stephan L. Ginell, Randy Alkire, Changsoo Chang, Marianne E. Cuff, Norma E. C. Duke,
Gofron Kazimierz, Youngchang Kim, Krzysztof Lazarski, Jack Lazarz, Mike Molitsky, Bogi
Nocek, Jurek Osipiuk, Soon Ok Park, Gerd Rosenbaum, Frank J. Rotella, Kemin Tan,
Rongguang Zhang, Andrzej Joachimiak

Argonne National Laboratory, Argonne, IL, United States

The Structural Biology Center (SBC) at Argonne National Laboratory operates two beamlines
- one insertion device (ID) and one bending magnet (BM) - at Sector 19 of the Advanced
Photon Source as a national user facility for macromolecular crystallography. These
beamlines continue to be one of the most powerful, capable and productive X-ray sources for
structural biology in the US. The beamlines can deliver very low angular divergence X-ray
micro-beams onto micrometer-size crystal samples mounted using robotic systems, thereby
permitting structural biologists to study the structures of large and complex molecular systems
at atomic resolution. Diffraction from these crystals is recorded on large, fast, and efficient
CCD area detectors, and is processed on high-performance, integrated computing systems
with advanced control and data analysis software designed specifically for the SBC.

Presentation will highlight new and upgraded advances to the SBC beamlines including: on
axis crystal viewing, beam visualization, point and click sample alignment, auto-loop
alignment, adjustable mini beam with apertures to 5m, ACTOR crystal mounting robotic using
either Rigaku or Uni-Puck/ALS sample pucks, remote data collections options, new
fluorescence scanning, auto energy changes, data bases and interfaces and advances found
in HKL3000 a program suite for data collection and processing, structure solution and model
building in near real time. Some recent important SBC developments and research highlights
from the sector 19’s PDB deposits will be presented.

The SBC beamlines offer the most efficient worldwide data collection and structure
determination systems currently available for protein crystallography and have demonstrated
record productivity (2727 PDB deposits (on average 389 per year in the past 3 years) and
1006 publications). In 2009, the Nobel Prize in chemistry was awarded for research on
ribosomes, a major part of which was performed at the SBC 19-ID beamline.

Beamtime on the sector 19 beamlines is available to the crystallographic research community

via the APS peer reviewed proposal system. The proposal evaluation is based upon the
projects’ scientific merit, need for synchrotron time, feasibility of conducting the experiments
at the SBC, and the probability for success of the project. The highest rated proposals will
receive beamtime first.

Information on the user program and the sector 19 beamlines will be provided and can also
be obtained from the SBC web site (http://www.sbc.aps.gov).

This work is supported by the U.S. Department of Energy, Office of Biological and
Environmental Research, under Contract DE-AC02-06CH11357.
T-224

Laser Induced Protein Crystallization

Neela Yennawar, Sava Denev, Venkataraman Gopalan, Hemant Yennawar

Pennsylvania State University, University Park, United States

Screening of proteins for crystallization under laser irradiation was investigated with six
proteins ribonuclease B, glucose dehydrogenase, lysozyme, sorbitol dehydrogenase, fructose
dehydrogenase and myoglobin. Shining 532nm green circularly polarized laser light with pico-
second pulse and 6mW power for 30 seconds, on newly setup protein drops, showed marked
improvement in the number of screen conditions amenable for crystal growth as compared to
the control drops under identical conditions but without laser exposure. In some proteins
bigger and better quality crystals were formed. The speed of crystallization increased in most.
During laser irradiation, the amount of precipitation in the screened drops increased indicating
a transient decrease in protein solubility. The resolution of x-ray diffraction of crystals grown in
drops with laser induced nucleations improved in some examples. At the optimised laser
settings, there was no deleterious effect of the laser on crystal growth. Crystal structure
solution confirmed that the protein had not degraded due to the laser radiation.
T-227

High-Brilliance Home-Lab X-Ray Sources: Status and Future

Carsten Michaelsen, Jürgen Graf, Jörg Wiesmann

Incoatec GmbH, Geesthacht, Germany

Modern microfocus X-ray sources define the state-of-the-art for a number of applications such
as protein crystallography and small-angle scattering in the home lab. These sources have
small source sizes of 100 µm or smaller. They are usually combined with multilayer mirrors as
beam-shaping devices that image the source spot onto the sample position, magnified to a
suitable size, and deliver a parallel or focused monochromatic beam.
11
Microfocusing rotating anode systems deliver flux densities in the range of 10 photons/(s
2 2
mm ) at power loads of up to 20 kW/mm when combined with synthetic multilayer mirrors.
However, these sources are expensive and need regular and, sometimes, time-consuming
maintenance.

Low power microfocus sealed tube sources such as the Incoatec Microfocus Source “IµS”
represent an interesting low-maintenance alternative to rotating anode generators. Power
loads of several kW/mm in anode spot sizes of Ò 50 µm deliver a small and highly brilliant
2
10 2
beam. The IµS delivers a flux density of up to 10 photons/(s mm ) in a focused beam
(FWHM = 0.11 mm, 7.6 mrad) suitable for most protein crystals.

Emerging microfocus X-ray sources based on liquid-metal-jet technologies show even higher
2
power loads up to 500 kW/mm , an order of magnitude higher than possible with solid target
12 2
sources, and intensities up to 10 photons/(s mm ) together with a relatively low power
consumption and reduced maintenance.

We will present selected results from several microfocus source systems to demonstrate their
potential for crystallography and small-angle scattering.
T-230
Complementary Technology To The Synchrotron

Pierre Le Magueres, Angela Criswell, Joseph Ferrara

Rigaku Americas Corporation, The Woodlands, Texas, United States

X-ray diffraction data collection at synchrotron beam lines is a critical tool for
crystallographers to resolve protein crystal structures. The characteristics of the x-ray beam
(high intensity, low divergence, very small size) and its tuneable wavelength are features
required for anomalous diffraction-based phasing methods, for high-resolution structure
refinements and for data collection on weakly diffracting samples or samples with long unit
cell parameters. In addition, the proliferation of synchrotron beam lines in many countries and
the increased availability beam time has made synchrotron facilities accessible to virtually
every crystallographic laboratory in the world.
To use the synchrotron most effectively, it is absolutely essential that crystallographers arrive
prepared with samples whose quality as well as cryo-conditions have been previously tested
and optimized at home. To address this, Rigaku has developed new instruments that will help
researchers screen large numbers of samples in their own lab and recover those suitable for
synchrotron data collection. We will first present the new ‘ScreenMachine’, a simple and self-
contained x-ray diffractometer optimized for fast and easy screening of macromolecular
samples. We will also report on the improvements made to the automatic sample changer
ACTOR , as well as on the latest technologies now offered to the home lab in the area of
hybrid pixel array detectors (Pilatus) and multilayer optics with a smaller beam size.
T-233

X-ray compatible microfluidic platforms for membrane protein crystallization

Sudipto Guha, Sarah Perry, Paul Kenis

Department of Chemical and Biomolecular Engineering, Univerity of Illinois at Urbana

Champaign, Urbana, IL, United States

Membrane proteins play a crucial role in many important biological processes including
energy and material transduction across cellular membranes, molecular recognition and
immune response. Efforts into understanding the function of these proteins have been
severely hampered by difficulty in obtaining high quality crystals. These proteins are
amphiphilic in nature and extremely sensitive to the surrounding environment. To obtain
crystals, the proteins have to be isolated from the cellular membrane into artificial membrane
1
like environments without denaturing them. We use a technique called in meso crystallization
which uses lipids to create mesophases in which proteins are stabilized.

We have created microfluidic platforms which allow creation of these mesophases by mixing
aqueous protein solution with highly viscous lipids. Addition of salt and precipitant leads to
nucleation and growth of crystals. We have successfully validated a polydimethylsiloxane
2
(PDMS) based microfluidic platform to crystallize Bacteriorhodopsin in meso. However
PDMS attenuates X-rays; hence on chip analysis of crystals is not feasible.

We present here an X-ray compatible chip comprising of cyclic olefin copolymer (COC) and a
thin PDMS layer needed for valve actuation. As proof of concept we have crystallized soluble
proteins on-chip and obtained a full data set for the same. We are currently working on
validating this platform with membrane proteins. Further uses of such a platform include on-
chip screening using minute quantities of protein, studying phase behaviour and interaction
between various lipids and using a cryocooled chip to minimize radiation damage to crystals.

References:

1. Cherezov V. et al., 2007, Science, 318, 1258–1265

2. Perry S.L. et al., 2010, CG&D, 9, 2566–2569

T-236

Testing Protein Crystals with X-rays in Crystallization Plates.

Tadeusz Skarzynski

Oxford Diffraction, Yarnton, Oxfordshire, United Kingdom

Protein crystals are usually difficult to grow and can suffer damage on
their way from the crystallization drop to the X-ray beam. The damage may be
caused by manual handling, change of environment during harvesting, adverse
effects of cryo-protection solutions and the dramatic change of temperature due
to cryo-cooling. Traditional X-ray experiments to test crystals take time and
effort and are often inconclusive.

Characterization of protein crystals with X-rays, in-situ, without needing

to extract crystals from the crystallization plate allows establishing a “base line”
for crystal quality, and evaluating resolution limits before crystals are subjected
to any manipulation. The in-situ testing also allows quickly distinguishing
between salt and protein crystals, test harvesting, soaking and cryoprotectant
conditions and selecting the best crystals for data collection.

We will show how the in-situ testing, both at synchrotron beam lines
and using the Oxford Diffraction PX Scanner system for home labs can be
used as a powerful tool providing valuable feedback at various stages of crystal
handling. Several examples of significant variation of diffraction properties of
crystals grown from the same conditions will be shown and discussed,
highlighting the importance of critical assessment of crystal quality at room
temperature in some cases.

Initial results of using the in-situ diffraction to detect ligand and heavy-atom binding will be
presented and discussed as well.
T-239

New micro-beam beamline at SPring-8, targeting at protein micro-crystallography

1 1 1 1 1
Masaki Yamamoto , Kunio Hirata , Go Ueno , Yoshiaki Kawano , Takaaki Hikima , Atsushi
1 1 1,2 1,2 1
Nisawa , Hironori Murakami , Nobutaka Shimizu , Takashi Kumasaka , Takashi Tanaka ,
1,2 1,2 1,2 1,2
Sunao Takahashi , Tomoyuki Takeuchi , Hirokatsu Yumoto , Haruhiko Ohashi , Shunji
1,2 1
Goto , Hideo Kitamura
1 2
RIKEN SPring-8 Center, 1-1-1 Kouto, Sayo, Hyogo 679-5198, Japan, SPring-8/JASRI, 1-1-1
Kouto, Sayo, Hyogo 679-5198, Japan

In order to collect proper diffraction data from outstanding micro-crystals, a brand-new data
collection system should be designed to provide high signal-to noise ratio in diffraction images.
SPring-8 and KEK-PF are currently developing two micro-beam beamlines for Targeted
1)
Proteins Research Program by MEXT of Japan. At SPring-8, a new undulator beamline
dedicated for protein micro-crystallography, named RIKEN Targeted Proteins Beamline
(BL32XU), is under construction, which will start user operation from May 2010.

The beamline is designed to provide the stabilized and brilliant micro-beam to collect high-
quality data from outstanding micro-crystals. A small sized and highly brilliant X-ray beam with
size of a micrometer will be providing high S/N data by both increasing reflection intensities
and reducing background scattering. An in-vacuum undulator and K-B mirrors fabricated with
2)
Elastic Emission Machinery technique will be equipped for the light source and the micro-
focusing optics, respectively. The beam size is variable from 1 to 20 Óm with high-precision
slits at virtual light source according to designed experiments. The initial result of beamline
commissioning showed the minimum beam size at sample position corresponds to 1 x 1 Óm
2

with 6 x 10 photons/sec/Óm .
10 2

At end station, R&D for high-precision diffractometer, high-efficiency area detector, sample
auto-changer, and sample environment suppressing background scattering are in progress.
Support of real-time damage monitoring system for radiation sensitive micro-crystals is also
being planned.

We will present the current status and the future prospects of protein micro-crystallography at
SPring-8.

This study was supported by Targeted Proteins Research Program from the Ministry of
Education, Science and Culture (MEXT) of Japan.

[1] http://www.tanpaku.org/e_index.html

[2] Mimura H. et.al, JAPANESE J. Appl. Phys. 44, L539-L542 (2005).

T-242

Polymorphism in molecular crystals via charge density distribution

1 2 2 1
Mikhail Antipin , Konstantin Lyssenko , Yulia Nelyubina , Tatiana Timofeeva
1 2
New Mexico Highlands University, Las Vegas, NM, United States, Institute of
Organoelement Compounds RAS, Moscow, Russian Federation

To consider different factors affecting the stabilization of a particular polymorph we used the
analysis of the electron density distribution function Ô(r) in crystal within the “Atoms in
Molecules” (AIM) theory. This experimental X-ray diffraction approach allows comparing the
topological characteristics of chemical bonds, atomic charges and effective atomic volumes.
Such way of description is substantially more sensitive to the difference in the intermolecular
interaction patterns in comparison with the classical approach based on the analysis of inter-
and intramolecular geometrical parameters. The usage of the AIM approach allows
distinguishing the bonding interactions from all other contacts in a crystal by means of bond
critical points, and estimating their energy values with high accuracy and, thus, to obtain the
energy of a crystal lattice. It should be noted that the difference between the crystal lattice
energies obtained from the X-ray diffraction data and sublimation enthalpy measured
experimentally in many cases are as small as 0.2 kcal/mol. In the current presentation the
results of experimental charge density analysis in the series of polymorphs of acetaminophen
(paracetamol), p-dichlorobenzene, triphenylphosphine oxide and sulfide and other will be
discussed and the benefits of this approach for the analysis of different factors, governing
stabilization of particular form, will be demonstrated. Data on relative stability of polymorphs
are important to define active pharmaceutical ingredient that satisfy drug manufacturers with
their high solubility, processability, and biological action.
T-245

More flux – Less background: New improvements in low power microfocus beam
delivery systems for diffraction and SAXS experiments

Vincent Roger, Sergio Rodrigues, Peter Hoghoj

XENOCS, Sassenage, France

Microfocus sealed tube systems are increasingly used in single crystal applications replacing
more and more traditional high power rotating anode sources for small crystal analysis. These
solutions out-perform traditional x-ray generators with higher brilliance x-ray beam despite low
power and benefit from low maintenance and low facilities requirements.

Nevertheless so far microfocus sealed tube systems’ performances remained significantly

lower compared to new generation of microfocus rotating anode sources.

We will present Xenocs new developments in the field of beam delivery and beam
conditioning systems enabling the optimum use of low power high brightness microfocus
sources. These developments include both new aspheric multilayer optics with increased
capture angle and improved focusing properties as well as new collimation devices for
reduced background signal.

Overall performance in terms of useful intensity is thus increased by a factor two or more
compared to previous generation of microfocus sealed tube systems narrowing the gap with
microfocus rotating anode generators.

Comparative data, for single crystal diffraction and SAXS applications, acquired in
collaboration with our academic partners will be shown to illustrate improved beam properties
impact.
T-248

Development and Performance of Microfocusing Source

and Multilayer Optics based Beam Solution for Crystallography

Bonglea Kim, Boris Verman, Doug Wilcox, Roman Samokyszyn, Mike Young, Licai Jiang

Rigaku Innovative Technologies, Auburn Hills, United States

Beam solutions based on microfocusing source and multilayer optics technology was first
developed and applied to protein crystallography and small angle x-ray scattering by Rigaku
more than ten years ago. The technology developed at Rigaku offers the best performance in
this product category, which includes the highest resolution and intensity while lowering cost
and making operation easier for users. Yet the technology is still evolving, and the
performance continues to improve. In this presentation, we will review major issues in the
development of this technology and illustrate the major system parameters which are key to
excellent performance. These issues include fundamental principles, major engineering
issues, past achievements and current status. Particularly, we will discuss the development
of x-ray sources, x-ray optics and the close integration of these two key technologies. These
discussions will offer some essential methodologies in evaluating the performance and
avoiding confusion. Applications to macro and small molecule crystallography and SAXS will
be discussed.
T-252

API crystallogenesis probed by second harmonic generation microscopy

Garth Simpson, Duangporn Wanapun, Umesh Kestur, Lynne Taylor

Purdue University, West Lafayette, United States

Second order non-linear optical imaging of chiral crystals (SONICC) is investigated as a

selective probe for characterizing crystallinitiy in active pharmaceutical ingredient (API)
formulations. Second harmonic generation, or the frequency doubling of light, is symmetry
forbidden in amorphous media, but allowed for all crystals with a chiral unit cell.
Consequently, SONICC provides excellent selectivity for trace crystallinity of APIs and can be
performed rapidly over large fields of view for diverse samples. Studies with model
compounds (griseofulvin and chlorpropamide) demonstrate detection limits of SONICC for
crystallinity better than 1 part in 100 billion by volume, corresponding to a >9 order of
magnitude improvement in % crystallinity compared to existing commonly used
conventional methods (e.g., x-ray diffraction). The absence of a background response from
disordered media allows the development of simple image analysis algorithms for automated
quantification of nucleation rates, crystal growth rates, and activation energies for nucleation
from a single set of measurements. Studies with powdered samples demonstrate the ability to
easily quantify the residual 0.05% crystallinity remaining after exhaustive cryo-milling (S/N
>1000).
T-258

X-RAY POWDER DIFFRACTION STUDY FOR THE Cu2Cd1-zFezSnSe4

ALLOY SYSTEM
1 1 2 2 2
Jose Henao , Mario Macias , Miguel Quintero , Ekadink Moreno , Manuel Morocoima ,
2 2 2 2
Eugenio Quintero , Pedro Grima , Rafael Tovar , Pablo Bocaranda
1 2
Universidad Industrial de Santander, Bucaramanga, Colombia, Universidad de Los Andes,
Merida, Venezuela

Room temperature X-ray powder diffraction (XRPD) measurements were carried out on
polycrystalline samples of the Cu2Cd1-zFezSnSe4 alloy system, in steps of approximately 0.1
in z. The diffraction patterns were used to show the equilibrium conditions and to derive
crystallographic parameters values. In each case, the XRPD reflections were indexed and the
calculated lattice parameters were refined, and then, the initial values were estimated.
Afterward, the XRPD patterns were refined by the whole pattern fitting using the Rietveld
method. The results confirmed that the tetragonal stannite α (I-42m) structure occurs across
the whole composition range at room temperature.

From analysed data, was found that line splitting, viz. [(220), (204)], [(312), (116)], etc., due to
tetragonal distortion c/a < 2 of the stannite structure, is observed across the whole
composition range, and the separation of the splittings decreases as the composition z is
increased. Furthermore, were found that the values of c/a increase nonlinearly from about
1.955 for z=0 to 1.975 for z=1. The deviation of the crystallographic parameter c from the
Vegard law was related to the nonlinear variation of the internal distortion parameter σ with z.

In the observed tetrahedrally coordinated stannite structure, each Se anion is surrounded by

four cationic sites, i.e. two Cu, one Sn and one M, where the mixed cation M is given as
M=(1-z)Cd + zFe, and each cation is similarly coordinated by four Se atoms.

The results shows that as z is increased, the size of the mixed cation M is reduced and this
resulting in an overall increase of the Se-M-Se and a reduction of the Se-Sn-Se angles.

Additionally, the magnetic measurements showed that the amounts of extra phases were
found to decrease considerably for samples which were re-melted in compressed form.
T-267

Diffraction Data from Liquid Crystal Elastomers: Versatile Display and Analysis
Techniques

John Konnert, Christopher Spillman, Jeffrey Deschamps, Jawad Naciri, Banahalli Ratna

Naval Research Laboratory, Washington DC, United States

Diffraction patterns of liquid crystal elastomers may contain broad features due both to short
range order and to rotational disorder of domains. Techniques have been developed for
interpolating, compressing or expanding the 3D diffraction data obtained with a CCD detector
and placing the resulting scaled diffraction pattern into either a (256,256,256) or a
(512,512,512) array. One then applies a rotation matrix to bring the pattern into the desired
orientation for analysis. Viewing and analyzing the properties of constant Intensity surfaces,
spherical half shells, and planes of data has proved useful. It is not necessary to rotate the
entire array of data ,x , by the rotation matrix A, x'=Ax to obtain the intensity values , x', of the
subset of data to be examined. The inverse of A need only multiply the elements x' of the
-1
subset, A x'=x, in order to retrieve the elements of x, to be placed in x'. Below is shown a
spherical half shell and a 3D surface plot with intense core of the 40A layer data for a LC
elastomer.
T-270

CRYSTAL STRUCTURE OF CCM3, A CEREBRAL CAVERNOUS MALFORMATION

PROTEIN CRITICAL FOR VASCULAR INTEGRITY

Xiaofeng Li, Rong Zhang, Haifeng Zhang, Weidong Ji, Wang Min, Titus Boggon

Yale Univ., New Haven CT, United States

CCM3 mutations are associated with cerebral cavernous malformation (CCM), a disease
affecting 0.1-0.5% of the human population. CCM3 (PDCD10, TFAR15) is thought to form a
‘CCM complex’ with CCM1 and CCM2, however, the molecular basis for these interactions is
not known. We have determined the 2.5Å crystal structure of CCM3. This structure shows an
all alpha-helical protein containing two domains, an N-terminal dimerization domain with a fold
not previously observed, and a C-terminal focal adhesion targeting (FAT)-homology domain.
We show that CCM3 binds CCM2 via this FAT- homology domain and that mutation of a
highly-conserved FAK-like hydrophobic pocket (HP1) abrogates CCM3-CCM2 interaction.
This CCM3 FAT-homology domain also interacts with paxillin LD-motifs using the same
surface, and partial CCM3 co-localization with paxillin in cells is lost on HP1 mutation.
Disease-related CCM3 truncations affect the FAT-homology domain suggesting a role for the
FAT-homology domain in the etiology of CCM.
T-273

Influence of Glycerol addition to Dispersed Liquid Crystalline Phases –

A Small Angle X-Ray Study

2 1,3 1 1
Heiner Santner , Sandra Engelskirchen , Reinhard Maurer , Otto Glatter
1 2 3
Karl-Franzens University, Graz, Austria, Anton Paar, Graz, Austria, University of Stuttgart,
Stuttgart, Germany

Phytantriol is a hydrophobic surfactant comprising a highly branched phytanyl-chain

with a tri-hydroxy headgroup. The binary Phytantriol – H2O system shows a complex phase
behavior featuring a variety of lyotropic liquid crystalline phases (lamellar, inverse hexagonal
and cubic structures) and a fluid isotropic phase (L2 phase). Above a certain composition
these phases coexist with an excess water phase, which allows dispersing the respective
nanostructure in a continuous water phase. The resulting sub-micrometer sized particles are
stabilized by the addition of the tri-block copolymer Pluronic F127.

Internally self-assembled dispersions represent self-assembly in confinement. Inside

the sub-micrometer sized particles liquid crystalline material is confined, which consists of
water and oil domains separated by an amphiphilic monolayer. While the internal
nanostructure was found to be in thermodynamic equilibrium the whole particle is kinetically
stabilized [1, 2].

The confined nanostructure is capable of solubilizing hydrophilic, hydrophobic or

amphiphilic substances offering many advantages as carrier systems for active substances.
Bridging the gap between basic research and technical application one often faces the need
to improve the properties of the respective formulation. In the present contribution we focus
on stability against cold through the addition of glycerol to the continuous water phase of the
internally self-assembled dispersion. The effect of glycerol on the confined nanostructure was
determined via Small Angle X-Ray Scattering revealing that the addition of glycerol induces
phase transitions from inverse hexagonal phases to water-in-oil microemulsions. In this
respect the addition of glycerol has a similar effect like raising temperature.

zP| ¡?b\«fi›K?kM?¡ ?\ M k\‹£«· ‒ GQOOSH QOK?TQTSLTQUPM

zQ| x\£⁄«·‒K?`M¡ ?\ M k\‹£«· ‒ GQOOTH QPK?TUXLTVVM

T-276

SWAXS Analysis on Multicompartment Micelles formed by The Newly Designed Ion

Pair Hybrid Surfactant
1 2 3 2
Semra Ide , Hande Unsal , E.Hilal Soylu , Nihal Aydogan
1
Hacettepe University , Faculty of Engineering, Department of Physics Engineering 06800
2
Beytepe, Ankara, Turkey, Hacettepe University , Faculty of Engineering, Department of
3
Chemical Engineering, 06800 Beytepe, Ankara, Turkey, Karadeniz Technical University,
Faculty of Science & Literatur, Department of Physics 61080, Trabzon, Turkey

Multicompartment micelles are aggregates of surfactants composed of a hydrophilic shell and

a multidomain hydrophobic core which makes it possible to cosolubilize and transport several
different and immiscible materials in different subdomains selectively and preventing any
undesired interactions before reaching the target. Hence, multicompartment micelles have
high potential to be used in controlled drug delivery, imaging technology, selective entrapment
and release of dyes,gene therapy agents, etc.Because of molecular structure, there is the
possibility of being arranged one after another for hydrocarbon and fluorocarbon-based
compartments in some of these aggregates, especially for segmented worms resulting in
small-volume micellar subdomains .Therefore, a novel hydrocarbon-fluorocarbon ion pair
+ +
hybrid surfactant CH3(CH2)11(OCH2CH2)23N (C2H5)3SO3-(CF2)7CF3 (C12E23N SO3-F8) was
designed in order to minimize some problems faced in previous studies such as low content
of hydrophobic groups in the aggregate, absence of common interface between two distinct
hydrophobic cores, low solubilization capacity, and overlapping of hydrophobic subdomains.
+
Having high solubilization capacities of C12E23N SO3-F8 for hydrocarbon-based and
fluorocarbon based probes both separately and simultaneously leads to the deduction of
achieving efficient compartmentalization inside the micellar core. SAXS study can be used for
yielding valuable structural information about nanostructured aggregations built by these type
surfactants. Depending on the molecular structure of the ion paired segments, diverse
morphologies can be expected for the formed compartments e.g. spheres in/on spheres,
open sandwich bread and mace shapes which may be composition of several rods / sphere
and rod. SAXS experiments were performed on the samples (with mM of 3%, 7% and 10%)
by using a Hecus SWAXS system (installed in the content of Hacettepe BAB Project fund:
06A602012) with CuKÕ , Ö= 1.54 Å. To define the possible multicompartment structure,
various models for the form factor were considered for fitting the SAXS patterns.
T-279

Structural Chemistry of Mono–substituted Nitrobenzenes With Pendant Ethylamino

Substituents

1 1 1 1 1 1
Philip Squattrito , Dillip Mohanty , Thomas Payne , Chad Thurman , Hao Yu , Qian Sun ,
2 2 2
Kristin Kirschbaum , Mark-Robin Giolando , Chris Brue
1 2
Central Michigan University, Mount Pleasant, Michigan, United States, University of Toledo,
Toledo, Ohio, United States

As part of our continuing study of model compounds for intermolecular interactions in

polyamine polymers, a series of mononitrobenzenes with one ortho ethylamino substituent
and either a para ethylamino substituent or a bridging sulfonyl have been synthesized and
structurally characterized. The compounds 4-nitro-N,N’-diethylbenzene-1,3-diamine (I) and
2,6-(bisethylamino)-3-nitrobenzonitrile (II) differ only in the absence or presence of a cyano
group in between the two ethylamino groups on the ring, allowing for an analysis of the effect
of the cyano group on the intermolecular interactions and crystal packing. The primary
interaction in (I) is an intermolecular N-H…O hydrogen bond between an amino hydrogen
atom and a nitro O atom that links molecules into one-dimensional chains. By contrast,
molecules of (II) are joined into dimers by bifurcated N-H…O hydrogen bonds between the
amino H atom and both a nitro O atom on the ortho nitro group of the same molecule and a
nitro O atom on the neighboring molecule. The third compound, di(4-ethylamino-3-
nitrobenzene)sulfone (III), contains the same ortho nitro/ethylamino pairing as in (I) with the
position para to the nitro group occupied by the sulfone instead of a second ethylamino group.
Molecules of (III) are linked into zigzag double chains through N-H…O hydrogen bonds
between the amino H atoms and sulfonyl O atoms. The detailed intermolecular interactions
and packing of (III) will be analyzed in relation to those observed for (I) and (II).
T-282

Functional group recognition in carboxyalkylammonium salts

Melanie Rademeyer, Belinda van der Westhuizen

University of Pretoria, Pretoria, South Africa

The carboxyalkylamine 4-aminobutanoic acid (GABA) is a commercially available supplement

1
against anxiety , while the longer chain 6-aminohexanoic acid is marketed as a treatment for
2
bleeding disorders . In the pharmaceutical industry the salts of active ingredients often show
improved physicochemical properties compared to the neutral ingredient. This study
investigated the structures and functional group recognition occurring in halide and oxoanion
salts of 4-aminobutanoic acid and 6-aminohexanoic acid, with a number of novel structures
reported. Monovalent counter anions including chloride, bromide, iodide, nitrate and
perchlorate were chosen. Packing trends were identified and non-covalent interactions and
the role of the anion highlighted. Emphasis was also placed on hydrogen bonding interactions
and recognition occurring between terminal functional groups and anions. A number of
permutations of hydrogen bonding donors and acceptors are possible. It was found that for
the anhydrous halide salts all three hydrogen bonding groups (anion, ammonium group and
carboxylic acid group) interact to form a complex, two-dimensional hydrogen bonding
network. However, in the case of the hydrated halide salts and the oxoanion salts the
carboxylic acid functional groups only interact with other carboxylic functional groups, while
the ammonium groups exclusively hydrogen bonds to the anions (and water molecules when
present). Thus, in the case of the hydrated halides and the oxoanion salts a certain degree of
“recognition” is displayed.

1. Abdou, A. M., Higashiguchi, S., Horie, K., Mujo, K., Hatta, H., Yokogoshi, H. BioFactors,
2008, 26, 201-208.

2. Thomas, D. C., Wormald, P. J., Am J Rhinol., 2008, 22, 188-191.

T-285

The influence of crystallographic symmetry elements on the observation of a uniform

stacking motif in ¼-filled one dimensional molecular systems.
1 2 3 4 1
Eric Reinheimer , Marc Fourmigue , Patrick Batail , Claude Coulon , Kim Dunbar
1 2
Texas A&M University, College Station, TX, United States, Universite Rennes 1, Rennes,
3 4
France, Universite d' Angers, Angers, France, Centre de Recherches Paul Pascal (CRPP-
CNRS), Pessac, France

The study of the structures and physical properties of conducting molecular solids have
spawned many fascinating discoveries in the realms of crystallography, chemistry and solid
state physics. Seminal discoveries such as TTF-TCNQ (TTF = tetrathiafulvalene; TCNQ =
tetracyanoquinodimethane) and the TMTSF (TMTSF = tetramethyltetraselenafulvalene) family
of electrocrystallized salts have been the subject of intense study and debate since their initial
syntheses more than thirty years ago. One unifying principle has dominated interdisciplinary
debate on these materials; that they suffer from a confluence of multiple physical phenomena
which serve to inhibit the complete understanding of each individual phenomenon. In order to
gain a greater understanding of the novel behavior that these materials display, physicists
and theoreticians have suggested that simpler systems be prepared. Among the approaches
advocated is to prepare charge transfer materials based on oxidized chalcofulvalene moieties
that exhibit equivalent intermolecular spacing between the moieties along the stacking axis.
These one dimensional, non-dimerized, systems are ¾-filled with electrons (1/4- filled with
holes) and are essential for testing theoretical work that predicts that such systems will be
Mott insulators.

To date, systems with uniform non-dimerized chains have been quite rare. Initial
examples with DMtTTF (DMtTTF = dimethyltrimethylene-tetrathiafulvalene) as well as its
selenium analogue were reported to show uniform stacking when first crystallized via
- -
electrochemical methods with the tetrahedral anions [ClO4] and [ReO4] in the 1980s. More
recent examples of exhibiting a uniform stacking motif were found to exist in samples
containing the non-centrosymmetric tetrathiafulvalene donors EDT-TTF-CON(CH3)2 and o-
-
Me2TTF when crystallized with [AsF6] and the halide anions respectively.

Closer inspection of each salt’s solid state structure reveals that the individual
crystallographic symmetry of each of the materials described above is critical for defining the
uniform stacking motif deemed necessary by theoreticians. This talk serves to illustrate the
contribution that X-ray crystallography has made in characterizing these chalcofulvalene salts
as unique solid state materials. In addition current understanding of their physical properties
will also be discussed.
T-289

Experimental and Theoretical Determination of the Electron Density Distribution of

Methyl- -Cellobioside
1 1 2 2
Edwin D. Stevens , Ryuta Sasabayashi , Michael K. Dowd , Glenn P. Johnson , Alfred D.
2
French
1
Department of Chemistry, University of New Orleans, New Orleans, LA 70148, United
2
States, Southern Regional Research Center, USDA, New Orleans, LA 70124, United States

×-Cellobiose is a disaccharide containing two glucose residues joined by a × 1,4 glycosidic

linkage and therefore it provides a model for the basic, structural repeat unit of cellulose. The
crystal structure of the methanol solvate of methyl-× -cellobioside shares a remarkable number
of structural similarities with the structure of cellulose IIII. High-resolution single-crystal x-ray
diffraction measurements of methyl-× -cellobioside collected at 120 K have been used to
determine the experimental electron density distribution of the molecule in the crystal. The
electron density has also been obtained from large basis set DFT calculations of the
molecular wavefunction using the experimental geometry. The crystalline environment has
been simulated in the theoretical calculations by including fragments of surrounding
molecules in a cluster calculation.
A topological analysis of both the
experimental and theoretical electron
distributions has been performed using the
Atoms in Molecules approach, and the
results are compared with recent low
temperature studies of the electron density
distributions of Ø,Ø-trehalose (Stevens,
Dowd, Johnson, and French, Carbohydrate
Research 2010, in press) and sucrose
(Jaradat, Mebs, Chęcińska and Luger,
Carbohydrate Research 342 2007, 1480–
1489).
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T-295

Synthesis, Characterization, and X-ray and Synchrotron Radiation Structure

Determination of Monoitaconate Esters.
1 1 1 2
Graciela Diaz de Delgado , Belkis Ramirez , William Velasquez , Maren Pink
1 2
Universidad de Los Andes, Merida, Merida, Venezuela, Indiana University, Bloomington,
Indiana, United States

Monomers derived from itaconic acid are widely used in the preparation of polymer
complexes of potential use in biomedicine, agriculture, as hydrogels, in drug-delivery
systems, fabrication of contact lenses, among other applications. In this work, the structure of
several itaconate monoesters is presented. The esters were prepared by reaction of itaconic
acid with alcohols in the presence of acetyl chloride. The materials prepared include the
methylitaconate, ethylitaconate, benzylitaconate, dodecylitaconate, among others. For
example, Methylitaconate crystallizes in the orthorhombic system, space group Pca21, with
3
unit cell parameters a=11.305(4), b=5.156(2), c=23.659(7) Å, V=1379.1(8) Å , Z=8. The
refinement converged to R=0.0753, wR=0.1878, S=1.057. In this structure, typical cyclic
dimer hydrogen bonds are observed between molecules in the bc plane. Data from
synchrotron radiation studies on other monoitaconates will also be presented.

Funding for this work was provided by CDCHT-ULA and by FONACIT, grant LAB-97000821.
T-299

The Molecular Structure Of The First Benzoindenone Compound Isolated From The
Roots of Psychotria prunifolia
1 2 1
Jose Ricardo Sabino , Christopher Ceccarelli , Laryssa Campos Ribeiro , Cecilia Maria Alves
1 1
de Oliveira , Lucilla Kato
1 2
Univ. Federal de Goias, Goiania GO, Brazil, Agilent Technologies, Blacksburg, VA, United
States

As part of our program to assess the chemical and biological diversity of native plants of the
Brazilian Cerrado, we have examined promising active extracts of Rubiaceae species
concerning their antitumoral potential. In this work, leaves, barks and roots from Psychotria
prunifolia was subjected to ethanolic extraction and successive chromatographic separation
to provide an unpolar compound which have not been identified before. The molecule, shown
below, is described as a benzoindenone, with no reference in CAS or other public database
sources. A crystal suitable for x-ray study was obtained by evaporation of a solution of
methanol-chloroform (1:1). The molecule crystalizes in the P21/c space group with cell
parameters: a = 9.7355(2) Å, b = 7.3271(2) Å, c = 19.6875(5) Å, β = 102.676(2)°, volume
3
1370.14(6) Å . Data collection were performed with a Varian Gemini Ultra diffractometer
controled by CrysAlisPro (Oxford Diffraction Ltd., Version 1.171.33.55), using Cu-Kα radiation
at 100 K. 16063 data points were collected of what 2447 are symmetry independent (Rint =
0,038). Structure solution was achieved with Direct Methods using Olex2 (J. Appl. Cryst.
2
(2009). 42, 339–341). Model refinement was performed with full matrix least squares on F
with final residuals R1 = 0.034, wR2 = 0.095 for observed data with I>2σ (I), and R1 = 0.042,
wR2 = 0.10 for all data.
T-309

Synthesis and Activity of Oxynitride Nanoparticles

Craig Bridges, Mariappan Paranthaman

Oak Ridge National Laboratory, Oak Ridge, TN, United States

Metal oxides have played a key role in the development of modern science and technology, in
large part due to the ability to manipulate chemical and electronic structures through chemical
doping and substitution. The position that mixed anion phases can occupy in the development
of materials with novel properties has recently been well demonstrated, through the flurry of
activity stimulated by the discovery of superconducing oxypnictide and oxychalcogenide
phases. While oxyanion materials have been well investigated in the bulk, they are relatively
underdeveloped as nanoparticle phases. The development of the chemistry of nanophase
oxyanion synthesis is an important area, as this may expand the role these materials play in
the future for both energy storage and conversion, as well as more fundamentally providing
insight into the impact of particle size on anion substitution. The synthesis of oxynitride
phases has typically required the formation of an amorphous or small particle precursor, due
to the relatively slow diffusion of the nitride anion at typical synthesis temperatures. Here we
examine in detail the correlation between particle size and crystallinity on the resulting
synthesis conditions in the formation of oxynitrides. Nanoparticles have been prepared
through a variety of solution phase methods to obtain well defined precursors for
ammonolysis. In situ diffraction results illustrating the important role of nanocrystal precursors
size on oxyanion nanoparticle formation will be presented, and the impact of particle size on
the resulting catalytic properties will be discussed. This work provides insight into the
underlying factors controlling anion transport for this particularly difficult class of oxyanion
synthesis.
T-313

Practical use of a bent perfect Si monochromator on a neutron four-circle

diffractometer at the HFIR.
1 1 1 2
Bryan Chakoumakos , Huibo Cao , Alexandru Stoica , Mihai Popovici†
1 2
Oak Ridge National Laboratory, Oak Ridge, TN, United States, Missouri University
Research Reactor, Columbia, MO, United States

The design of doubly-bent perfect crystal monochromators has steadily improved, making
them competitive with mosaic crystal monochromators. A multi-wafer neutron
†
monochromator, designed by M. Popovici and A.D. Stoica, has been commissioned on the
HB-3A Four-Circle Diffractometer at the HFIR, ORNL. The unit is made from a bent packet of
silicon wafers of almost [110] orientation with the <1-10> zone axis vertical. The reflection
planes of practical interest are (220)/(440), (331), and (111)/(333), accessible by rotation of
o
the unit, which give lambda = 1.56, 1.01, and 2.5 Å, respectively at the fixed 48 take-off angle
-1
for the instrument. The horizontally curvature, rho = 1/R (m ), of the monochromator is
-1
variably adjustable, from essentially flat to a curvature of 0.7 m . The neutron flux incident on
the sample and the Bragg peak width at the detector are highly dependent on rho, such that
the incident flux on the sample passes through a maximum, increasing by ×1.8 for 1.01 Å and
by ×3.3 for 1.56 Å, as compared to the flat condition. The flux increase is due to the delta-
lambda/lambda increasing, but eventually the incident beam mask cannot handle the
increasing divergence and the intensity drops off. The Bragg peak width increases and the
width versus scattering angle flattens as rho increases Given these effects, rho be adjusted
to deliver high intensity primarily for crystal structure refinements, or high resolution for
resolving symmetry changes, e.g., charge order with lattice distortion and complex magnetic
orders. Traditional step scanning and more efficient continuous scanning modes are possible.
This research is supported by UT Battelle, LLC under Contract DE-AC05-00OR22725 for the
U.S. Dept. Energy, Office of Science.



T-317

X-ray studies of the photoexcitation of Zn[4-

Cl-PhS]2phenanthroline

and Zn[4-Me-PhS]2bathocuprine
1,2 1
Mette Schmoekel , Jason Benedict , Radoslaw
1,3 1
Kaminski , Philip Coppens
1
University at Buffalo, SUNY, Buffalo, NY, United
2
States, Aarhus University, Aarhus C, Denmark,
3
University of Warsaw, Warsaw, Poland

Photo-crystallographic studies of light-induced

short-lived species in molecular crystals are part
of a relatively unexplored but rapidly increasing area of crystallography. It is a powerful tool
for increasing the understanding of the processes that occur when crystals are exposed to
laser light. Depending on the type of compound, time scale, and the lifetimes of the
metastable species in question such studies can be performed by either in-house pseudo-
steady state or synchrotron-based pump-probe experiments using either monochromatic or
1
polychromatic sources. Here we present photo-crystallographic liquid-helium temperature
2
studies of two ~100 μ s lifetime Zn-complexes, Zn[4-Cl-PhS]2phen and Zn[4-Me-
PhS]2bathocuprine. The in-house experiments used a high rep-rate Nd-vanadate laser at both
100 and 20 kHz. The structural changes are analyzed with photo-difference plots showing the
changes in electron density due to laser exposure, and subsequent least-squares refinement
3 4
with the program Laser05. The refinement is based on the RATIO method in which the
ratios, R(h)=Ilight ON(h)/Ilight OFF(h) rather than the absolute intensities are refined. The results
are interpreted in terms of both a molecular change and a slight motion of the unconverted
ground state molecules. Parallel theoretical calculations suggest the excitation to correspond
to formation of a weak S-S bond in the excited triplet states of both complexes.

1
W. K. Fullagar, G. Wu, C. Kim, L. Ribaud, G. Sagerman and P. Coppens, J. Synchr. Rad. 7,
2
229-235 (2000). R. G. Highland, J. G. Brummer, and G. A. Crosby, J. Phys. Chem., 90,
3
1593-1598 (1986). Y. Ozawa, S. Pillet, I. Vorontsov, R. Kaminski, University at Buffalo,
4
Crystallographic Programs. P. Coppens, M. Pitak, M. Gembicky, M. Messerschmidt, S.
Scheins, J. B. Benedict, S. Adachi,T. Sato, S. Nozawa, K. Ichiyanagi, M. Chollet and S.
Koshihara, J. Synchrotron Rad., 16, 226-230 (2009).

Acknowledgements: Research supported by the National Science Foundation (CHE0843922).

T-331

Characterization of the crystal of protein CTB and protein -environmental ligand

complexes by using a X-ray diffraction

Huong Tran, Jeffrey Wolt, Edward Yu

Iowa State University, Ames, IA, United States

Transgenic plants may play an important role in the cost-effective large scale production of
biopharmaceutical proteins and industrial enzymes. The introduction of novel plant-made
proteins in agro-ecosystems poses concerns about the potential harm of crop residuals to the
surrounding ecosystem. Few studies have considered how the environmental fate and
activities of transgenic proteins may impact soil processes. Protein reactivity with
environmental ligands are investigated through crystallization and x-ray diffraction as a means
for understanding environmental degradation and persistence of transgenic protein. Vibrio
cholera enterotoxin subunit B (CTB) is studied as a model system under laboratory
conditions. In order to have a full understanding of changing conformation of protein with
humic acid, three quinones [lawsone (2-hydroxy-1, 4- naphthoquinone), juglone (5-hydroxy-1,
4 –naphthoquinone) and AQDS (anthraquinone-2, 6- disulfonate)] were used to model
reactive sites found in humic acid. The crystals of protein –ligand complex will be analyzed by
X-ray diffraction at Agronne National Lab with detector ADSC Q315. The molecular
replacement is used to obtain phase information for determining the structure. The results
from this research will provide the novel insight into the mechanism of interaction between
protein and environmental ligands.
T-200

Cathepsin K and the regulation of bone resorption

1 1 1 2 2,3
Michael James , Martin Kienetz , Maia Cherney , Zhenqiang Li , Dieter Bromme
1 2
Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada, Department
3
of Human Genetics, Mount Sinai School of Medicine, New York, NY, United States, Faculty
of Dentistry, University of British Columbia, Vancouver, BC, Canada

The regulated interplay between bone resorption and bone formation leads to a complete
replacement of the human skeleton every seven to ten years. The inorganic demineralization
stage is accomplished by the release of HCl from specialized cells called osteoclasts. These
cells also secrete large quantities of the papain-like lysosomal cysteine peptidase, cathepsin
K. The organic matrix of bone consists of > 90% type I collagen and cathepsin K is the
predominant peptidase that degrades collagen. The lack of a cathepsin K activity leads to a
severe bone resorption defect known as pycnodysostosis, a disease that Henri Toulouse-
Lautrec was believed to have suffered from. Excessive cathepsin K activity leads to
orteoporosis. Collagen consists of three polypeptide chains each ~ 1000 amino acids in
length. At the N- and C-termini of each chain there are ~50 amino acids that form the globular
telopeptides; the remaining ~900 amino acids of each chain are twisted into left-handed
helices that intertwine to form a right-handed triple helix. The triple-helical region of each
chain is especially resistant to general peptidase degradation. The unique triple-helical
degrading activity of cathepsin K depends on the formation of complexes with
glycosaminoglycans such as chondroitin 4-sulfate (C4-S). We have determined the structure
of the 1:n complex between cathepsin K and C4-S to 1.8Å resolution. The C4-S molecule (17
kDa) adopts a cosine wave-shaped oligomeric conformation and each cathepsin K molecule
interacts tightly with three disaccharides (an alternating copolymer of β -D-glucuronic acid and
2′ -deoxy-2′ -acetamido-β -D-galactose-4-sulfate) of the C4-S. The binding sites of C4-S are
located in the R-domain of cathepsin K and are distant from its active site. Our poster will
present the structure this interesting complex between cathepsin K and the oligosaccharide of
chondroitin 4-sulfate.
T-332

Crystal Structure of the Catalytic Domain of Rlf

1 1 1 1 1,2 1
Peng Wei , Xu Jiwei , Sun Yao , Liu Ian , Rao Zihe , Li Xuemei
1 2 3
Chinese Academy of Sciences, Beijing, China, Nankai Univ., Tianjin, China, tsinghua Univ.,
Beijing, China

Ral, the subfamily of the small GTPase Ras superfamily, plays essential role in many different
cellular processes including cell growth, differentiation and vesicular transport. Ral family
includes two members in human, named RalA and RalB. As other members of the Ras family,
Ral cycles between its activated GTP-bound form and inactivated GDP-bound form in cells,
controlling by the guanine nucleotide exchange factors (GEF), which catalyse the substitution
of Ral bound GDP by GTP, and the GTPase activating proteins(GAP), which enhance the
GTPase activity of Ral, leading to hydrolysis of the Ral bound GTP to GDP. Six different
GEFs for Ral GTPase have been identified up-to-date; they are Rlf, RalGDS, Rgl, Rsc,
RalGPS1 and RalGPS2. All those GEF share a common cdc25 domain responsible for their
GEF activity and bear different regulatory domain. RalGDS, Rgl, Rlf, Rsc have a Ras-GTP
binding domain(RBD) and their GEF activity are promoted by Ras-GTP, while RalGPS1 and
RalGPS2 lack the RBD but have a GRB2 binding PXXP motif, suggesting their different
activation pathway. We have determined the three dimension structure of the cdc25 domain
of Rlf. The cdc domain of Rlf shows high similarity with that of Son Of Sevenless (SOS) but
differ in the conformations of a long loop close to the active site. Unlike that of SOS, Rlf cdc25
domain alone has GEF activity when it separated from the whole protein, and this structure
variety between Rlf and SOS may cause their functional differences.
T-333

Structural Diversity of the Proline Utilization A Family Revealed by SAXS, Light

Scattering, and Analytical Ultracentrifugation

Ranjan K. Singh, Travis A. Pemberton, John J. Tanner

Univ. Of Missouri, Columbia, Mo, United States

The proline catabolic enzymes catalyze the 4-electron oxidation of proline to glutamate. The
1
reaction involves two enzymes, proline dehydrogenase and ∆ -pyrroline - 5-carboxylate
dehydrogenase. Some bacterial organisms have both of these enzymes fused together, and
the fused bifunctional enzymes are called Proline utilization A (PutA). In addition to these
bifunctional enzymes, some PutAs are trifunctional, because they moonlight as transcription
repressors of their own gene.

Our lab recently reported that the quaternary structure of the bifunctional PutA from B.
japonicum is a ring-shaped tetramer [1]. However, the structural organization of PutAs from
other organisms is still unknown. In particular, there are no structures available for
moonlighting trifunctional PutAs. This poster will focus on an examination of the diversity of
the quaternary structure within the PutA family. Small angle X-ray scattering, analytical
ultracentrifugation, and light scattering experiments have been done to reveal the oligomeric
states and shapes of several bifunctional and trifunctional PutAs. Our current data suggest
that bifunctional and trifunctional PutAs have substantially different quaternary structures. We
suggest that these structural differences are related to the specific set of functions that PutAs
must fulfill in utilizing proline in different organisms.

[1] Srivastava D, Schuermann JP, White TA, Krishnan N, Sanyal N, Hura GL, Tan A, Henzl
MT, Becker DF, Tanner JJ. Crystal structure of the bifunctional proline utilization A
flavoenzyme from Bradyrhizobium japonicum. Proc. Natl. Acad. Sci. U. S. A. (2010)
107(7):2878-83.

This research is supported by NIH grants GM065546 and GM061068.

SP.03

From the Structure of the Ribosome to New Antibiotics

Thomas Seitz

Yale Univ., New Haven CT, United States

Over the past decade, we have determined the structures of the Haloarcula marismortui large
ribosomal subunit and many of its complexes with substrate analogues and antibiotics, which
have led to an understanding of the mechanism of peptide bond formation and its inhibition by
antibiotics. More recently, we have established the structure of the Thermus thermophilus
fmet
70S ribosome complexed with tRNA in the P site and elongation factor P (EF-P) bound
next to it between the E and P sites which suggests that EF-P stimulates the formation of the
fmet
first peptide bond by properly positioning the fmet-tRNA . The structures of the 70S
ribosome and three bound tRNA molecules and either the anti-TB antibiotics viomycin or
capreomycin show how they inhibit protein synthesis and suggest how new anti-TB drugs
might be designed. Further progress is being made in our pursuit of the structures of the 70S
ribosome with arresting polypeptide captured in the exit tunnel.
SP.04

The Amazing Ribosome, Its Tiny Enemies And Thoughts About Its Origin

Ada Yonath

Weizmann Inst., Rehovot, Israel

Ribosomes, the universal cellular machines, possess spectacular architecture

accompanied by inherent mobility, allowing for their smooth performance as polymerases that
translate the genetic code into proteins. The site for peptide bond formation (PTC) is located
within a universal internal symmetrical region connecting all of the remote ribosomal features
involved in its functions. The elaborate architecture of this region positions ribosomal
substrates in appropriates stereochemistry for peptide bond formation, for substrate-mediated
catalysis, and for substrate translocation. The high conservation of the symmetrical region
implies its existence irrespective of environmental conditions and indicates that it represents
the ancient ribosome. The PTC is located above an elongated tunnel along which nascent
chains progress until they emerge out of the ribosome. This tunnel may also be involved in
chaperoning function, provides the binding site of the first cellular chaperone that encounters
the emerging nascent chain, and hosts a major family of antibiotics.

Crystallographic analysis of complexes of ribosomes and antibiotics targeting them revealed

the structural bases for synergism, selectivity, induced fit; remote interactions, secondary
conformational rearrangements and cross-resistance to ribosomal antibiotics; the common
and specific pathways of resistance and cross resistance; the minute chemical differences
that can turn competition into synergism; and the factors leading to resistance acquired by
secondary conformational alterations.
05.01.1

Solid-State Conformational Differences Between “Bridge-Flipped” Isomeric Organic

Molecules
1 1 1 1 1
William Ojala , Jonathan Smieja , Dana Newman , Jeremy Leavell , Anthony Gerten ,
2
Charles Ojala
1 2
University of St. Thomas, St. Paul, Minnesota, United States, Normandale Community
College, Bloomington, Minnesota, United States

We have designated as “bridge-flipped” isomers those organic molecules related by a

reversal of a bridge of atoms connecting two major parts of the molecule. Families of
compounds in which this isomerism is found include the benzylideneanilines, for which the
isomerism is Ar-CH=N-Ar’ vs. Ar-N=CH-Ar’ , and the phenylhydrazones, for which the
isomerism is Ar-CH=N-NH-Ar’ vs. Ar-NH-N=CH-Ar’ (Ar = aryl). In these two families, steric
differences between the isomers are minor regardless of bridge orientation, raising the
question of whether the two isomers might assume the same molecular packing arrangement
in the solid state and be readily co-crystallized. We have found such isostructuralism to be
rare, but several examples do exist. In a continuing effort to identify more isostructural pairs
and to examine structural factors such as hydrogen bonding or molecular conformation that
would facilitate or discourage isostructuralism, we have been examining pairs in which one of
the isomers has been found to be nearly planar, preparing the bridge-flipped isomer and
determining its crystal structure to find out whether the isomer is planar as well and the
packing arrangements thus potentially isostructural. We have determined and describe here
the crystal structures of two benzylideneanilines, 4-methoxy-N-[(3-
nitrophenyl)methylene]benzenamine and N-[(3-bromophenyl)methylene]-4-
chlorobenzenamine, nonplanar molecules for which the isomer has been found to be planar in
each case by previous workers. We also describe the structures of 4-bromo-N-
(phenylmethylene)benzenamine and N-[(4-bromophenyl)methylene]benzenamine,
compounds for which cell constants but not coordinates have been reported in the literature
previously. Here as well, one isomer is nearly planar in the solid state, but the other is
significantly nonplanar and the compounds are not isostructural. We discuss here also the
crystal structures of two bridge-flipped, non-isostructural phenylhydrazones, 4-
chlorobenzaldehyde-4-nitrophenylhydrazone (nearly planar) and 4-nitrobenzaldehyde-4-
chlorophenylhydrazone (nonplanar). Finally, we describe the structures of two bridge-flipped
symmetrical bis-benzylideneanilines, both of which would be capable of occupying a
crystallographic inversion center by assuming a centrosymmetric molecular conformation but
only one of which actually does.
05.01.2

To bridge or not to bridge? Reversible bridging and terminal carbonyl ligand

configurations in some unusual tricobalt carbon cluster derivatives of a tripodal
phosphine ligand.

Jim Simpson, John McAdam, Brian Robinson, Roderick Stanley

University of Otago, Dunedin, New Zealand

In simple methinyltricobaltnonacarbonyl clusters, RCCo3(CO)9, six of the carbonyl ligands

occupy equatorial sites, close to the plane of the triangle of cobalt atoms with the other three
carbonyl groups approximately orthogonal to that plane in axial positions. Monodentate or
bidentate phosphine or phosphite ligands almost invariably substitute carbonyl groups from
equatorial sites [1,2]. Furthermore, the remaining equatorial carbonyl ligands generally adopt
terminal conformations. A search of the Cambridge Database [3] reveals that carbonyl
bridging of the Co—Co bonds is unusual and is generally found only in situations where the
apical substituent R, or the substituting ligands, significantly increase the electron density on
the CCo3 cluster core.

Me

We have prepared a number of derivatives of methinyltricobaltnonacarbonyl

CO
clusters with the potentially tridentate 1,1,1- OC Co CO
Co
tris(diphenylphosphinomethyl)-ethane, CH3C(CH2PPh2)3, (triphos) Co

ligand. In all cases, the resulting compounds have bridging carbonyl OC OC CO

ligands. This paper will report the structure of these complexes and P
P
examine the factors that influence carbonyl bridging in these and Ph P Ph Ph
Ph
Ph Ph
related cluster systems. In particular, remission of the build-up of H C
2
H C
2 CH2
electron density on the cluster core by oxidation of the triphos derivatives
returns the carbonyl configurations to fully terminal.
CH3

Acknowledgements: We thank the New Economy Research Fund; Grant No. UOO-X0808 for
support of this work and the University of Otago for purchase of the diffractometer.

[1] Matheson, T.W., Robinson, B.H. & Tham, W.S. (1971) J Chem Soc A 1457-1464.

[2] Downard, A.J., Robinson, B.H. & Simpson, J. (1986) Organometallics 5, 1122—1131.

[3] Allen, F. H. (2002). Acta Cryst. B58, 380—388.

05.01.4
2+
Crystallography of Complexes of the [Re6(μ -Se)8] Core-Containing Clusters

Gary Nichol, Xiaoyan Tu, Zhiping Zheng

The University of Arizona, Tucson, AZ, United States

2+
Cluster complexes which contain an [Re6(μ -Se)8] core attract interest from synthetic and
computational chemists due to their facile chemical transformations, ease of control of
1
stereochemistry and notable electrochemical and luminescent properties. In almost all cases
single crystal X-ray diffraction is used to characterize these complexes, with supporting
information provided by spectroscopic techniques.

However, such cluster complexes frequently present a challenge to the crystallographer.

Disorder, twinning, highly solvated structures, weak diffraction and unstable crystals are all
common problems encountered in the course of this research. The dominance of the
2+
diffraction pattern by the [Re6(μ -Se)8] core, and the use of counter ions which are
synthetically suitable but crystallographically horrific, often make resolving some of these
problems a serious challenge. Here, problems, challenges and solutions will be discussed in
2+
the context of our recent work on the use of [Re6(μ -Se)8] core-containing clusters in so-
2
called “click” chemistry and the resulting unexpected formation of imino complexes .

1. X. Tu and Z. Zheng. CrystEngComm, 2009, 11, 707—719.

2. X. Tu, E. Boroson, H. Truong, G. S. Nichol and Z. Zheng. Inorg. Chem. 2010, 49,
380–382
05.01.3
IV V 9-
X-ray Crystal Structure of [(As6V 12V 3O51) ]∞ .

Carla Slebodnick, Victoria Soghomonian, Elinor Spencer

Virginia Tech, Blacksburg, VA, United States

A number of recently reported anionic 3D oxo-vanadium arsenate frameworks have shown

promising electronic properties. Expanding on this effort, a new anionic framework,
IV V 9-
[(As6V 12V 3O51) ]∞ , is reported. The material crystallizes in the cubic space group Im3m.
Original efforts to solve the structure yielded what appeared to be ten edge-shared VO5
square pyramids to
form V10O26 'balls'
with bidentate
corner- sharing AsO4
tetrahedra
linking two separate
V10O26 balls to form a
3D network.
Unfortunately,
a satisfactory
refinement of
this structure model
was not achieved after
trying many space groups and twinning models. Constraining specific vanadium and oxygen
occupancies to instead form disordered 'half-balls', however, gives a model that is both
chemically and crystallographically reasonable. In addition to summarizing the 3-year effort to
achieve this satisfactory model, the title structure will be compared and contrasted with a
number of very similar structures from the literature.
05.01.5

Be Careful What You Wish For: A Service Crystallographer’ s Lament

Patrick Carroll

University of Pennsylvania, Philadelphia PA, United States

This is the tale of a “bored” service crystallographer who wished for some interesting
problems with which he could entertain his colleagues at the ACA meeting. The result is a
series of twinned structures, disordered structures, unexpected and difficult-to-solve
structures and other forms of crystallographic misery.
05.01.6

Investigating Hexafluroacetylacetone (HFAA) by X-ray Diffraction – Solid-State and

Theoretical Studies of a Volatile Liquid
1 1 2 1
Christopher Incarvito , Chandrima Chatterjee , Lori Burns , Patrick Vaccaro
1 2
Yale University, New Haven, CT, United States, Georgia Institute of Technology, Atlanta,
GA, United States

Despite numerous efforts to unravel the ground-state structure of HFAA, its detailed geometry
is yet to be ascertained. While most theoretical endeavors point to the Cs structure as the
global minimum configuration, gas-phase electron diffraction studies [2,3] have suggested a
symmetric (C2v) enol tautomer. Motivated by such contradictory conclusions, the ground-
state manifold of HFAA has been re-examined using low-temperature single-crystal X-ray
diffraction techniques. HFAA has a normal melting point of 177K and, therefore, exists as a
liquid at room temperature. After introducing the liquid in a glass capillary, it was mounted
vertically on a Rigaku R-AXIS SPIDER diffractometer and cooled to 93K by a cryogenic
stream of nitrogen vapor. A single crystal was grown 'in situ' by the zone-melting method [1],
with a heated filament producing a molten zone that was slowly translated along the length of
the capillary. The HFAA crystal structure emerging from collected diffraction data clearly
favors an asymmetric H-bond. In addition, the wider separation of 2.683 Å between the donor
and acceptor oxygen atoms implies a weaker hydrogen bond compared to acetylacetone. Our
X-ray analysis has been corroborated by high-level quantum chemical calculations, a detailed
discussion of which may be presented in this paper.

[1] R. Boese, M. Y. Antipin, D. Blaser, and K. A. Lyssenko, J. Phys. Chem. B, 102, 8654
(1998).
[2] K. IIjima, Y. Tanaka, and O. Shigeki, J. Mol. Struc., 268, 315 (1992).
[3] A. L. Andreassen, D. Zebelman, and S. H. Bauer, J. Am. Chem. Soc., 93, 1148 (1971).
05.01.7

Similar Shapes and Isomer Disorder in the Structure of C86•Ni(OEP)•2toluene

1 1 2 2 2 2
Marilyn Olmstead , Alan Balch , Ziyang Liu , Hua Yang , An Jiang , Zhimin Wang
1 2
University of California, Davis, United States, Zhejiang University, Hangzhou, China

Since the structure of the fullerene with 60 carbons, Ih-C60 was determined 18 years ago, the
structures of few pristine empty cage fullerenes have been successfully characterized by X-
13
ray crystallography. In most cases their structures have been inferred from C NMR and IR
measurements or by derivatization.. The largest higher fullerene characterized by X-ray
1
crystallography to date is that of D5h-C90, reported by us earlier this year . Some of the
reasons for the paucity of these structures stems from the increasing number of possible
isomers as the number of carbon atoms increases and the difficulty in the separation of the
isomers that are preferentially formed in the carbon soot of the vaporized graphite. Other
reasons are the very small amount of material isolated, together with the propensity for
disorder in the structures of these nearly spherical molecules. We recently succeeded in
obtaining the structure of an empty cage C86 fullerene, obtained by co-crystallization with
Ni(OEP) in toluene. In this structure, the eight ethyl arms of the porphyrin molecule
encapsulate the fullerene and enable the determination of the structure with less rotational
disorder. However, in this instance, we were surprised to be able to detect the presence of
two concomitant isomers of C86 that reside in the same crystallographic position. The two
isomers, Cs(16)-C86 and C2(17)-C86, differ by a 90 ° rotation of a set of two pentagons and
two hexagons, the so-called Stone-Wales transformation. The presence of both isomers is in
agreement with theoretical calculations. Structural similarities between the two isomers are
compared in this report.
1
H. Yang, C. M. Beavers, Z. Wang, A. Jiang, Z. Liu, H. Jin, B. Q. Mercado, M. M. Olmstead
and A. L. Balch. Angew. Chem. Int. Ed., 2010, 49, 886.
06.01.1

40 Years Synchrotron X-Radiation in Biology

Gerd Rosenbaum
1 2
University of Georgia, Athens, GA, United States, Argonne National Laboratory, Argonne,
IL, United States

2010 marks the 40th anniversary of the recording of the first x-ray diffraction with synchrotron
radiation [1]. The small angle diffraction pattern of insect flight muscle proved that the
calculated 100-fold gain in flux from the DESY synchrotron over a state-of-the-art fine focus
rotating anode X-ray generator could, indeed, be achieved. Time resolved diffraction of the
cross-bridge cycle in muscle, the driving force behind the synchrotron adventure, would be a
big step closer to reality.

The first part of the presentation will commemorate this event. The main part will trace the
tremendous advances synchrotron radiation has enabled in biology: from small angle
diffraction and solution scattering to macromolecular crystallography, spectroscopy,
microscopy and imaging.

References:

1. G Rosenbaum, K C Holmes, J Witz: “Synchrotron radiation as a source for X-ray diffraction

(a preliminary report)”, Nature, 230, 434-37, 1971
06.01.2

Femtosecond Protein Nanocrystallography at the LCLS

John Spence

ASU, Tempe Az, United States

First results will be reported from experiments at the world's first hard X-ray laser (the Linac
Coherent Light Source) at Stanford using membrane protein nanocrystals. X-ray pulses at 2
kV of femtosecond duration were used to read out 30 diffraction patterns per second from a
liquid jet of protein nanocrystals sprayed across the beam. We have evaluated the idea that a
sufficiently short pulse will terminate before radiation damage begins, yet contain sufficient
photons to produce a useful diffraction pattern. Details of the protein-beam injector, which
must provide full hydration, will be given, and the method of data analysis discussed. This
involves merging diffraction data from millions of patterns from sub-micron nanocrystals of
photosystem one , of varying size and in random orientations. Many terrabytes of data were
collected over several days. Plans for pump-probe experiments will also be outlined. This
approach allows structure analysis of proteins which do not produce large crystals, possibly
without radiation damage, directly from solution and without need for cooling. This project is a
large international collaboration, involving the CAMP group from three Max Plank Institutes
and ASU physics. PIs include H. Chapman, P. Fromme, I. Schlichting, B. Doak, U. Weierstall,
J. Uhlich, A. Barty, L. Struder, D. Rolles, the LCLS staff and the ASG team.
06.01.3

Aligning Molecules with Polarized Lasers for X-ray Diffraction Analysis

Linda Young

Argonne National Laboratory, Argonne, IL, United States

Single molecule imaging with atomic resolution endures as a dream for crystallographers who
often have difficulties producing crystals of suitable size and quality. Initial successes with the
world's first x-ray free-electron laser in imaging nanometer-sized crystals of biomolecules are
a step toward this dream. Such nanocrystallography studies retain the N^2 Bragg intensity
enhancement and extending coherent diffractive imaging to the single-molecule limit will be
challenging. One limitation is the unknown orientation of the molecule at the instant of x-ray
diffraction. In this talk, I will describe laser-based techniques to align molecules that can
constrain the rotational degree-of-freedom to form an effective laser-based goniometer. From
a fundamental perspective, the molecule will be aligned by the presence of a strong laser field
and understanding the ensuing structure distortion is of interest.
06.01.4

Coherent X-ray diffraction from sub-micron protein crystals

1 2,1 1 3
Francesco Gramiccia , Céline Besnard , Sebastian Basso , Cameron Kewish , Phil
4,1 5,3 1
Pattison , Franz Pfeiffer , Marc Schiltz
1 2
Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland, UNIversité de GEnève,
3 4
Geneva, Switzerland, Paul Scherrer Institut, Villigen, Switzerland, European Synchrotron
5
Radiation Facility, Grenoble, France, TU München, Münich, Germany

X-ray diffraction is the most widely-used technique to investigate the structure of biologically
important molecules. However, the growing of crystals of sufficient size and quality for
diffraction experiments remains one of the major bottlenecks. Crystals of sizes of about 20-30
microns have been studied successfully on dedicated microfocus beamlines, but radiation
damage is thought to be the ultimate factor that puts a lower limit on the size of crystals that
can be used.

With future X-ray Free Electron Laser sources, the high flux provided by a single pulse may
be sufficient to record the scattering of the object before the onset of radiation damage
effects. Moreover, in a diffraction pattern of very small crystals, the complex and coherent
maxima in between Bragg peaks may be used to solve the phase problem by oversampling.

Using existing third generation synchrotron sources which provide sufficient coherent flux to
investigate macromolecular nanosized crystals, we have recorded coherent diffraction
images on D-xylose isomerase in order to explore the effects of coherence in the diffraction
patterns.

The diffraction pattern recorded on micron-sized crystals of D-xylose isomerase reveals star-
shaped aspects around the Bragg spots. They are interpreted as crystal-shaped truncation
features which are characteristic effects in coherent scattering. Simulated data, computed by
using an analytical expression for the shape amplitude, agree well with the observed patterns.
06.01

Temperature- and cooling rate-dependence of protein conformation: the active site flap
of urease as a model system.
1 1 2
Robert Thorne , Matthew Warkentin , Andrew Karplus
1 2
Cornell University, Ithaca, NY, United States, Oregon State University, Corvallis, OR, United
States

Urease from Klebsiella aerogenes is an ~240 kDa (ð )3 heterotrimer, with the ð-subunits
having a TIM-barrel domain that contains a bi-nickel active site. The unit cell is cubic I213,
with ~178 Å edges (Jabri et al., 1995). Comparison of structures at room temperature and at
T=100 K shows a dramatic change in the ~20 amino acid "flap" covering the active site
(Pearson, MA & PAK, unpublished).. At room temperature the flap is more mobile than the
rest of the molecule, but has an average position placing a key residue, His320, in the active
site properly positioned to act as a catalytic acid. At T=100 K half of the flap becomes a more
highly ordered ð-helix that pulls His320 out of the active site; the other half (starting at
His320) becomes so disordered that it cannot be modeled.

We have examined how these changes develop as a function of temperature by solving the
structure at 11 temperatures between T=300 K and 100 K. The conformational transition
occurs between 270 and 240 K. The disorder in the flap goes through a maximum at T~210
K, and at still lower temperatures remains larger than at room temperature.

We have also investigated how the cooling rate affects the behavior of the flap at T=100 K.
For conventional flash cooling (~100 K/s) and slow cooling (~0.1 K/s), the T=100 K structure
is as previously described. However, for very rapid cooling (~10,000 K/s) using the
"hyperquenching" protocol (Warkentin et al., 2006), the T=100 K structure more closely
resembles the 270 - 300 K structure: the B-factors are lower and the conformation is more
"uncoiled" than for the smaller cooling rates. This suggests that biologically relevant
information lost during cooling for urease (and in principle other proteins) might be made
available by increasing cooling rates to 10,000 K/s or larger.

Jabri, E., M. B. Carr, R. P. Hausinger, and P. A. Karplus. 1995. The crystal structure of
urease from Klebsiella aerogenes. Science 268:998-1004.

Warkentin, M., Berejnov, V., Husseini, N. S. & Thorne, R. E. (2006). Hyperquenching for
protein cryocrystallography. J. Appl. Cryst. 39, 805-811.
06.01.6

Liquid metal jet micro-focus x-ray source: Highest brilliance for home lab
instrumentation

Christoph Ollinger, Lutz Bruegemann

Bruker AXS, Karlsruhe, Germany

For a wide variety of x-ray applications the depth of accessible information is limited by the
brilliance of the x-ray source. Recent break throughs in x-ray source technology push the
limits further. By using liquid metal jet targets (e.g. a Gallium alloy) instead of fast spinning
solid metal targets power loads of the order of 500 kW/mm^2 can be provided. Enabling focal
spot sizes below 20 microns at an x-ray energy of 9.2 keV pave the way for applications with
a brilliance comparable to bending magnet sources in a home-lab instrument.

Combining such a source with state-of-the-art multilayer mirrors, allows to transport the
brilliance, enabling highest flux-densities at the sample suitable for e.g. diffraction and
scattering investigations on very small samples or with very high spatial resolution.

During the course of the presentation dedicated data will demonstrate the potential of such a
source integrated into a laboratory x-ray diffraction instrument. The investigations will be
compared with results obtained with common micro-focus x-ray diffraction instrumentation.
07.17.1

Charge-density-wave crystals and low-dimensional magnets

Sander van Smaalen

University of Bayreuth, Bayreuth, Germany

Long-range order in solid matter can be achieved without three-dimensional translational

symmetry [1]. Such incommensurate crystal structures have been observed in all classes of
compounds, from the elements to protein crystals. Key to the understanding of aperiodic
order is competing interactions, which individually would favor mutually incommensurate
periodicities. Metals and insulators supporting low-dimensional valence bands are prone to
the formation of superstructures due to electron-phonon interactions. Charge-density-waves
(CDWs) and low-dimensional magnetic interactions may lead to incommensurate and
commensurate superstructures, depending on the values of the Fermi wavevector. Here, the
implications are discussed of these superstructures for understanding the phase transitions
and physical properties of CDW materials and other low-dimensional electronic crystals.

References

[1] S. van Smaalen: Incommensurate Crystallography, Oxford University Press (2007).

07.17.2

2D-noncommensurate Modulated Misfit Layer Structures of Franckeite and Cylindrite

1 2 2 3
Emil Makovicky , Václav Petříček , Michal Dušek , Dan Topa
1 2
University of Copenhagen, Copenhagen, Denmark, Czech Academy of Sciences, Prague,
3
Czech Republic, University of Salzburg, Salzburg, Austria

Crystal structures of these triclinic Pb-Sn-Sb-Fe-S compounds have a pronounced 1D

transversal wave-like modulation and a non-commensurate layer match in two dimensions.
They consist of alternating pseudohexagonal (H) and pseudotetragonal (Q) layers and form a
homologous pair: franckeite has Q layers of double thickness compared to cylindrite.
Franckeite from San José, Bolivia, is Pb5.2Ag0.2Sn2.4Sb2.2Fe1.0S14.5, Q layer is an MS layer (M
2+ 2+ 3+
= Pb ,Sn ,Sb ..) four atomic planes thick, with a 5.815 Å, b 5.873 Å, (the layer-stacking
o o o *
vector) c 17.366 Å, α 94.98 , β 88.43 , γ 89.97 ; the modulation vector q = -0.001 a + 0.1282
* * 4+ 2+ 3+
b - 0.0295 c . H layer is a single-octahedron MS2 layer (M = Sn , Fe , Fe ..) with a 3.672 Å,
o 0 o *
b 6.275 Å, c 17.447 Å, α 95.26 , β 95.45 , γ 89.97 ; the modulation vector is q = -0.001 a +
* *
0.1374 b - 0.031 c . The modulation wave has λ = 45.80 Å; the match of centred (sub)cells in
this b direction, 15.5 Q : 14.5 H, occurs at 91.01 Å, what makes 2 λ minus a structurally
important Δ = 0.59 Å. The a and b vectors of both subsystems are parallel; the stacking c
vectors diverge; divergence between modulation wave-front and the stacking c directions is
present as well. 5D superspace refinement was performed in the superspace group C-1,
using 7397 observed reflections; overall R(obs) = 0.094. The Q layers are composed of two
tightly-bonded double-layers; their interspace hosts lone electron pairs. Average composition
for the outer surface is Pb0.74(Sn,Sb)0.26S; for the interspace lining it is (Sn,Sb)0.74Pb0.26S.
Local variations in the Pb:(Sn,Sb) ratios produce the transversal modulation of the Q layer. It
re-establishes a one-dimensional commensurate contact along [010] between the curved Q
and H surfaces to the greatest extent possible. Layer-stacking disorder is important and
omnipresent. Cylindrite forms large cylindrical aggregates, franckeite contorted crystals
because of the increased thickness of the Q layer. The modulated b direction becomes
cylinder axis and the unmodulated a direction of non-commensurate mismatch becomes
cylinder tangent. The existence of these structures depends on the radius ratios for the
cations involved. They cannot exist for a pure Pb-Bi combination that results in the structure
type of cannizzarite.
07.17.3

Electron microscopy and its application to the structural characterization of

incommensurate, compositely modulated and 'disordered' crystal structures.

Ray Withers

The Australian National University, Canberra, A.C.T, Australia

Very many crystalline materials are sensibly inflexible - locked into a fixed stoichiometry, a
conventional 3-D space group symmetry and in a unique dominant free energy minimum.
However, not all materials like to be nailed down! Close inspection of a large and
everincreasing number of materials has shown that many do not in fact fit into such a neat
pigeon hole and are modulated in one form or another. These modulations can be short or
long range ordered, of large or small amplitude while the associated occupational and
displacive Atomic Modulation Functions (AMF’s) required for complete structural
characterization in superspace can be essentially sinusoidal, inherently square wave or saw
tooth in form. Whatever the particular characteristics, an understanding of the local crystal
chemistry as well as the associated physico-chemical properties of such phases can not be
had until such modulations are recognized and properly taken into account.
The Transmission Electron Microscope (TEM) is an extremely well-adapted instrument for the
detection as well as the symmetry and structural characterization of such modulated
structures as a result of the sensitivity of electron diffraction to weak subtle features of
reciprocal space, the ability to obtain such information from small local regions as well as the
capacity to image in various modes with excellent spatial resolution and over a considerable
range of temperature. In this contribution, the application of electron microscopy to the study
of interface, composite, compositionally and/or displacively modulated structures (including
nominally ’disordered’ structures) will be discussed. The characteristic diffraction signatures
associated with these different types of modulated structure will be highlighted along with the
practical application of transmission electron microscopy to problems such as pseudo-
symmetry and twinning, to indexation in (3+D)-dimensional superspace and to overall
superspace symmetry and structural characterization.
07.17.4

A new table of (3+d)-dimensional superspace groups

1 1 2
Harold T. Stokes , Branton J. Campbell , Sander van Smaalen
1 2
Physics & Astronomy, Brigham Young University, Provo, UT, United States, Laboratory of
Crystallography, University of Bayreuth, Bayreuth, Germany

The symmetry of an incommensurately modulated structure is described by a superspace

group in (3+d)-dimensional space, where d is a positive integer that gives the number of
independent modulation waves. Superspace groups are a subset of the general (3+d)-
dimensional space groups that is restricted to point symmetries that do not interchange or mix
the three external dimensions with the d internal dimensions. Here, we present a table of
superspace groups for (3+1), (3+2) and (3+3) dimensions that resolves errors in previous
tables and also contains a variety of previously unpublished symmetry data. After discussing
the use of alternative superspace-group settings, we will demonstrate tools for comparing two
sets of operators to determine whether they correspond to distinct superspace groups or to
two different settings of the same superspace group, which make it possible to quickly identify
symmetry and setting given an arbitrary set of operators. The focus will be on practical
examples.
07.17.5

Pr2(MoO4)3 and Nd2(MoO4): Partially Disordered Scheelite-Like Structures?

Cristina González-Silgo, Candelaria Guzmán-Afonso, Manuel E. Torres, Antonio D. Lozano-

Gorrín, Javier González-Platas, Ulises R. Rodríguez-Mendoza, Juan Rodríguez-Carvajal

Universidad de La Laguna, La Laguna, Tenerife, Spain

Due to a large number of possible cationic substitutions and polymorphisms, the spectrum
of physical properties displayed by scheelites is very broad. They are important host crystals
for a variety of inorganic phosphors-converted light emitting diodes [1], tunable solid state
laser materials [2] and nonlinearities for second harmonic generation and stimulated Raman
scattering [3]. Several authors have recently published new ordered scheelite superstructures
and partially disordered scheelite-like modulated structures. The effects of the different cation
order and the superstructures symmetry, in what concerns to the optical properties analysis,
present some puzzeling challenges [4]. Very recently, the structure of Pr2(MoO4)3 (where 1/3
of the A-site positions are vacant) has been solved, with an incommensurate structure from
synchrotron data [5]. In this work Pr2(MoO4)3 and Nd2(MoO4)3 have been prepared by solid-
state synthesis. We have collected neutron and X-ray powder diffraction data, at room
temperature, in the D2B diffractometer at ILL and in our laboratory. All the structures have
been obtained by multipattern Rietveld refinement using a new symmetry modes procedure:
by AMPLIMODES [6] and FULLPROF [7] programs; and using the advantages of the neutron
3+ 3+
radiation. We have found that Pr and Nd cations are not completely ordered within the
La2(MoO4)3 superstructure with possible new ordering similar to the Eu2(MoO4)3 structure.

[1] Su, Y.; Li, L; Li, G.; Chem. Mater. 2008, 20, 6060-6067.

[2] Méndez-Blas, A.; Rico, M.; Volkov, V.; Zaldo, C.; Cascales. C.; Phys. Rev. B, 2007, 75,
174208-174222.

[3] Zverev, P.G.; Basiev, T.T.; Prokhorov, A.M.; Opt. Mater. 1999, 11, 335-352.

[4] Arakcheeva, A.; Chapuis, G.; Acta Crystallogr. B, 2008, 64, 12-25.

[5] Logvinovich, D.; Arakcheeva, A.; Pattison, P., Eliseeva, P.; Tomes, P.; Marozau, I.;
Chapuis, G.; Inorg. Chem. 2010, 49, 1587-1594.

[6] Orobengoa, D., Capillas, C., Aroyo, M. I., Perez-Mato, J. M.; J. Appl. Cryst. 2009, 42, 834-
845.

[7] Rodríguez-Carvajal, J. (1993). FULLPROF. Program for Rietveld analysis of X-ray and
Neutron powder diffraction patterns. ( http://www.ill.eu/sites/fullprof/).

[]
07.17.6

Positional average structure from an incommensurately modulated crystal of

profilin:actin
1 1 2 1
Jason Porta , Jeff Lovelace , Antoine Schreurs , Gloria Borgstahl
1 2
University of Nebraska Medical Center, Omaha, NE, United States, Utrecht University,
Utrecht, Netherlands

Modulation of protein crystals is seldom reported, mainly due to a lack of methods for solving
these unique structures. We report here the incommensurately modulated average structure
of profilin:actin and the methods that were used to carry out the analysis. Crystal modulation
is characterized by a loss of short-range translational symmetry, where a single unit cell is no
longer sufficient to accurately describe the structure. Such a loss of periodicity is often caused
by dynamic processes within the crystal arising from, for example positional modulations.
Experimentally, the incommensurately modulated state is characterized by the appearance of
distinct satellite reflections surrounding the main Bragg reflections on the diffraction pattern
that cannot be indexed with a supercell. In order to fully describe a modulated structure, and
hence the dynamic processes within the crystal, one must explore higher-order space over
multiple unit cells. By careful examination of atomic positions over higher dimensional space,
a modulation function can be calculated that traces the atomic disorder. Such a function is
periodic, but incommensurate with the crystal lattice. As a first step to solving this function for
PA crystals, we have determined the average structure of the modulated state. Main and
satellite reflections were integrated with Eval15 and scaled with SadAbs to 3.0 Å. The
average structure was then solved using the Phenix crystallographic software suite. Fourier
electron density maps indicate the whole structure moves with major motion in actin
subdomains 2 and 4. Superposition with the periodic profilin:actin structure and analysis by
DYNDOM confirm these observations by showing the rotation of these domains. The
modulation is in the b direction, which corresponds to the ribbon of actin molecules along this
crystallographic axis. Analysis of these domain movements give insight into the long sought
after globular (G) to fibrous (F) actin transition.
07.17.7

Extending the 14-electron rule beyond the Nowonty Chimney Ladder phases: a 14-
electron series based on defect structures of the MoSi2 structure type.
1 2 3 4
Daniel Fredrickson , Magnus Bostroem , Yuri Grin , Sven Lidin
1 2
University of Wisconsin-Madison, Madison, WI, United States, Sandvik Materials
3
Technology, Sandviken, Sweden, Max-Planck-Institut fuer Chemische Physik fester Stoffe,
4
Dresden, Germany, Lund University, Lund, Sweden

The Nowotny Chimney Ladder phases (NCLs) provide one of the clearest examples of
electron count control of structure stability in intermetallic phases. The NCLs are compounds
formed between transition metal (T) and main group elements (E) with stoichiometries of the
form TE2-x. The structures of these phases are based a beautiful variation on the TiSi2
structure type in which E atom helices thread through T atom helical channels. For NCLs
with late transition metals (groups 7 and higher), the E atom deficiency, x, is tuned (often
quite precisely) so that the total number of valence electrons divided by the number of T
atoms is fourteen -- a criterion for stability known as the fourteen electron rule. In this
presentation, we will explore the possibility of extending this rule beyond the TiSi2 defect
structures. Electron microscopy investigations of the Re4Si7 phase and ternary derivatives
by a number of researchers have revealed complex and incommensurate superstructures
based on another transition metal disilicide structure type: the MoSi2 type. The
superstructures appear to be correlated with electron count, suggestive of a close connection
to the fourteen electron rule of the NCLs, but the details in terms of both geometry at the
atomic level and the electron structure remain unresolved. In an effort to complete this
picture, we have synthesized Re4Si7 and several Os- or Al- substituted derivatives, and
analyzed their crystal structures with single crystal X-ray diffraction. These phases exhibit
complex diffraction patterns that can be resolved into strong reflections arising from a MoSi2
basic cell, and weaker satellite reflections. Structure solution and refinement using
superspace methods reveal that satellites arise from Si vacancies occurring as edge-
deletions, with the neighboring Si atoms relaxing to smooth out the deletions. The result
resembles a crystalline ordering of Si edge-dislocations in a MoSi2-type crystal. Electronic
structure calculations on these phases, at both the ab initio and semi-empirical levels, show
features analogous to the band structures of the NCLs, strongly suggestive of a common
origin of stability for both families of phases. These results suggest that domain of the
fourteen electron rule extends beyond the NCL phases to incorporate defect structures of the
MoSi2 type.
07.17.8

To What Extent Does the Zintl-Klemm Formalism Work? The Eu(Zn1-xGex)2 Series

1 2 3 4 5
Tae-Soo You , Sven Lidin , Olivier Gourdon , Yaqiao Wu , Gordon Miller
1 2
University of Delaware, Newark, DE, United States, Stockholm University, Stockholm,
3 4
Sweden, Oak Ridge National Laboratory, Oak Ridge, TN, United States, Ames Laboratory,
5
Ames, IA, United States, Iowa State University, Ames, IA, United States

The series of ternary polar intermetallics Eu(Zn1-xGex)2 (0 ≤ x ≤ 1) has been investigated and
characterized by powder and single crystal X-ray diffraction as well as physical property
measurements. For 0.50(2) ≤ x < 0.75(2), this series shows a homogeneity width of
hexagonal AlB2-type phases (space group P6/mmm, Pearson symbol hP3) with Zn and Ge
3
atoms statistically distributed in the planar polyanionic 6 nets. As the Ge content increases in
this range, a decreases from 4.3631(6) Å to 4.2358(6) Å, while c increases from 4.3014(9) Å
to 4.5759(9) Å, resulting in an increasing c/a ratio. Furthermore, the Zn-Ge bond distance in
the hexagonal net drops significantly from 2.5190(3) Å to 2.4455(3) Å, while the anisotropy of
the displacement ellipsoids significantly increases along the c direction. For x < 0.50 and x >
0.75, respectively, orthorhombic KHg2-type and trigonal EuGe2-type phases occur as a
second phase in mixtures with an AlB2-type phase. Diffraction of the x = 0.75(2) sample
shows incommensurate modulation along the c direction. Temperature-dependent magnetic
susceptibility measurements for two AlB2-type compounds show Curie-Weiss behavior above
40.0(2) K and 45.5(2) K with magnetic moments of 7.98(1)μ B for Eu(Zn0.48Ge0.52(2))2 and
7
7.96(1) μ B for Eu(Zn0.30Ge0.70(2))2, respectively, indicating a (4f) electronic configuration for Eu
2+
atoms (Eu ). The Zintl-Klemm formalism accounts for the lower limit of Ge content in the
AlB2-type phases, but does not identify the observed upper limit.
07.17.9

Charge-Density Waves and Structural Modulations in Layered Polytelluride

Compounds

Christos Malliakas, Mercouri Kanatzidis

Northwestern University, IL, Evanston 60201, United States

The generation of charge density waves (CDW) produces modulated structures and is
believed to be a competing process to unconventional superconductivity and other quantum
ground states. Incommensurate to commensurate or near-incommensurate phase transitions
associated with CDW can give rise to unusual and often unexplained phenomena. CDW
states can be created through a Fermi surface nesting effect and the creation of a new
ground state with broken translational symmetry. As a result of the new periodicity, a band
gap opens at the Fermi surface and an overall electron energy lowering is achieved.

In this presentation, a systematic study of layered polytelluride CDW compounds will be

reported in order to understand and elucidate their structural modulations. Since these
structural distortions are generally incommensurate with respect to the underlying crystal
sublattice the use of multidimensional crystallographic methods in superspace is necessary
for solving their structures. The general approach for solving aperiodic crystals and details for
the crystallographic refinement and solution in (3+1) and (3+2) dimensions will be also
discussed.

The family of compounds RETe3 (RE = Rare-Earth), revealed for the first time that the nature
of the CDW in these compounds varies subtly but significantly with RE element. Furthermore,
investigation of AMRETe4 (A = Na, K; M = Cu, Ag; RE = La, Ce) that is structurally related to
RETe3 showed that cross-plane interactions play an important role in defining the CDW
distortions in these materials. Additionally, RE2Te5-x that is composed of the substructures of
RETe2-x and RETe3 represents a rare example of composite structure with two different Te
nets which exhibit a hybrid CDW distortion with two independent modulation vectors. Finally,
a unique double modulated Te net in Cu0.66EuTe2 will be also presented. CDWs in these
materials originate from the extended hypervalent Te∙ ∙ ∙ Te bonding that occurs in the planar
square nets of tellurium.
07.17.10

Ga square nets – Modulation and Twinning, Oh My!

Danielle Gray, Mercouri Kanatzidis

1 2
University of Illinois, Urbana, IL, United States, Northwestern University, Evanston, IL,
United States

Compounds with electrons confined to low dimensional structures are prone to distortions
known as charge density waves (CDWs). A series of RENi1-xGa3Ge (RE = Tb, Dy, Ho, Er,
Tm; 0.04 ≤ x ≤ 0.16, but is fixed +/- 1% for a given RE composition) compounds with 2-D Ga
square nets exhibit CDWs. Examination of the average tetragonal (I4/mmm) structure for the
RENi1-xGa3Ge materials revealed structural problems despite very good fitting statistics. The
Ga in the square nets had large thermal parameters as compared to the rest of the atoms,
and the Ni atoms had occupational disorder on one site that induced a positional disorder on
the Ge sites. Upon close inspection of reciprocal space, 4 pairs of additional reflections are
visible around each main reflection. A (3+1)D superspace approach was used with
application of 4-fold pseudomerohedral twinning in order to model the incommensurate
structure. RENi1-xGa3Ge compounds crystallize in the superspace group I2/m(α β 0)0s with the
c-axis as the unique axis. The structural models show coupling between the CDWs in the Ga
nets and the Ni/Ge site occupancy waves.
07.18.1

Snapshots into HIV-1 capsid maturation: structural insights derived from mutations in
the HIV-1 Capsid Assembly Inhibitor binding site
1 1,2 3 3 1
Luis R. Castillo , Vanda Lux , Sebastien Igonet , Felix Rey , Hans-Georg Kräusslich
1 2
University Hospital Heidelberg, Heidelberg, Germany, Universität Duisburg-Essen, Essen,
3
Germany, Institut Pasteur, Paris, France

HIV-1 maturation from a non-infectious into an infectious agent is accompanied by

morphological changes in its capsid shell which undergoes a protease-mediated conversion
from a spherical into a conical shape. We previously identified a Capsid Assembly Inhibitor
(CAI) that blocks immature and mature HIV-1 capsid assembly in vitro. CAI binds to a
hydrophobic pocket in the C-terminal domain of the capsid protein (C-CA), which promotes a
conformational change. X-Ray structures from mutants in the C-CA binding pocket showed
that while some keep the unliganded C-CA conformation, others adopt the conformation of C-
CA in complex with CAI(C-CA/CAI). More extensive mutagenesis work has allowed us to
identify two C-CA mutants that adopt an intermediate phenotype between the C-CA and C-
CA/CAI conformations. Recently, cryo-electron tomography of the HIV-1 immature capsid
lattice showed that a C-CA/CAI conformation mutant fits very well in the immature shell.
Besides, an NMR structure of a C-CA with two amino acids extension at the N-terminus
showed a distinct quaternary conformation from the X-Ray structures, but which fitted better
into a cryo-electron reconstruction of a mature-like capsid assembled particle. X-ray
structures from the construct used in NMR showed the N-terminus extended C-CA
conformation in the crystal is the same as that found in the unliganded C-CA. Nevertheless,
the C-CA NMR structure showed a similar conformation of the mutants with an intermediate
phenotype. Since more than one C-CA construct structure could be classified in a defined
state based on the CAI-induced conformational change and based on the fact that each
defined conformation could fit into EM reconstructions from capsid shell particles; these
structures might represent snapshots of a conformational change pathway that the HIV-1
capsid undergoes during maturation.
07.18.2

Receptor Recognition Mechanisms of Coronaviruses

Fang Li

University of Minnesota, Minneapolis, MN, United States

Coronaviruses recognize a variety of receptors and infect many hosts. NL63 coronavirus
(NL63-CoV), a prevalent human respiratory virus, is the only group-I coronavirus known to
use angiotensin-converting enzyme 2 (ACE2) as its receptor. Curiously, ACE2 is also used by
group-II SARS coronavirus (SARS-CoV). Defined receptor-binding domains (RBDs) on the
spike proteins of NL63-CoV and SARS-CoV bind ACE2 with high affinity. We have
determined the crystal structures of NL63-CoV RBD complexed with human ACE2 and of
SARS-CoV RBD complexed with human ACE2, revealing for the first time how two different
viruses can recognize the same receptor protein. Specifically, NL63-CoV and SARS-CoV
RBDs have no structural homology in cores or receptor-binding motifs (RBMs) that directly
contact ACE2, but recognize the same “virus-binding hotspot” on ACE2. Among group-I
coronaviruses, RBD cores are conserved, but RBMs are variable, explaining how these
viruses recognize different receptors. Moreover, we have also determined the crystal
structures of the RBDs from various SARS-CoV strains complexed with ACE2 proteins from
humans and palm civets, revealing the structural mechanisms whereby SARS-CoV
transmitted from palm civets to humans and caused the worldwide SARS epidemic in year
2002-2003. Specifically, SARS-CoV strains from palm civets became adapted to human
ACE2 through stepwise mutations in their RBMs, gaining affinity for human ACE2 and
infectivity in human cells. Overall, our studies provide the molecular and structural basis for
understanding viral evolution, virus-receptor interactions, viral host ranges and cross-species
infections. They also guide the development of novel antiviral strategies against coronavirus
infections.
07.18.3

The crystal structure of E. coli fimbrial tips at 2.7 Angstroms resolution: Structural
views of fimbrial assembly, donor strand complementation, and force-mediated cell
adhesion

Ronald Stenkamp, Isolde Le Trong, Pavel Aprikian, Brian Kidd, Wendy Thomas, Evgeni
Sokurenko

University of Washington, Seattle, WA, United States

Fimbriae and pili are macromolecular structures on the surface of Gram negative bacteria that
are important for cellular adhesion. We have solved a 2.7 Å resolution crystal structure of a
complex of E. coli fimbrial proteins containing FimH, FimG, FimF, and FimC. This provides
the most complete model to date for how subunits assemble into these non-flagellar adhesive
appendages. The first three subunits form the tip of the fimbriae while FimC is the chaperone
protein involved in the usher-chaperone assembly process. The fimbrial subunits form an
extended structure in the two complexes per asymmetric unit that can serve as a model for
native fimbrial tips. The subunits are held together through donor strand complementation
where a β -strand from one subunit completes the β -sandwich structure of the subunit closer
to the tip of the fimbria. FimC provides a surrogate donor strand before delivery of each
subunit to the FimD usher and the growing fimbria. Structures of several of the subunits in
complex with FimC have been seen before. Comparison of the subunits in this structure with
those complexes show that the lectin and pilin domains of FimH change their relative
orientation and position in forming the tip complex. The pilin domain also compresses one
end of the lectin domain β -sandwich which loosens the mannose-binding pocket at the
opposite end. This provides a model for FimH’ s force mediated mannose binding properties.
In addition to this structural change, the non-chaperone subunits undergo a conformational
change in their first β -strand when the chaperone is replaced by the native donor strand.
Some residues differ by as much as 14 Å between their positions in the tip complex and their
positions in their FimC complexes. Donor strand complementation is used in the assembly of
many bacterial sub-structures, and this structural shift has not been described elsewhere.
The structure of the tip complex provides new views into the allosteric effects of mechanical
force on receptor-ligand interactions and into how multiple subunits can bind the same
chaperone and still bind specifically to particular subunits in the larger fimbrial structure.
07.18.4

Crystallographic and cryo-EM studies of the 750-kD 6 6 holoenzyme of propionyl-

coenzyme A carboxylase
1 1 1 2 2,3
Christine Huang , Kianoush Sadre-Bazzaz , Yang Shen , Binbin Deng , Z. Hong Zhou ,
1
Liang Tong
1 2
Columbia University, New York, NY, United States, University of Texas Medical School at
3
Houston, Houston, TX, United States, University of California, Los Angeles, Los Angeles,
CA, United States

Propionyl-CoA carboxylase (PCC) is essential for the catabolism of several amino acids,
cholesterol, and odd-carbon fatty acids. Deficiencies of PCC activity in humans are linked to
propionic acidemia, an autosomal recessive disorder that can be fatal in infants. The PCC
holoenzyme is an 66 dodecamer, with a molecular weight of 750-kD. The subunit contains
the biotin carboxylase (BC) and biotin carboxyl carrier protein (BCCP) domains, while the 
subunit supplies the carboxyltransferase (CT) activity. We describe the crystal structure at
3.2Å resolution of a bacterial PCC 66 holoenzyme as well as cryo-EM reconstruction at 15Å
resolution demonstrating a similar structure for human PCC. The structure defines the overall
architecture of PCC and reveals unexpectedly that the subunits are arranged as monomers
in the holoenzyme, decorating a central β 6 hexamer. A hitherto unrecognized domain in the α
subunit, formed by residues between the BC and BCCP domains, is crucial for interactions
with the  subunit. We have named it the BT domain. The BC and CT active sites in the
holoenzyme are separated by approximately 55Å, indicating that the entire BCCP domain
must translocate during catalysis. The BCCP domain is located in the active site of the 
subunit in the current structure, providing insight for its involvement in the CT reaction. The
structural information establishes a molecular basis for understanding the large collection of
disease-causing mutations in PCC, and also has important relevance for the holoenzymes of
other biotin-dependent carboxylases, especially MCC and eukaryotic ACC.
07.18.5

Crystal structure of the Head module of Mediator in gene expression, and its
interactions with the RNA polymerase II transcription machinery
1 1 2 2 3
Yuichiro Takagi , Tsuyoshi Imasaki , Gang Cai , Kuang-Lei Tsai , Guillermo Calero , Kentaro
1 1 4 3 2
Yamada , Francesco Cardelli , Imre Berger , Roger Kornberg , Francisco Asturias
1 2
Indiana University School of Medicine, Indianapolis, IN, United States, The Scripps
3
Research Institute, La Jolla, CA, United States, Stanford University School of Medicine,
4
Stanford, CA, United States, EMBL Grenoble, Grenoble, France

Mediator is a large multi-protein complex essential for eukaryotic gene expression by

transducing the regulatory DNA information through DNA-bound transcription activators to
RNA polymerase II (pol II). Mediator from yeast is complex composed of 21 subunits that are
organized into three distinct modules, termed Head, Middle/Arm and Tail. The Head module
(7 subunits, 223 kDa) is an essential sub-complex of Mediator as it controls Mediator-pol II
and Mediator-promoter interactions in vivo. Thus, understanding structural basis of the
Mediator Head module, and the Head-RNA pol II machinery is required to elucidate the
mechanism of transcription initiation. We have determined the structure of the Head module
of Mediator at 4.5 Å resolution by X-ray crystallography. A use of Tantalum cluster SIRAS, as
well as Se-Met SAD was the key to obtain significant phase information. Combined with
biochemical and EM studies, the X-ray module of the Head reveals three distinct domains:
fixed jaw, movable jaw, and handle domain. The three domains are predominantly rich in
apha helices. They are connected to the less-well ordered central hinge domain, which
appears to provide flexibilities to the three domains. To reveal how the Head module interacts
with the pol II machinery, using single particle cryo-EM, we have determine the structure of
the Head-the minimum preinitiation complex (Head-mPIC) composed of Head, pol II, IIF, IIB,
TBP, and promoter DNA. In the EM structure, the fixed and movable jaws clearly interact with
Rpb4/7 subunits of pol II Notably, the clamp of pol II appears widely open in the presence of
the Head module, providing the structural evidence to support our model in which Mediator
modulates access of promoter DNA to the pol II cleft in part through the Head-Rpb4/7
interaction.
07.19.1

Combined Ultrasmall-Angle X-ray Scattering and X-ray Photon Correlation

Spectroscopy

Studies of Nonequilibrium Dynamics in Polymer Composites

1 1 1 2 2
Fan Zhang , Andrew Allen , Lyle Levine , Jan Ilavsky , Gabrielle Long
1
National Institute of Standards and Technology, Gaithersburg, MD, 20877, United States,
2
Argonne National Laboratory, Argonne, IL, 60439, United States

While scattering and imaging techniques have enjoyed great success in studies of the
microstructure over the full nanometers-to-micrometers size range in advanced materials
such as composites and alloys, the dynamics of these materials, especially the response to
abrupt changes in the sample environment, largely remains elusive. Knowledge of the
incipient dynamical behaviour inevitably leads to a better understanding of the processing–
structure–property relationships, and to a consequent improvement in material design and
performance.

Recently, we have developed combined ultrasmall-angle X-ray scattering / X-ray photon

correlation spectroscopy (USAXS/XPCS) studies to probe the slow equilibrium and
nonequilibrium dynamics of optically opaque materials with prominent scattering features in a
q range between that of dynamical light scattering and conventional XPCS. Using
USAXS/XPCS, we have studied the dynamical, nonequilibrium structural variation of polymer
composites to an extent beyond what has been currently available. Upon heating, these
polymer composites, synthesized as advanced dental materials, undergo irreversible
dynamical structural change that is insensitive to either small angle X-ray scattering or X-ray
diffraction.

On exploiting the coherent beam properties of the Advanced Photon Source in combined
USAXS/XPCS studies, a distinctive variation in the coherent scattering patterns is observed.
Detailed analyses show systematic changes under different starting conditions, which we
attribute to polymer creep arising from effects such as thermal mismatch on heating, or a
change in particle density associated with an incipient amorphous-to-crystalline
transformation in the “amorphous” calcium phosphate (ACP) filler material used in advanced
dental nanocomposites. With the polymer composites as a prototype for a future class of
materials study, we hope to establish the USAXS/XPCS technique as a unique tool to follow
slow dynamics in disordered materials.
07.19.2

Nanocrystalline sodalites and intermediate structures between sodalite and cancrinite

grown under high NaOH and NaOH/TEA concentrations at 333K

Josef-Christian Buhl, Sandra Cramm, Karsten Schuster

Leibniz University Hannover, Hannover, Lower Saxony, Germany

The systems Na2O-SiO2-Al2O3-H20-NaCl/NaNO3 were investigated within reaction periods of

1 - 96 h under superalkaline low temperature conditions at 333 K. Mixtures of aluminosilicate
gels with NaOH and NaCl or NaNO3 were used as educts. Nanoparticles of NaCl-sodalite
(SOD) or nanocrystalline nitrate enclathrated intermediate phases (INT) between the
structures of SOD and cancrinite (CAN) were successfully synthesized already after 3 h
reaction time. Triethanolamine (TEA) was added in further series of syntheses to study the
influence on formation of SOD, CAN and INT, because deceleration of nucleation rate is well
known in the zeolite A and X system due to the complexing effect of TEA on the reactive
aluminium-species in the solution [1].

The products were characterized by X-ray powder diffraction, scanning electron microscopy,
FTIR spectroscopy and thermal analysis in comparison with the microcrystalline samples
NaCl-SOD and NaNO3-CAN. Both were synthesized at 473 K and 48 h from kaolinite, i.e. the
common conditions of hydrothermal formation of microcrystalline SOD and CAN.

Analyses data show formation of nanoparticles in each case already after 3 hours with sizes
not exceeding 50 nm and total batch compositions of about 50% nanocrystalline material
beside 50% amorphous parts. Whereas the experiments with NaCl yielded the SOD-structure
nitrate addition favours nanocrystalline intermediates (INT) between the structures of SOD
and CAN as previously found in the carbonate system under common synthesis conditions
from kaolinite [2]. INT is characterized by one dimensional stacking disorder of aluminosilicate
layers along hexagonal c-axis. In CAN framework these layers show AB-stacking and SOD
exhibits cubic ABC-sequence [2].

In the reaction series with TEA in principle the same phases were observed but SOD
appeared in form of unusual big board-like aggregates of small spheres which in turn consist
of numerous nanocrystaline particles. INT was found as big spheres formed by countless
nanocrystalline rods.

[1] Charnell, J.F.: J. Crystal Growth 8 (1971) 291 – 294.

[2] Hermeler, G., Buhl, J.-Ch., Hoffmann, W.: Catalysis Today 8 (1991) 415 – 426.
XMR

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Porous materials are the subject of intense research. The most familiar porous materials are
zeolites, which have numerous applications including molecular sieves for selective
absorption (e.g. for gas storage or water purification) and catalysis.

Although zeolites are well known, they form only one class of porous material. Molecular-
based porous materials form an important growth area in crystal engineering; one class of
these materials are metal organic frameworks (MOFs). These are an attractive alternative to
zeolites because they are not restricted to tetrahedral network topologies, and are much more
amenable to incorporation of chemical modifications, such as chirality. (MOFs) contain metal
ions linked by rigid organic linkers. One of the first MOFs to be studied was (Zn4O(C8H4O4)3),
otherwise known as MOF-5 (Fig. 1). MOF-5 is the parent structure of a series of iso-reticular
MOFs (IRMOFs) whereby the overall repeating structure is conserved while allowing changes
in the pore size and functionality by alteration of the organic linker.

Fig. 1 MOF-5 structure shown as ZnO4 tetrahedra joined by benzene dicarboxylate linkers.
The large sphere represents the largest van der Waals sphere that would fit in the cavity.

Here, the effect of pressure on MOF-5 to 4 GPa is presented, where hydrostatic fluid entering
the pore volume and pressure induced contraction and expansion of the framework can be
observed.
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07.19.5

Combined neutron- and X-ray diffraction study of borosilicate glasses relevant to the
immobilization of nuclear waste

1 2 3 1
Margit Fabian , Thomas Proffen , Uta Ruett , Erzsebet Svab
1 2
Research Institute for Solid State Physics and Optics, Budapest, Hungary, Los Alamos Natl.
3
Lab., Lujan Neutron Scattering Ctr, Los Alamos, United States, Deutsches Elektronen-
Synchrotron (DESY), Hamburg, Germany

Borosilicate based glasses are generally accepted as proper high-level radioactive waste
isolating media [1]. In spite of their importance, relatively few diffraction investigations have
been performed because of the large number of constituent elements.

Here we report our results on three borosilicate based glassy series prepared under the same
melt quenching preparation technique [2], starting with the 3-component material up to the 6-
component one, with the composition of (75-x)SiO2•xB2O3•25Na2O,

(65-x)SiO2•xB2O3•25Na2O•5BaO•5ZrO2 [2] and

70wt%[(65-x)SiO2•xB2O3•25Na2O•5BaO•5ZrO2)]+30wt%UO3 (x=5,10,15,20 mol%). For the

study of the short- and intermediate range order we have performed neutron- and X-ray
diffraction measurements. Neutron diffraction measurements were carried out at the 10 MW
Budapest research reactor using the PSD diffractometer [3] and the time-of-flight NPDF
instrument at the LANSCE/Los Alamos pulsed neutron source [4]. The high-energy X-ray
diffraction measurements were performed at the BW5 experimental station at Desy/Hamburg
using 109,5 keV radiation energy [5]. Both the traditional Fourier transformation technique
and the reverse Monte Carlo (RMC) simulation of the experimental data have been applied to
get structural information. Simultaneous RMC simulation of the two data sets was applied to
generate reliable 3-dimensional atomic configurations and to calculate the partial atomic pair
correlation functions and coordination number distributions. It was established that the basic
network structure consists of tetrahedral SiO4 units and of mixed tetrahedral BO4 and trigonal
BO3 units for the three investigated glassy systems. The multi-component glasses proved to
be stable and capable of hosting uranium. For the U-O first neighbour correlations two
distinct peaks were resolved at 1.84 Å and 2.24 Å, and for higher distances intermediate-
range correlations were observed. Significant correlations have been revealed between
uranium and the network former Si and B atoms, indicating that uranium ions take part in the
network forming. Details of the structural characteristics will be presented.

[1] Chun K S, Kim S S and Kang C H 2001 Journal of Nuclear Materials 298 150

[2] Fábián M, Sváb E, Proffen Th and Veress E 2008 J. Non-Cryst. Solids 354 3299

[3] Sváb E, Mészáros Gy and Deák F 1996 Materials Science Forum 228 247

[4] Proffen Th, Billinge S J L, Egami T and Louca D 2003 Zeitschrift für Kristallographie
18 132

Poulsen H, Neuefeind J, Neumann H B, Schneider J R and Zeidler M D 1995 J. Non-Cryst.

Solids 188 63
07.19.6

Advances in Deep Space Nuclear Reactor Design

Boris Udovic

Private, Sezana, Slovenia

Lightweight thermo-emissive nuclear reactor hot cathode bodies with thick shells of highly
12 3
thermally conductive monoisotopic Cfsp CVD diamond and high-density tetrahedral
amorphous ta-C CVD carbon phases are projected to acquire severe long-life properties with
high temperature self-repairing sealant capabilities over encapsulated nuclear fuel kernels in
deep space flight missions. The unavoidable leakage of the white spectrum of striking
neutrons, fissile particles and the continuous emission of and rays gives rise to fission
spikes by cascades of primary, secondary and higher order displacements of ballistically
knocked-on atoms via gradual transitions from “Rutherford” to “hard sphere” collisions inside
the monoisotopic matrix of the whole onion carbon envelopment. As the thermo-emissive
current sharply intensifies with the squared value of the absolute temperature, the operative
regime targeted near 2500 K matches to a considerable extent some multiple requirements: -
to avoid greater effects of sublimation phenomena at the diamond surfaces, -to promote
structural changes that can overcome the threshold energy barrier of the settling-
displacements inside the superheated solid phase because of localized excitations produced
12 3
by hot thermal spikes. Once formed, the Cfsp ta-C CVD carbon phase network prevents
any re-hybridization of the whole diamond unit cell and makes impossible the relaxing states
to graphite allotrope backwards. Namely, the compressive shrinkage of the carbon matrix in
3
the fashion of a self-pressing nutcracker jaw, which squeezes every displaced sp carbon
atom out from its tightly strained inter-atomic cage, hinders any size enlargement of the
disordered ta-C phases by self organization phenomena, raising a higher degree of symmetry
of the whole tetrahedral network bone. The high temperature and low pressure working gas of
free caesium atoms Cs saturates the nearest inter-space around the hot cathode boundary
layer. Congruently, the driving force of the electron transfer reaction from the nearest thermo-
12 2
ionized caesium atom Cs to each Cfsp carbon atom at the top of the ta-C coating epi-layer
promptly promotes the quick rehybridization change of the appearing planar carbo-olefinic
units into pyramidalized free radicals, which rebuild and keep safe the diamontoid 3D
network.
07.19.7

Evolution of copper-rich precipitates in reactor pressure vessel steels under irradiation

1 1 1 1 1
Mikhail Sokolov , Michael Miller , Randy Nanstad , Ken Littrell , Lee Robertson , Enrico
2
Lucon
1 2
ORNL, Oak Ridge, United States, SCK-CEN, Mol, Belgium

Current fleet of nuclear power plants is poised for operating life time extension. It means that
main structural components, including reactor pressure vessel, will be subject to higher
neuron exposure than originally planned. This raises serious concerns regarding our ability to
predict reliability of reactor pressure vessel steels at such high doses. In this study, several
representative reactor pressure vessel steels (RPVS) were irradiated at high doses to study
degradation of mechanical properties and related microstructural changes of RPVS. It is well
known that copper-rich precipitates are key microstructural futures that are responsible for
radiation hardening of RPV steels. In this study, the evolution of copper-rich precipitates
(CRP) is studied by means are small-angle neutron scattering and atom-probe tomography.
These techniques are used to measure number density, volume fraction, and radius of
precipitates. Evolution of these microstructural features is cooperated to degradation of
fracture toughness and hardening of these steels.
07.19.8

Correlating Small Angle Scattering with Electrical Resistivity Changes in the Nickel-
Base Superalloy Waspaloy
2 2 2 3 1
Ricky Whelchel , V. S. K. G. Kelekanjeri , Rosario Gerhardt , Jan Ilavsky , Ken Littrell
1 2
HFIR/NSSD Oak Ridge National Laboratory, Oak Ridge, TN, United States, School of
Materials Science and Engineering, Georgia Institute of Technology, Atlanta, GA, United
3
States, 2X-ray operations division, Argonne National Laboratory, Argonne, IL, United States

Waspaloy is a nickel-base superalloy used primarily in disc rotors in gas turbine engines.
Waspaloy is ideal for this purpose due to the material's increased high temperature strength
and creep resistance due to the formation of nanometer-scale precipitate phases (γ ') within a
solutionized matrix phase (γ ) via heat treatment. The pre-existing precipitate phases formed
via heat treatment can evolve with in-service thermal exposure in gas turbine engines,
resulting in evolving mechanical properties of the bulk superalloy components. Consequently,
it is desirable to monitor this evolution in mechanical properties non-destructively.

Electrical resistivity is one method by which the precipitate microstructural evolution may be
sensed. Electrical resistivity is sensitive to the formation of small phases and the removal of
precipitate phase solute [1]. Small precipitates, on the order of 1nm, have significant electron
scattering ability and are the dominant features affecting electrical resistivity changes at early
stages of the aging process. Solute removal due to precipitation results in a more pure, and
thus more conductive, matrix phase.

Waspaloy specimens aged at 800°C from 0.5h to 88.5h were evaluated via small angle
neutron scattering (SANS), ultra small angle X-ray scattering (USAXS), electrical resistivity,
and SEM. The average γ ' precipitate size and volume fraction obtained from modeling the
small angle scattering data was used to calculate a figure of merit of electron scattering. This
figure of merit is designed to correlate the electron scattering ability of the material with the
precipitate microstructure.
07.21.1

A Career is What You Make of It

Joseph Ferrara

Rigaku Americas Corp, The Woodlands, TX, United States

My professional odyssey started while I was an undergraduate at Case Institute of

Technology. For my senior research project I used ion cyclotron resonance to study collision
quenching of photoexcited iodobenzene ions. This is where I started my love of
instrumentation; I worked in a lab with very old equipment, some handmade and even some I
made myself. I then continued on at Case for my graduate degree thinking I would finish
quickly continuing my undergraduate research. That was not to happen.

I heard a new faculty mumble something about room temperature superconductors (this was
5 years prior to the discovery of cuprate superconductors). That phrase took me down a path
of synthetic organic chemistry and organometallic chemistry, physical chemistry and finally,
my true calling, crystallography,

Upon completing my dissertation in 1987, my wife and I moved to College Station, TX, where
I started as a service crystallographer for Molecular Structure Corporation. In 1996, Rigaku
bought MSC lock, stock and barrel. Currently, I am currently CSO of Rigaku Americas
Corporation, VP of the X-ray Research Laboratory in Tokyo, and a Rigaku Innovative
Technologies board member.

Over the years I have solved some pretty tough structures, written and rewritten lots of
FORTRAN, dealt with European compliance issues, started a quality assurance department,
managed software and hardware development teams, learned some Japanese and watched
some people blossom. I get to meet lots of very interesting people and really do have fun.
07.21.2

Beamline Scientist

James Holton
1 2
University of California, San Francisco, CA, United States, Lawrence Berkeley Laboratory,
Berkeley, CA, United States

Probably the best thing about working at a beamline is the sheer number of
“collaborators” you work with (we call them “users”). Most crystallographers will only work
directly with a few dozen other scientists in their entire career, but a typical beamline scientist
works side-by-side with more than a hundred different investigators each year. Each user
has their own favourite tools, techniques and software, but it is only by seeing lots of different
approaches that one can fully understand what works and what doesn’ t in protein
crystallography. This is not just a good way to accumulate expertise, but also a good way to
become widely recognized as an expert. Even if your career goals eventually lead you away
from the synchrotron, you can hardly do worse than having hundreds of crystallography labs
recognize you as that one person who was really helpful and knowledgeable in their time of
need. Becoming an expert in methods is not just a luxury of beamline science, it is a job
requirement, and although there are those who regard methods development as secondary to
“real science”, there are few who do not recognize the importance of data. There is nothing
quite like beautiful data. Even the most cantankerous reviewers can’ t argue with it nor the
conclusions that follow obviously from it. But, the trick to cutting edge science is knowing
what you can and cannot conclude from “decent” data (the kind you get early in a project),
and making these calls requires someone who knows the method inside and out.

1995 B. S. in Biology, Caltech

2001 Ph. D. Molecular and Cell Biology – Tom Alber, UC Berkeley

2001 Staff Scientist, Beamline 8.3.1 ALS

2005 Assistant Adjunct Professor, UCSF

07.21.3

An adventure: Odyssey or Oddity?

David Rose

University of Waterloo, Waterloo, Ontario, Canada

Well, it certainly has been an adventure. After a “traditional” training (BA, Univeristy of
Pennsylvania; DPhil, Oxford; Postdoc, MIT), my career has been divided between three
research sectors: government, research institute, academia. They each have somewhat
different cultures, plusses and minuses. I would be happy to share with the audience my
perspectives on these, as well as other issues such as: making your own breaks, what size
“pond”, how to be an attractive candidate for research positions, etc. I would also encourage
young scientists to be “citizens of science” both within your institutions and through
international societies like the ACA.
07.22.1

Multiple Conformations of human glucokinase in solution: Insights into enzyme

cooperativity and activation of glucokinase

Shenping Liu, Mark Ammirati, Xi Song, Xiayang Qiu

Pfizer Inc, Groton, CT 06385, United States

Glucokinase (GK) plays an important role of glucose sensor in controlling plasma glucose
level. To fulfill this role, the kinetic properties of GK are essential: it shows low affinity for
glucose and displays positive enzyme cooperativity. Inactivating GK mutations cause
diabetes, while activating GK mutants have decreased cooperativity and cause hypoglycemia,
underscoring GK's importance in glucose homeostasis. The positive cooperativity of
glucoskinase, a monomeric enzyme with one active site, was hypothesized to be caused by
either a mneumonica or a slow-transition mechanism, both of which involve two
conformations with different glucose affinity. Using small angle X-ray scattering (SAXS)
method, we determined the conformations of wild type GK and activating mutants in solution,
in presence of glucose and GKA. Our results show that glucose dose dependently shift the
population of GK from inactive to an active conformation, clearly supporting the mneumonic
model of GK's coorperativity. Compared to wild type GK, activating mutants requires less
glucose concentration to be activated. GKAs decrease the level of glucose required for GK
activation, and different GKAs demonstrated different GKA activation profiles. Our findings
provide an insights in GK’s coorperativity, activation as well as design GKA with different
profiles.
07.22.2

From biochemical and structural studies of soluble guanylate cyclase (sGC) toward
drug design

Emmanuelle Laffly, Jane Macdonald, Franziska Seeger, Elsa Garcin

UMBC, Baltimore, MD, United States

The overall goal of my research project is to understand the structural basis for assembly and
activation of soluble guanylate cyclase (sGC). sGC is the direct sensor and mediator of nitric
oxide (NO) signal transduction via cyclic GMP (cGMP). NO-induced vasodilation depends on
the activation of sGC. Compounds activating cGMP production by sGC have outstanding
clinical potential for treating cardiovascular diseases.

Structural information on sGC is limited to x-ray structures of homologous single domains.

Despite these pieces of information, a detailed understanding of the events leading to
activation in full-length sGC (Fl-sGC) is missing. To elucidate the structural details of Fl-sGC
assembly and determine the dynamic events associated with NO-induced Fl-sGC activation, I
have initiated a multidisciplinary approach. The combination of biochemical and mutagenesis
studies with low- (SAXS), medium- (DXMS) and high-resolution (X-ray crystallography)
structural methods will yield critical information to design novel sGC activators.

I have developed the first heterologous bacterial overexpression system for bovine Fl-sGC,
expressed and purified soluble and active Fl-sGC, and collected preliminary SAXS data. I am
now docking high-resolution x-ray structures from homologous single domains into the
calculated ab initio low-resolution SAXS envelopes to build a 3D model for Fl-sGC.

To pursue this work, we plan further developments including purification under a controlled
atmosphere to prevent oxidation damage and the use of Baculovirus or Pichia pastoris that
may be better suited to obtain larger quantities of Fl-sGC necessary for our structural
characterizations.
07.22.3

Crystal structure and thermodynamic analysis of Fab 106.3 complexed with BNP 5-14
reveal molecular details of mAb for clinical diagnosis.

Kenton Longenecker, Qiaoqiao Ruan, Elizabeth Fry, Sylvia Saldana, Susan Brophy, Paul
Richardson, Sergey Tetin

Abbott, Abbott Park, United States

Plasma concentration of the B-type natriuretic peptide (BNP) is a clinically recognized

biomarker for cardiovascular disease. Diagnostic immunoassays can measure BNP levels
using the monoclonal IgG1 antibody 106.3, which binds with high affinity to an epitope
spanning residues 5-13 of the mature bioactive peptide. To understand this molecular
recognition, we crystallized the Fab fragment of mAb106.3 complexed with the peptide
epitope, and determined the structure with X-ray diffraction to 2.1 Å resolution. The crystal
structure reveals detailed interactions that five of the complementary-determining regions
(CDRs) make with the partially folded peptide. Thermodynamic analysis of fluorescence
spectroscopy measurements suggests the interaction is driven by enthalpy changes with an
overall free energy of binding, Δ G = -54 kJ/mol. We interpret parameters based on the
structural information, and the kinetics suggest a rapid diffusion-limited mechanism of binding.
Comparative analysis with alanine-scanning studies of the epitope explains the basis of
mAb106.3 selectivity for BNP over other related natriuretic peptides.
07.22.4

Structural and Functional Insights into a Cardiac Specific Histone Methyltransferase

1 2 1 1 1 1
Nualpun Sirinupong , Joseph Brunzelle , Jun Ye , Ali Pirzada , Lindsey Nico , Zhe Yang
1 2
Wayne State University, Detroit, MI, United States, Advance Photon Source, Argonne, IL,
United States

SmyD1, a histone H3K4 methyltransferase, was found specifically expressed in heart and
skeletal muscle, important for cardiac development and related to heart diseases. The unique
domain structure characterized by a split SET domain, a conserved MYND zinc finger, and a
novel C-terminal domain (CTD) distinguishes SmyD1 from other SET domain
methyltransferases. Here we report the crystal structure of full-length SmyD1 in complex with
AdoHcy at 2.3 Å. The crystal structure reveals that SmyD1 folds into five distinct structural
domains, and the shape of the protein resembles an open-ended wrench, where the two thick
grips are separated by a large, deep concave opening. Substantial structural differences exist
between SmyD1 and other SET proteins, which reflect the unique structural and functional
properties of this protein. The crystal structure reveals that SmyD1 does not contain the pre-
SET domain that is necessary for methylation by other SET proteins. In addition, SmyD1 has
a nearly buried cofactor binding site due to the bulky SET-I domain, which appears to restrict
the exchange during catalysis between cofactor and its product as suggested by our mutation
studies. Moreover, the structure reveals an unusually spacious target lysine binding site,
which provides structural basis for the low histone binding affinity and weak methylation
activity of SmyD1. The remarkable feature of the SmyD1 structure is the presence and
location of the CTD domain, whose function was unknown. The structure reveals that the
CTD domain is located adjacent to the histone binding site and contributes to the formation of
a unique Y-shaped binding cleft. The structural and functional analysis suggest that the CTD
domain may be involved in the direct interaction with histone H3, and form an extended
substrate binding site that is likely to recognize the residues far from the C-terminal side of
target lysine 4. The structure determination of SmyD1 also offers important functional
implications of SmyD1 in cardiac development. The structure reveals that the proline-rich
peptide binding site in the MYND domain is fully exposed and can be readily accessed by
skNAC, a cardiac transcription factor. The structure and function studies suggest that the
MYND domain may primarily serve as a protein interaction module, and cooperate SmyD1
with skNAC to regulate cardiomyocyte growth and maturation. In addition, the structure
explains why both the MYND domain and the S-sequence are required for the interaction with
skNAC, and whether this interaction can affect SmyD1 structure and function, which in turn
may alter the histone methylation profile in heart and influence gene expression in cardiac
development. Overall, the crystal structure of SmyD1 provides structural insights into the
novel mechanism of SmyD1 regulation, and also provides structural basis for further
understanding the role of SmyD1 in heart development and cardiovascular diseases.
07.22.5

DPPIV Inhibitors – A Structural Biologists Perspective

Sridhar Prasad G.

CalAsia Pharmaceuticals, Inc., San Diego, CA 92121, United States

Dipeptidyl peptidase IV (DPPIV) is a member of the prolyl oligopeptidase family of serine

proteases. DPPIV removes dipeptides from the N terminus of substrates chemokines,
neuropeptides and peptide hormones including Glucagon-like peptide-1 (GLP-1). GLP-1
enhances the glucose-dependent secretion of insulin from pancreatic beta-cells following the
ingestion of food. Infusion of exogenous GLP-1 in type 2 diabetes patients has shown to
lower the plasma glucose and improve the beta-cell function. However, the rapid clearance
of GLP-1 in vivo by DPPIV has diminished the prospect of exogenous GLP-1 as a potential
therapy. An alternate approach has been to reduce the activity of the GLP-1 processing
enzyme, DPPIV, by small molecule inhibitors. A number of small-molecule DPPIV inhibitors
have shown to have beneficial effects in animal models and additionally proven clinically
benefits in human trials. As a result, DPPIV inhibitors have now become marketed drugs.
Given the importance of this new class of drugs, a large number of pharmaceutical and
academic groups have been pursuing to discover more safe and efficacious compounds
using multiple approaches, including fragment and structure-based hit identification and lead
optimization methods. The availability of the three-dimensional structural information of the
enzyme DDPIV has played an important role in the discovery and development of safe, potent
and novel clinical compounds in record time. These “drug candidates” have now successfully
entered human trials and are awaiting approval of the regulators. A comprehensive analysis
of the inhibitors discovered using different approaches will be presented, with an emphasis on
programs that involved the use of three-dimensional structural information.
07.22.6

Inhibition of recombinant maltase-glucoamylase by acarbose, salacinol, kotalanol, and

de-O-sulfonated kotalanol
1 2 4 3 1
Kyra Jones , Lyann Sim , Buford Nichols , B. Mario Pinto , David Rose
1 2
University of Waterloo, Waterloo, Ontario, Canada, University of Toronto, Toronto, Ontario,
3 4
Canada, Simon Fraser University, Burnaby, British Columbia, Canada, Baylor College of
Medicine, Houston, Texas, United States

Inhibition of intestinal α -glucosidases and pancreatic α -amylases is an approach to controlling

blood glucose and serum insulin levels in individuals with type II diabetes. An important target
enzyme is maltase-glucoamylase, a Family 31 glycoside hydrolase responsible for the final
step of starch hydrolysis releasing free glucose in the small intestine. Here we examine the
inhibition of the C-terminal catalytic subunit of maltase-glucoamylase by salacinol, acarbose,
kotalanol, and de-O-sulfonated kotalanol and present the inhibition profile of the catalytic
domain with respect to these compounds. We determined enzymatic activity using a coupled
assay measuring glucose hydrolyzed from substrate in the presence of each inhibitor. The
results facilitate comparison of the active site requirements of the N- and C-terminal subunits,
as the N-terminal catalytic domain has previously been characterized. The structure of the N-
terminal domain has been used as a basis for understanding these results, enhancing the
understanding of the role of each catalytic subunit in starch digestion. Ultimately, this will help
to guide the development of new compounds with anti-diabetic activity. Further, this research
can be applied to nutritional diseases such as obesity and cardiovascular disease.
07.22.7

Intestinal Glucosidases: Structure/Mechanistic studies towards clinical applications for

diabetes and obesity.
1 2 1
David Rose , Lyann Sim , Kyra Jones
1 2
University of Waterloo, Waterloo, Ontario, Canada, University of Toronto, Toronto, Ontario,
Canada

The human system for digesting starch to nutritional glucose has evolved to handle diverse
sources of starch. Initial processing by salivary and pancreatic amylases breaks starch
polymers down into “limit dextrins”, which include both g-1,4 and 1,6 linked glucose
saccharides. These are further processed by the small intestinal glucosidases, maltase-
glucoamylase (MGAM) and sucrase-isomaltase (SI).

This presentation will review our results on investigating the crystal structures of the 4
glycoside hydrolase family 31 enzymes that make up MGAM and SI. We are participating in
an international collaboration to understand the structural basis for the inhibitory properties of
these enzymes, the development of novel inhibitors, and the investigation of the
complementary roles of MGAM and SI in the processing of limit dextrins of starch. Our
hypothesis is that the enzymes each play key roles in different circumstances of diet,
starvation and gorging.

Anti-diabetic compounds on the market currently are broadly specific inhibitors of

glucosidases. We suggest that an improvement of their affinities and specificities for the most
relevant glucosidase activities, through structural and synthetic studies, will improve their
efficacy and reduce side effects. The action of these compounds to control blood glucose
levels has relevance not just to diabetes but also to nutritional disorders such as obesity, and
to cardiovascular diseases.

Our progress on testing novel compounds in a diabetic rat model will be presented.

See also Poster presentation by Kyra Jones on the inhibition profiles of MGAM domains.
07.22.8

Structural and Functional Studies of Gα q-mediated Signaling

1 1 1 3 1,2
Angeline Lyon , Valerie Tesmer , Krishna Suddala , John Northup , John Tesmer
1 2
Life Sciences Institute, University of Michigan, Ann Arbor, MI, United States, Department of
3
Pharmacology, University of Michigan, Ann Arbor, MI, United States, Laboratory of Cell
Biology, National Insitutes of Health, Bethesda, MD, United States

G protein-coupled receptors (GPCRs) are the largest family of membrane receptors and
serve to integrate extracellular signals with intracellular responses. Upon activation by
hormone, neurotransmitter, or other agonist binding, GPCRs facilitate nucleotide exchange on
G proteins (Gα β γ ), which in turn signal through downstream effectors. The Gq-coupled class
of GPCRs are linked to platelet activation and heart development, as well as pathologic
processes such as the onset and maintenance of arrhythmias, hypertrophy, and heart failure.
In an effort to develop a structural understanding of these pathways, we have pursued studies
of Gα q with GRK2 (a G-protein coupled receptor kinase) and p63RhoGEF (a guanine
nucleotide exchange factor for RhoA). New results in our lab on other Gα q effectors, such as
phospholipase Cβ (PLCβ ), indicate that these effector interactions occur through highly
conserved structural elements. Through biochemical and structural studies, we are defining
the mechanisms of Gα q-mediated activation of effector.
07.22.9

Surprises and explications in two projects: Discovery of high quality dual

thrombin/factor Xa inhibitors and renin inhibitors
1 2 5 3 3
Zsolt Bocskei , Gary McCort , Steinhagen Henning , Scheiper Bodo , Matter Hans , Thiers
2 4 4 2 2
Bérangère , Lassalle Gilbert , Meneyrol Jerome , Altenburger Jean-Michel , Petit Frederic ,
4 3 4 3
Herault Jean-Pascal , Wehner Volkmar , Alet Nathalie , Schreuder Hermann , Bono
4
Françoise
1 2 3
sanofi-aventis research, Strasbourg, France, sanofi-aventis research, Paris, France, sanofi-
4
aventis research, Frankfurt, Germany, sanofi-aventis research, Toulouse, France,
5
Grunenthal, Aachen, Germany

Two cardiovascular drug discovery projects will be presented with considerably different
scopes:

Thrombin-Factor Xa dual antithrombotic inhibitors were developed on the basis of the

expectation that simultaneous targeting of multiple coagulation enzymes may offer an
improved efficacy and therapeutic index. Optimization started from an internally established
selective thrombin inhibitor series and crystal structures helped to explain the SAR and to
achieve increased affinity on FXa, while keeping activity low on other serine proteases like
trypsin. Crystal structures explained how it was possible to maintain high FXa activity despite
a bulky P2 group on the inhibitor. Replacement of basic P1 groups with neutral groups was
also followed and rationalized.

In the renin project most interesting hits of an HTS as well as backscreening campaigns were
characterized by co-complex protein crystallography and these crystal structures contributed
to the selection of hits and the optimization strategies. While studying these early hits we met
a number of unexpected structural features and binding modes due to the high flexibility and
plasticity of the renin active site. These structures and their use will be described along with
the optimization of two chemical series (acyl-guanidines and indol piperazines) into highly
specific drug-like renin inhibitors using structure assisted drug design.
07.23.1
2
Coordination Polymers with Surface Areas exceeding 5000 m /g: Should Traditional
Sorbents Worry?

Adam Matzger

University of Michigan, Ann Arbor, United States

Adsorbents play a critical role in a variety of industrial, laboratory and consumer applications.
Materials such as silica gel, zeolites, and activated carbon have been investigated for
centuries and represent the most commonly used adsorbents. In the last decade new high
surface area materials based on coordination chemistry have emerged. These inorganic-
organic hybrid materials are porous crystals that promise to redefine the types of processes
and applications that can be enabled by adsorption. Synthetic challenges and novel
approaches to the synthesis of crystalline microporous coordination polymers (MCPs) will be
discussed. Recent progress with coordination copolymerization will be discussed in the
context of producing structurally defined ultrahigh surface area materials. Application of MCPs
for gas separations and storage will be briefly discussed.
07.23.2

Metal organic frameworks as CO2 capture materials and as fuel cell electrolytes

George Shimizu, Ramanathan Vaidhyanathan, Jeff Hurd, Simon Iremonger

University of Calgary, Calgary, Alberta, Canada

Metal organic frameworks (MOFs) represent a tunable molecular scaffolding that can be
adjusted for a breadth of applications. This presentation will concern our efforts towards
tailoring the properties of MOFs towards two globally relevant energy challenges.

The first concerns our efforts to make MOFs with amine lined pores for CO2 capture. In
contrast to liquid amines which chemisorb CO2 and have high energy costs for regeneration,
the MOF approach gives physisorbed gases and hence more facile release. Despite the
weaker binding mode, we will show that high selectivities are possible owing to heats of
adsorption over 40 kJ/mol. We will also present molecular level insights to the CO2 capturing
ability of these solids.[1]

The second topic concerns new electrolyte membranes for PEM and direct methanol fuel
cells. A major hurdle in these technologies is an electrolyte capable of conducting protons
above 100˚C. Higher operating temperatures will enhance electrode kinetics and decrease
electrode poisoning among several critical operational benefits. In contrast to the
macromolecular approaches typically employed towards these electrolytes, we have used a
MOF strategy to generate crystalline networks with acidic pores. These MOFs can include
amphoteric N-heterocycles, to act as non-volatile proton carriers in their pores to give proton
-4 -3 -1
conduction from 10 -10 Scm at 150˚C without humidification. Moreover, we show that
PCMOF materials can be incorporated into gas-tight membrane electrode assemblies to give
open circuit voltages > 1.0 V at 100˚C in a H2/air fuel cell.[2]

[1] R. Vaidhyanathan et al. “An amine-functionalized metal organic framework for preferential
CO2 adsorption at low pressures,” Chem. Commun. 2009, 5230.

[2] J. A. Hurd et al. “Anhydrous proton conduction at 150˚C in a crystalline metal organic
framework,” Nature Chem. 2009, 1, 705.
07.23.3

Material-based hydrogen and methane storage: Understanding the storage mechanism

using x-ray/neutron diffraction and computational modeling

Wei Zhou

National Institute of Standards and Technology, Gaithersburg, United States

The storage of energy carrier gases (e.g., H2 and CH4) based on novel materials has
attracted much research attention in recent years. For physisorptive systems (such as porous
metal-organic frameworks), it is important to identify the gas adsorption sites and elucidate
the gas-host interaction mechanism. For chemisorptive systems (such as complex hydrides),
obtaining accurate structural information and understanding the reaction pathway are critical.
In this talk, I am going to present some of our recent work on both metal-organics frameworks
and complex hydrides. Combing the strength of x-ray/neutron diffraction and computational
modeling, we are able to understand the storage mechanism of fuel gases in several new
material systems. Rational strategies are proposed to improve the material storage
performance.
07.23.4

Polyhedra Stabilized Metal-Organic Frameworks and Their Applications in Hydrogen,

Methane, and Carbon Dioxide Storage
1 1 1,2 1
Daqiang Yuan , Dan Zhao , Daofeng Sun , Hong-Cai Zhou
1 2
Texas A&M University, College Station, Texas, United States, Shandong University, Jinan,
China

A previously described approach towards stable metal-organic frameworks (MOFs) with high
surface area by incorporating microwindows within mesocavities was advanced by using
longer hexatopic ligands. One of the generated MOFs (PCN-68) has a BET surface area of
2 -1
5109 m g , which is among the highest so far. Their hydrogen, methane and carbon dioxide
storage for clean energy applications were systematically studied.
07.23.5

Structure and Dynamics of Proton-Conducting Metal-Organic Frameworks

1 2 1,2
Jamie Ford , Jason Simmons , Taner Yildirim
1 2
University of Pennsylvania, Philadelphia, PA, United States, NIST Center for Neutron
Research, Gaithersburg, MD, United States

Vehicles powered by polymer electrolyte membrane (PEM) fuel cells are an exciting
alternative to current fossil fuel technology. The membranes in these cells serve as both
charge transporter, ferrying protons from the anode to the cathode, and gas diffusion barrier,
preventing the backflow of oxygen to the anode. Currently, hydrated sulfonated polymers are
the preferred material for these membranes. The presence of water, however, limits the
operating temperature to 100 C, reducing the electrode kinetics and CO tolerance of the
entire system. In an effort to increase the efficiency and operating temperature of these fuel
cells, we are investigating the proton conductivity of new host/guest materials based on
metal-organic frameworks (MOFs) loaded with proton conducting small molecules. Through
choice of organic linker and inorganic metal salts, the geometry, size, chemistry and
interconnectivity of the MOF frameworks can be tuned. These thermally stable structures
provide well-defined pores for the guest molecules to form proton-conducting pathways. Here,
we will discuss the crystallographic structures of the bare and loaded frameworks as well as
the dynamics of the guest molecules and protons within in the systems.
07.23.6

Mn2-xFexP1-yGey , a potential system used for room temperature magnetic refrigeration

1 1 2 2
Qingzhen Huang , J.W. Lynn , Danmin Liu , Ming Yue
1 2
NIST Center for Neutron Research, Gaithersgurg, MD 20899, United States, Beijing
University of Technology, Beijing, 100022, China

Magnetic refrigeration, a clean and energy saving technology, offers a solid-state

alternative to the traditional gas-compression-based cooling, and will eliminate the harmful
refrigerant gases and reduce energy requirement. Mn2-xFexP1-yGey is a good candidate
material used for room temperature magnetic refrigeration. It has a first order ferromagnetic
transition with large entropy change in a wide and adjustable temperature range. To
understand the materials’ crystal and magnetic structures and their relationships with the
magnetic and thermal properties is of central interest for materials scientists. We use the
neutron powder diffraction technique to determine, in situ, the crystal and magnetic structures,
combined with magnetic and thermal properties measurements, to obtain the information
necessary for understanding the relevant structural details and their relation to the physical
properties at different temperatures and/or under magnetic fields. Our research results have
demonstrated that changes of magnetization and the entropy are in proportion to the changes
of the ferromagnetic phase fraction when decreasing the temperature or increasing an applied
magnetic field. The determination of the crystallite size indicates that small crystallite sizes
inhibit the paramagnetic to ferromagnetic transformation, suggesting that it is possible to
achieve 100% transformation by increasing the crystallite size, and, therefore, obtain the
maximum entropy change. We found that the compositional homogeneity is one of the main
issues for the system performing under a low magnetic field, because that the Curie
temperature Tc is very sensitive to the Mn and Ge contents and their distributions and,
therefore, the chemical inhomogeneities are responsible the existence of an interval of Tc in
which the two phases coexist.. By improving the chemical homogeneity we have made
samples with as low as 1.2 tesla magnetic field requirements to achieve more than 80%
ferromagnetic transformation. We expect that it is possible to improve the system so that the
magnetocaloric effect will to exceed 100 J/kg-K under a magnetic field as low as less of 1
tesla.
07.23.7

Experimental and theoretical studies of the Magnetocaloric Effect (MCE) in the Mn5-
xFexSi3 series

1,2 1,2 1,2 2 2

Michael Gottschlich , Olivier Gourdon , Michael Ohl , Joerg Persson , Thomas Brueckel
1 2
Oak Ridge National Laboratory, Oak Ridge, Tennessee, United States, Research Center
Juelich, Juelich, Nordrhein-Westfalen, Germany

The magnetocaloric effect (MCE) based on entropy changes of magnetic materials in an

applied magnetic field, holds the potential of applications for refrigeration without moving
mechanical parts. Therefore it has recently attracted the attention of many scientific research
groups. Although MCE was discovered a long time ago (1881) by Warburg in iron, we are still
investigating new usable materials low cost and chemically stable and safe.

Recently, after the characterization concerning magnetization measurements and refinements

on x-ray data, neutron measurements on polycristalline samples Mn5-xFexSi3 have been
collected on the HB2A neutron powder diffractometer at HFIR under various fields. These
preliminary measurements emphasize a unique atomic distribution as well as a ferromagnetic
ordering along the c axis which is in some extends “forced” by the field in the vicinity of the
magnetic transition. Such effect is certainly directly linked to the MCE measured.

Preliminary theoretical calculations which support our findings will also be presented.
07.23.8

Structure and Thermoelectric Properties of Selected Alkaline-Earth Cobalt Oxides

1 1 2 1 1
Winnie Wong-Ng , Guangyao Liu , Tana Luo , Joshua Martin , Yonggao Yan , Evan
3 1 4
Thomas , Qing Huang , James Kaduk
1 2
National Institute of Standards and Technology, Gaithersburg, MD, United States, University
3
of Maryland, College Park, MD, United States, Air Force Research Laboratory, Wright
4
Patterson, OH, United States, Poly Crystallography, Inc., Naperville, IL, United States

In recent years, thermoelectric research has attracted considerable attention partly because
of the green house gas emission problem and the ever-increasing gas price problem. There is
a desperate need for efficient energy conversion materials and environmentally friendly
technologies over the next twenty years. For energy conversion applications using waste
heat, oxide materials of high temperature stability are potential candidates. This paper
discusses our phase equilibria/structural/property studies of selected series of cobaltites,
including those in the SrO-CaO-CoOx and CaO-ZnO-CoOx systems.
07.23.9

Crystallographic and theoretical studies of a family of new arsenides ACd4As3 (A = Na,

K, and Rb)

Hua He, Svilen Bobev

University of Delaware, Newark, DE, United States

In recent years, binary and ternary d-block metal pnictides have attracted much attention from
the solid-state community. This intense research interest is due to the discoveries of
superconductivity and high figure of merit for thermoelectrics among such compounds. In this
presentation, we discuss the new arsenides, ACd4As3 (A = Na, K, and Rb), which have been
synthesized by reactions of the elements at high temperature; and their crystal structures
have been established by single-crystal X-ray diffraction. Despite of the large differences
among the radii of the alkali metals, the title compounds are isostructural and crystallize with
a new structure type in the rhombohedral space group R 3 m (No. 166) with the cell parameter
c increasing dramatically on going from Na to Rb. The crystal structures can be rationalized
+ –
as A cations and [Cd4As3] layers, which are made up of corner- and edge-shared CdAs4
tetrahedra. The potential application as thermoelectric material of the title compounds is
discussed in the context of their electronic structure calculated by the density-functional
method, as well as their close structural relationship with the layered alkaline-earth arsenides
and antimonides EZn2Pn2 and ECd2Pn2 (E = Ca, Sr, Ba; CaAl2Si2 type structure).
AW.03

From Paper Tape Input to Forensic Crystallography. Forty years of Small Molecule
Computing

A.L. Spek

Utrecht University, Utrecht, Netherlands

The lecture will largely follow the historical development of the program package PLATON.
This program is probably best known and most used as part of the IUCr CheckCIF facility.
Recently, it was instrumental in uncovering a large scale fraud with 'invented' structures that
were published in Acta Cryst. E.

PLATON has a history of 30 years. Development started as a second attempt to automate

various procedures as part of our National Single Crystal Structure Service. The earlier
attempt was based on software written in the ALGOL60 language that became obsolete with
the introduction of a Control Data/Fortran mainframe in Utrecht.

PLATON started off as a geometry calculations and molecular graphics program for local use.
Over time, algorithms for the detection of solvent accessible voids in a structure and missed
symmetry were added. This attracted the attention of Syd Hall, at that time Section Editor of
Acta Cryst. C. Both tests were included as part of the development of an early version of a
project to automate structure validation of structures to be published in Acta Cryst. C. Over
time, more than 400 various tests have been implemented including warnings for missed
twinning, problems with the reflection data or difference density map etc. One of the most
powerful tests turned out to be the Hirshfeld Rigid Bond Test.

PLATON also includes a large number of tools such as space group determination from
systematic absences, the SQUEEZE algorithm to handle disordered solvent in the refinement
and a version of the 'Charge Flipping' algorithm as an alternative tool to solve crystal
structures.

PLATON, in its native LINUX incarnation, also includes a tool named 'SYSTEM S'. The latter
aims at solving, completing, refining and validation a crystal structure automatically. For that,
it makes use of excellent external software such as SHELXL, SHELXS, SIR and DIRDIF.
01.04.1

Using Raman Microscopy to Map Reaction Pathways in Crystals in Real Time

Paul Carey

Case Western Reserve University, Cleveland, United States

By combining the methods of Raman microscopy and Raman difference spectroscopy

populations of reaction intermediates can be tracked in single crystals. Reactions are started
using soak-in conditions at room temperature and the Raman data as a function of time
provide both structural and kinetic data. These can be used to inform the crystallographer of
the optimal conditions for flash freezing and trapping target intermediates for X-ray data
collection. At the same time the X-ray structures provide reference points that are invaluable
for interpreting the Raman spectra. Two examples will be given: the complex branched
reaction pathway traversed by clinical drugs reacting with beta-lactamase drugs in antibiotic
therapy (1); following the first step in RNA synthesis in the active site of an RNA polymerase
(2). Advantages and limitations of the approach will be given.
I am indebted to my collaborators listed in references (1) and (2).
(1) P. S. Padayatti, A. Sheri, M. A. Totir, M. S. Helfand, M. P. Carey, V. E. Anderson, P. R.
Carey, C. R. Bethel, R. A. Bonomo, J. D. Buynak and F. van den Akker. J. Amer. Chem. Soc.
128, 13235-13242 (2006).
(2) M. L. Gleghorn, Y. Chen, E. K. Davydova, R. Basu, L. B. Rothman-Denes, P. R. Carey
and K. S. Murakami, submitted for publication (2010).
01.04.2

Structural evidence for the staging of active site configurations that control diiron
monooxygenase reactivity

Brian Fox, Lucas Bailey, Justin Acheson

University of Wisconsin, Madison, WI, United States

Diiron monooxygenases are multi-protein complexes that catalyze the oxidation of

hydrocarbons including methane, butane, toluene, ethene, and other compounds. Because of
this powerful reactivity, microbes readily use these chemicals as the sole source of carbon
and energy. Toluene-4 monooxygenase is a representative example from this enzyme family.
It is composed of an electron transfer chain between an NADH oxidoreductase and a Rieske-
type [2Fe-2S] protein, a large hydroxylase protein containing the diiron center active site, and
a small protein called the effector protein. Numerous studies have shown the effector protein
is required for catalysis, but until recently, the structural bases of these observations were not
known. High-resolution crystal structures of the stoichiometric complex of effector protein and
hydroxylase have begun to give new insight into the role of protein-protein interaction in
controlling the active site configuration, the reactions with substrates, and the formation of
intermediates along the reaction pathway. Key findings from this work will be presented.

This work supported by NSF MCB 0316232 and MCB 0843239.

01.04.3

Structural and mechanistic basis for hypoxia inducible factor hydroxylation by the
oxygen sensing prolyl hydroxylases

Rasheduzzaman Chowdhury, Michael A McDonough, Christopher J Schofield

Chemistry Research Laboratory, University of Oxford, Oxford, OX1 3TA, United Kingdom

Oxygen dependent prolyl-4-hydroxylation of the h-subunit of hypoxia inducible transcription

factor (HIFh) plays an essential role in the hypoxic response. Hydroxylation of prolines in the
N- or C-terminal oxygen dependent degradation domains (NODD or CODD) increases the
affinity of HIFh to the von Hippel-Lindau protein (pVHL) by ~1000 fold so signaling for HIF h
degradation. With limiting oxygen, HIFh hydroxylation slows, it dimerizes with HIF and
activates the transcription of a gene array. Prolyl-4-hydroxylation also stabilizes the triple helix
structure of collagen, the most abundant human protein. Both the collagen and the HIF prolyl
hydroxylases (PHDs) are Fe(II) and 2-oxoglutarate (2OG) dependent oxygenases. We
determined crystal structures of PHD2 in complex with CODD. With biochemical analyses, the
results demonstrate that catalysis involves a mobile region of PHD2 that encloses the
hydroxylation site and stabilizes the PHD2.Fe(II).2OG complex. When bound to PHD2 the
non-hydroxylated proline-residue adopts the C4-endo conformation. Evidence is provided that
4R-hydroxylation enables a stereoelectronic effect that changes the proline conformation to
the C4-exo state, as observed when hydroxylated HIFh is bound to pVHL and in collagen.
The results rationalize selective recognition of NODD/CODD by three PHD isoforms and the
effects of clinically observed mutations on PHD2 catalysis; they will be of use in the design of
new types of PHD inhibitor aimed at treating anemia and ischemic disease.
01.04.4

Consequences of Exposure of Ferric Myoglobin Nitrite Crystals to Synchrotron X-ray

Radiation
1 1 2
George Richter Addo , Jun Yi , Allen Orville
1 2
University of Oklahoma, Norman, OK, United States, NSLS-BNL, Upton, NY, United States

The nitrite anion is ubiquitous in the environment and is an important component of the
global nitrogen cycle. The conversion of nitrite to nitric oxide (NO) has normally been
associated primarily with the nitrite reductase (NiR) enzymes in the bacterial denitrification
pathway. Recently, there has been a resurgence of interest in nitrite reduction by heme
enzymes, due in a large part to literature reports that the mammalian proteins myoglobin (Mb)
and hemoglobin (Hb) covert nitrite to NO in an apparent NiR reaction. We reported the X-ray
crystal structure of the nitrite derivative of ferric horse heart Mb at 1.2 Å resolution and
showed that the nitrite ligand was bound to the heme Fe via the nitrito O-binding mode. We
extended the work to the nitrite derivative of ferric human Hb, and showed that the O-binding
mode of nitrite was also extant in this Hb(ONO) complex (crystal structure at 1.8 Å resolution).
Importantly, however, nitrite reduction by Mb (and Hb) occurs via the ferrous derivative and
not the ferric derivative. Despite several attempts using different preparation routes, we were
unable to prepare crystals of the ferrous Mb-nitrite complex derivative for crystal structural
determination.

We were intrigued by the new capabilities of the X26-c beamline at the National
Synchrotron Light Source that allowed for correlated X-ray diffraction and UV-vis
spectroscopy experiments. We thus pursued the goal of attempting to generate the ferrous
Mb-nitrite complex from X-ray induced photoreduction of the ferric precursor. The results of
this work will be presented and discussed.
01.04.5

ijxrs`kknfq`oghb? `mc? rhmfkd? bqxrs`k? rodbsq`k? `m`kxrhr? ne? sgd? odqnwhc`rd? edqqxk
hmsdqldch`sd
1 2 2 1 2
Tzanko Doukov , Yergalem Meharenna , Huiying Li , S. Michael Soltis , Thomas Poulos
1 2
SSRL, Menlo Park, CA, United States, UCI, Irvine, CA, United States

The ferryl (Fe(IV)O) intermediate is important in many heme enzymes and thus the precise
nature of the Fe(IV)-O bond is critical in understanding enzymatic mechanisms. The 1.40 Å
crystal structure of cytochrome c peroxidase Compound I has been solved as a function of x-
ray dose while monitoring the visible spectrum at BL9-2 at SSRL. Data were collected with an
open air Helium cryostat at 65 K, the lowest safe temperature before nitrogen solidifies.
Ninety six crystals were mounted by the SAM robot and were screened or data collected
locally or remotely from Irvine, CA. Data from 25 crystals were collected, from which the best
19 were used in the experiment. For each crystal, data collections were carried out in 15
o
separate runs. Run 1 consisted of 5 of data, representing the first 0.035 MGy of x-ray
o
exposure. Then the same 5 scanning angle were recollected 12 more times giving runs 2
o
through 13 with increased x-ray dose. In run 14 a full 120 of data were collected in order to
o
fully reduce the crystal followed by run 15 which again repeated the same 5 representing the
highest x-ray dose. The same 15-run data collection protocol was adopted for similarly sized
crystals and the scanning angles were chosen to optimize the completeness of the data.
o
Each composite data set was assembled by merging 5 of data with identical run numbers
from 19 crystals. Making the composite dataset in such manner ensures measuring the same
reflections in every separate run and allows their monitoring as a function of time/dose. In
addition the Fo-Fo difference maps are less noisy and more reliable. A total of 15 structures
at 1.40 Å resolution were refined providing a picture of the structural changes associated with
increasing x-ray dose.

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Conformational Flexibility and Catalysis in a Phosphohexomutase: Crystallography,

Kinetics, and NMR
1 1 2
Lesa Beamer , Steven Van Doren , Cristina Furdui
1 2
Univeristy of Missouri, Columbia, MO, United States, Wake Forest University Health
Sciences, Winston-Salem, NC, United States

A critical role for protein dynamics and conformational flexibility in catalysis has been long
suspected and often proposed, but difficult to demonstrate directly. However, powerful new
methodologies, particularly NMR, can now greatly enhance the static “snapshots” typically
provided by crystal structures. One system currently being studied by the complementary
techniques of X-ray crystallography and NMR is the enzyme
phosphomannomutase/phosphoglucomutase (PMM/PGM), from the human pathogen P.
aeruginosa. PMM/PGM has been well characterized in our laboratory by X-ray
crystallography, kinetic studies, and site-directed mutagenesis. Ten crystal structures of the
enzyme and various enzyme-ligand complexes have shown that the enzyme changes from an
open to closed conformational state upon ligand binding. Recent studies characterizing the
effects of mutants in a hinge region at a domain-domain interface show that increased
conformational freedom is unfavorable for catalysis due to entropic factors. These results
highlight the importance of conformational flexibility of the polypeptide backbone in catalytic
efficiency. To further explore the role of protein dynamics and conformational change in
enzyme mechanism, PMM/PGM is presently being investigated by NMR. A major
accomplishment in this effort is successful completion of the backbone assignments of this 50
kD protein using triple resonance methods. Additional characterization of apo and ligand-
bound protein dynamics is underway via relaxation dispersion methods. A synthesis of the
insights gained from this multi-disciplinary approach will be presented, with an emphasis on
understanding the multi-step catalytic reaction of PMM/PGM.
01.05.2

A master switching motif with multiple effects on differential conformational stability

2+
and Mg -assisted catalysis suggests a general mechanism for transducing enzymes.

Violetta Weinreb, Li Li, Brian Kuhlman, Charles Carter

University of North Carolina at Chapel Hill, Chapel Hill, NC, United States

B. stearothermophilus Tryptophanyl-tRNA synthetase (TrpRS) uses a sequence of different

conformational states to catalyze tryptophan activation. Like other Class I aminoacyl-tRNA
2+
synthetases it also requires one Mg ion for optimal catalysis. The metal favors catalysis by -
2+
6.5 kcal/mol. However, catalytic assist by Mg occurs if, and only if, it interacts with the
protein. We are trying to identify the metal-protein interactions that produce this catalytic
2+
effect. Physical interactions between Mg and TrpRS are mediated indirectly via active-site
lysines K111, K192 and K195. Mutations of these lysines showed that they all stabilize the
2+
transition state. However, their interactions with the Mg have the opposite effects and
significantly reduce their catalytic effects. This suggests that the balance of catalytically
2+
productive interactions between TrpRS and the Mg ion must arise from outside the active
site. We identified a set of core residues we call the D1 switch because they move during the
catalytic conformational transition. The D1 switch lies at the corner of the N-terminal ß-α -ß
crossover opposite the HIGH sequence and KMSKS loop, forming links to I4 in the N-terminal
ß strand and I140 in the Trp binding site from three of its side chains F26, Y33 and F37.
These residues are prime candidates for mediating synergistic, catalytically-critical coupling to
2+
the Mg ion at the active site. The Rosetta Design program suggested mutations I4V, F26L,
Y33F and F37I to ‘‘hyperstabilize’’ the PreTS along the structural reaction profile. Using
2+ 2+
multimutant thermodynamic cycles together with substitution of Mn for Mg and [ATP]-
dependent Michaelis-Menten kinetics we used these mutants to demonstrate long-range
2+ 2+
synergistic coupling between the D1 switch and the Mg ion. Thus, protein-Mg interactions
within the active site oppose catalysis, while synergistic long-range interactions to the metal
2+
drive catalysis indirectly, by changing an inactive Mg coordination into one that can stabilize
2+
the transition state. In this way transition-state stabilization by Mg occurs if, and only if,
conformational changes reposition it. This description may apply to a large number of NTPase
2+
enzymes that transduce chemical free energy by using control of Mg coordination to link
catalyzed hydrolysis of their purine triphosphate substrates to conformational changes for
cellular work and signaling. Supported by NIGMS 78227.
01.05.3
∆EFG
Structure of HydA , a key intermediate in [FeFe]-hydrogenase H-cluster
biosynthesis

David Mulder, Eric Boyd, Ranjana Sarma, Rachel Lange, James Endrizzi, Joan Broderick,
John Peters

Montana State University, Bozeman, MT, United States

The [FeFe]-hydrogenase (HydA) contains a complex FeS cluster active site, termed the H-
cluster, which exists as a [4Fe-4S] subcluster linked by a cysteine thiolate to a modified 2Fe
subcluster with unique non protein ligands. Although the 2Fe subcluster is thought to be
synthesized by the activities of the hydrogenase maturation enzymes HydE, HydF, and HydG,
the precise mechanism by which it is assembled remains unclear. Here, we report the
Δ EFG
structure of HydA (HydA expressed in a genetic background devoid of the active site H-
cluster biosynthetic genes hydE, hydF and hydG) determined to 1.97 Å resolution. The
structure reveals the presence of a [4Fe-4S] cluster and an open channel for the insertion of
the 2Fe subcluster. This indicates that H-cluster synthesis occurs in a stepwise manner, first
with synthesis and insertion of the [4Fe–4S] subcluster by generalized host-cell machinery
and then with synthesis and insertion of the 2Fe subcluster by specialized HydE, HydF, and
HydG maturation machinery. 2Fe subcluster insertion presumably occurs via a cationically
charged channel that collapses upon insertion through conformational changes in two
conserved loop regions. Loop region conservation in organisms containing the 2Fe
subcluster biosynthetic genes coupled with evolutionary analysis of HydA together indicate a
bacterial origin for HydA that postdates the emergence of eukarya. By establishing parallels to
FeMo-cofactor biosynthesis in nitrogenase maturation, general unifying themes from complex
FeS cluster biosynthesis are revealed.
01.05.4

Mitochondrial and Cytosolic Phenylalanyl-tRNA Synthetases Catalyze Incorporation of

ROS-damaged Amino Acid into Eukaryotic Proteins.
1 2 1 1
Liron Klipcan , Nina Moor , Naama Kessler , Mark Safro
1 2
Weizmann Institute of Science, Rehovot, Israel, Institute of Chemical Biology and
Fundamental Medicine, Novosibirsk, Russian Federation

The accumulation of proteins damaged by reactive oxygen species (ROS), conventionally

regarded as having pathological potentials, is associated with age-related diseases such as
Alzheimer's, atherosclerosis, and cataractogenesis. Exposure of the aromatic amino acid
phenylalanine to ROS-generating systems produces multiple isomers of tyrosine: m-tyrosine
(m-Tyr), o-tyrosine (o-Tyr), and the standard p-tyrosine (Tyr). Previously it was demonstrated
that exogenously supplied, oxidized amino acids could be incorporated into bacterial and
eukaryotic proteins. It is, therefore, likely that in many cases, in vivo-damaged amino acids
are available for de novo synthesis of proteins. Although the involvement of aminoacyl-tRNA
synthetases in this process has been hypothesized, the specific pathway by which ROS-
damaged amino acids are incorporated into proteins remains unclear. We provide an
evidence that mitochondrial and cytoplasmic phenylalanyl-tRNA synthetases in tandem
catalyze direct attachment of m-Tyr to tRNA-Phe, thereby opening the way for delivery of the
misacylated tRNA to the ribosome and incorporation of ROS-damaged amino acid into
eukaryotic proteins. Crystal structures of human mitochondrial, human cytoplasmic and
bacterial PheRSs complexed with m-Tyr and other ROS-damaged and medically important
amino acids will be discussed.
01.05.5

Applying structure-based drug design to the moonlighting enzyme dihydrolipoamide

dehydrogenase to target detrimental oxidative stress

Donald Berkholz, Rachael Vaubel, Christina Andrist, Grazia Isaya, James Thompson

Mayo Clinic College of Medicine, Rochester, MN, United States

Numerous mitochondria-related diseases including Friedreich ataxia cause

neurodegeneration and lack effective treatments. Evidence suggests changes in
oligomerization of the abundant mitochondrial enzyme dihydrolipoamide dehydrogenase
(DLD) result in a significant yet unappreciated source of disease symptoms including
mitochondrial iron imbalance and oxidative stress. Dimeric DLD exhibits dehydrogenase
activity that is an essential component of large complexes involved in energy metabolism.
Critically, it can dissociate into monomers with proteolytic and diaphorase activities not seen
in the physiological dimer that degrade vital mitochondrial proteins in vitro and increase
oxidative stress in vitro and in vivo. These detrimental activities oppose DLD’s primary
activity, with changes in its oligomeric state regulating whether DLD promotes oxidative
metabolism or oxidative damage.

We hypothesized that stabilizing dimeric DLD would decrease proteolytic and diaphorase
activities of monomeric DLD. To test this hypothesis, we are using structure-based drug
design to create high-affinity compounds from fragment-sized molecules with a combination
of crystallographic and computational approaches. In vitro screening of a chemically diverse
fragment library discovered 16 chemically similar compounds that bound to and stabilized
DLD – 4 of which bound with micromolar affinity – amounting to a 4% hit rate. Analysis of the
crystallographic dimer suggested favorable binding pockets within the dimer interface.
Intriguingly, docking studies of substrate-bound enzyme suggest that all of these compounds
bind to the same site within the interface pocket, which is being confirmed
crystallographically. This provides the basis for further drug design to target mitochondrial
diseases involving iron imbalance or oxidative stress.
01.05.6

Structure-guided therapeutic targeting of the essential protein farnesyltransferase from

the AIDS-associated pathogen Cryptococcus neoformans
1 2 2 1
Michael A. Hast , Connie B. Nichols , J. Andrew Alspaugh , Lorena S. Beese
1
Duke University Medical Center, Department of Biochemistry, Durham, NC, United States,
2
Duke University Medical Center, Department of Medicine, Durham, NC, United States

Cryptococcus neoformans is a human fungal pathogen that causes life-threatening

respiratory and neurological infections in immunocompromised individuals, including
transplant recipients and HIV patients. An ortholog of protein farnesyltransferase (FTase) that
is essential for viability has been identified as a potential drug target in C. neoformans.
FTase catalyzes a critical post-translational lipid modification of over 60 important signal
transduction proteins in the eukaryotic cell. We present a series of crystal structures of the
essential C.neoformans FTase (CnFTase) in complex with substrates and inhibitors and
identify dominant structural determinants of ligand selection. We show that previously
identified FTase inhibitors exhibit fungicidal activity against C. neoformans. Furthermore, we
show that the most potent fungicidal FTI causes mislocalization of Ras1 in the organism,
suggesting inhibition of prenylation of this important signaling molecule. Our combined
structural and functional studies provide a foundation for the design of a new generation of
antifungal FTase inhibitors (FTIs). This work was supported by NIH grants GM052382 to
L.S.B. and AI050128 to J.A.A.
01.05.7

Error-free DNA replication opposite an oxidative DNA lesion by Human Y family

polymerase iota

Kevin Kirouac, Hong Ling

University of Western Ontario, London, On, Canada

Human Y family DNA polymerase iota (polι ) is one of a few DNA polymerases that
incorporate the correct cytosine nucleotide with high specificity opposite 8-oxo-guanine; the
most abundant oxidative DNA lesion in cells. The structural basis of preferential cytosine
incorporation opposite 8-oxo-guanine by a eukaryotic DNA polymerase is currently unknown.
We present four crystal structures of polι in complex with DNA containing an 8-oxo-guanine
lesion, paired with correct dCTP or incorrect dATP, dGTP, or dTTP nucleotides. A narrow
active site restricts the 8-oxo-guanine in a syn conformation, which favours incoming dCTP
due to hydrogen bonding potential, base stacking interaction and optimal conformer
orientation. We demonstrate the importance of the finger domain residue Gln59 in template
8-oxo-guanine orientation and polι enzymatic activity using site directed mutagenesis. Lastly,
we propose a model for how polι may protect cells against oxidative DNA damaged by
reversing replication induced mutagenesis via a Base Excision Repair pathway.
02.04.1

Crystal Structures Viewed as Dynamical Systems

1 1 2
Carroll Johnson , Michael Burnett , Bryan Chakoimakos
1
Chemical Sciences Division, Oak Ridge National Laboratory, P.O. Box 2008, Bldg 4500S,
2
Oak Ridge, Tennessee 37981-6197, United States, Neutron Scattering Sciences Division,
Oak Ridge National Laboratory P.O. Box 2008, Bldg 7962, Oak Ridge, Tennessee 37831-
6393, United States

The excellent 800 page book [1] describes ergodic theory, topological dynamics, and theory
of smooth dynamical systems in great theoretical detail, but the applications are also mainly
theoretical. However, most of what they cover is directly applicable to crystal structures, but
that fact is seldom mentioned. Our massive databases of space group symmetry, unit cell
parameters, chemical contents, atom ID, positional anisotropic thermal parameters may
provide reality tests for some of that theory. All we need to do is provide a few good examples
and in the process we might learn things that could expand our own horizons. Below are
some areas of crystallography where these theories apply.
Flows, vector fields (of vector valued functions) and ordinary differential equations are basic
operations in dynamical systems theory. In crystallography, these translate to a thermal
motion flow [2] along a sequence of bonded thermal ellipsoids in an ORTEP drawing, and is
determined by the Radon-Nikodym derivative ratio (a cocycle [3]) for each ellipsoid pair. The
flow is from smaller to larger equiprobability ellipsoids to form a unidirectional network. The
calculations are made directly from the atoms' thermal and positional parameters, perhaps
using a future version of the ORTEP program. Also, dynamical flow theory gives the
possibility of calculating rotation torsions about bonds, an unsolved problem in
crystallography, using a cylindrical filter flow network derived from the above network.
[1] A. Katok, B. Hasselblatt (1995) Introduction to Modern Theory of Dynamical Systems,
Cambridge.
[2] T. Izer (1991) Theories of Intramolecular Vibrational Energy Transfer; Physics Reports
199, no.3, 73-146, North-Holland.
[3] V. Kaimanovich, K. Schmidt (2001) Ergodicity of Cocycles. 1: General Theory;
unpublished preprintThis research is supported in part by UT Battelle. LLC under Contract
No. DE-AC05-00OR22725 for the U.S. Department of Energy, Office of Science.
02.04.2

Substituent and Solvation Effects on N-H---O Hydrogen Bonds.

Ronald See, Justin Hileman

Indiana University of PA, Indiana, PA, United States

A database and computational study was done to determine the factors which effect hydrogen
bond strength in N-H---O hydrogen bonds. The importance of hydrogen bonding in general
has been well established, but N-H---O hydrogen bonds remain an understudied system.
These interactions dictate the form of the a-helix and the b-sheet, are crucial for base pairing
in DNA, and play a crucial role in the differential binding of O2 in the active site of myoglobin.
However, studies that detail the factors causing variation in the energy of N-H---O hydrogen
bonds are not available. Using the imidazole---OCX2 (where X = CH3, NH2, H, F and others)
system, N-H---O hydrogen bonds were studied computationally (B3LYP/6-31G**) to quantify
the effects of substituents and solvation. These hydrogen bonds varied in gas-phase energies
from 34-17 kJ/mol, and solvents decreased the attractive interaction based on a function of
the dielectric constant of the solvent. Database studies were also carried out on N-H---O
hydrogen bonding species, focusing on the effect of substituent groups. The results will be
interpreted in the context of I.D. Brown's Bond Valence Model of hydrogen bonds.
02.04.3

A List of Organic Kryptoracemates.

1 2
Carolyn Brock , László Fábián
1 2
University of Kentucky, Lexington, KY, United States, Pfizer Institute for Pharmaceutical
Materials Science, Cambridge Crystallographic Data Centre, Cambridge, United Kingdom

A list of 181 organic kryptoracemates has been compiled.** This class of crystallographic
oddities is made up of racemic compounds (i.e., pairs of resolvable enantiomers) that happen
to crystallize in Sohnke space groups (i.e., groups that include only proper symmetry
operations); the two enantiomers are crystallographically independent. Most (151) of the 181
structures could have crystallized as ordered structures in non-Sohnke groups; the remaining
30 do not fully meet this criterion but would have been classified as kryptoracemates by
previous authors.

Examples were found and checked with the aid of available software for searching the
Cambridge Structural Database, for generating and comparing InChI (IUPAC International
Chemical Identifier) strings, and for validating crystal structures. The pairs of enantiomers in
the true kryptoracemates usually have very similar conformations; often the match is near-
perfect. There is a pseudosymmetric relationship of the enantiomers in about 60% of the
kryptoracemate structures but the deviations from inversion or glide symmetry are usually
quite easy to spot.

Kryptoracemates were found to account for 0.1% of all organic structures containing either a
racemic compound, a meso molecule, or some other achiral molecule. The centroid of a pair
of enantiomers is more likely (99.9% vs. 99% probability) to be located on an inversion center
than is the centroid of a potentially centrosymmetric molecule, probably because the overall
shape and size of the van der Waals surface of an enantiomer pair is more variable than is
the corresponding surface of a single molecule.

*Current address for L. Fábián: Department of Chemistry, University College Cork, Cork,
Ireland’

* Fábián , L & Brock, C. P. (2010). Acta Cryst. B66, 94–103

02.04.5

The 3.4 Å structure of the 122 kDa Mtr4 protein: refinement and revelation of a novel
arch domain
1 1 1 2 2
Sean Johnson , Ryan Jackson , Bradley Hintze , Alejandra Klauer , Ambro van Hoof
1 2
Utah State University, Logan, Utah, United States, University of Texas Health Science
Center-Houston, Houston, Texas, United States

The RNA helicase Mtr4 performs a critical role in RNA processing and degradation as an
activator of the nuclear exosome. The molecular basis for this vital function is not understood
and detailed analysis is significantly limited by the lack of structural data. Here we present a
3.4 Å crystal structure of the 122 kDa Mtr4 protein from S. cerevisiae. The structure reveals a
novel arch-like domain that is specific to Mtr4 and Ski2 (the cytosolic homolog of Mtr4). In vivo
and in vitro analyses demonstrate that the Mtr4 arch domain is required for proper 5.8 S rRNA
processing, and suggest that the arch functions independently of canonical helicase activity.
Additionally, extensive conservation along the face of the putative RNA exit site highlights a
potential interface with the exosome. These studies provide a molecular framework for
understanding fundamental aspects of helicase function in exosome activation, and more
broadly define the molecular architecture of Ski2-like helicases.

Because of the limited resolution of the Mtr4 diffraction data, extensive use of secondary
structure restraints was required to complete refinement. To aid in definition of these
restraints, interactive python-based tools ("ResDe" - Restraint Definer) were developed to
rapidly define and edit distance restraints using the PyMol graphical interface. These tools
alleviate the daunting task of manually editing an extensive text file when a large number of
restraints are needed. We believe this tool will be of general interest to the crystallographic
community.
02.04.6

Nanovolume Optimization of Protein Crystal Growth Using the Microcapillary Protein

Crystallization System
1,2 1 1,2 3,4 3,4
Cory Gerdts , Glenn Stahl , Peter Nollert , Alberto Napuli , Wes Van Voorhis , Peter
4,5 1,4 1,4
Myler , Bart Staker , Lance Stewart
1 2
Emerald BioStructures, Inc., Bainbridge Island, WA, United States, Emerald BioSystems,
3
Inc., Bainbridge Island, WA, United States, University of Washington, Seattle, WA, United
4
States, Seattle Structural Genomics Center for Infectious Disease, Seattle, WA, United
5
States, Seattle Biomedical Research Institute, Seattle, WA, United States

The Microcapillary Protein Crystallization System (MPCS) is a microfluidic, plug-based

crystallization technology that generates diffraction-ready protein crystals in nanoliter
volumes. Using proteins from the Seattle Structural Genomics Center for Infectious Disease
(SSGCID), we sought to determine the rate of crystallization success using MPCS
nanovolume microbatch methods, relative to the more traditional microliter volume vapor
diffusion methods. SSGCID proteins underwent random sparse matrix crystallization
screening by vapor diffusion methods and crystallization hit conditions were applied in a
chemical gradient optimization using the MPCS. 120 different protein/precipitant
combinations were optimized using the MPCS technology, with a 75% crystallization success
rate. Our results also demonstrate recapitulated crystallization success for 93% of the 29
proteins tested. Moreover, the resulting crystals produced high quality X-ray diffraction data
leading to 6 novel protein structure determinations from crystals harvested directly from the
MPCS CrystalCards. These results suggest that the MPCS can be used to achieve chemical
gradient based optimization of vapor diffusion crystallization hits with high probability of
success using ~50-fold less protein than would be used in vapor diffusion based optimization
experiments. The MPCS technology has now been encapsulated within a new automated
instrument called the MPCS Plug Maker, winner of the 2010 Lab Automation New Product
Award.

This work was supported in part by the NIGMS-NCRR co-sponsored PSI-2 Specialized
Center Grant U54 GM074961 and by federal funds from the National Institute of Allergy and
Infectious Diseases, National Institutes of Health, Department of Health and Human Services,
under Contract No. HHSN272200700055C.
02.04.7

Solvent and Temperature effects upon the resulting Crystal Structure

James Fettinger

Univ. of California, Davis, CA, United States

During the past few decades low temperature data collections have become the preferred
method of choice for many desirable reasons; however there are structures that exhibit
undesirable changes when the data collection temperature is set too low. These effects are
frequently due to the temperature itself and/or the solvent of choice being incorporated into
the lattice itself resulting in an unexpectedly large Z' and/or twinning. A series of structures
will be described that exhibit these effects.
04.01.1

Combined X-Ray Scattering (SAXS) and Crystallography to Accurately Characterize

Dynamic DNA Repair Complexes in Solution

2 2 2 2 1,2
Robert Rambo , Greg Hura , Susan Tsutakawa , Michal Hammel , John Tainer
1 2
The Scripps Research Institute, La Jolla, CA, United States, aLawrence Berkeley National
Laboratory, Berkeley, CA, United States

Protein, DNA, and RNA shapes, as well as their detailed structural chemistry, encode key
information about connections needed to define the “interactomes” critical to biological
outcomes in cell biology. We are developing SAXS combined with crystallography as a
premiere tool for defining macromolecular conformations and connections at the proteomic
1-4
scale . Crystallography supplies unparalleled structural detail for mechanistic analyses;
however, it is restricted to describing conformations of macromolecules within crystal lattices.
In principle, SAXS can provide reliable complementary data on small and large
macromolecules. Our results on dynamic DNA repair protein and DNA complexes show that
SAXS has great potential to provide accurate shapes, conformations, and assembly states in
5-7
solution and inform biological functions in fundamental ways .
04.01.2

A Conformational Switch in the Scaffolding Protein NHERF1 Controls Autoinhibition

and Complex Formation
1,2
Zimei Bu
1 2
City College of New York, New York, NY, United States, Fox Chase Cancer Center,
Philadelphia, PA, United States

Scaffolding proteins are molecular switches that control diverse signaling events. A
particularly important example is the scaffolding protein NHERF1, which assembles and
regulates the localization and intracellular trafficking of a number of important membrane
proteins. At its N-terminus, NHERF1 begins with two modular protein-protein interaction
domains-PDZ1 and PDZ2-and ends with a C-terminal (CT) domain. The CT domain binds to
ezrin, which, in turn, interacts with cytosekeletal actin. Previously we have shown that ezrin
binding to NHERF1 increases the binding capabilities of both PDZ domains. Our solution
small angle neutron scattering and NMR experiments reveal the autoregulated intramolecular
domain-domain interactions, as well as much longer range conformational changes in
NHERF1 upon activation by ezrin binding. The results provide a structural explanation, at
both mesoscopic scales and atomic resolution, of the allosteric control of NHERF1 by ezrin as
it assembles protein complexes. We propose that this long-range allosteric regulation of
NHERF1 by ezrin enables the membrane-cytoskeleton to assemble protein complexes that
control cross-talk and regulate the strength and duration of signaling.
04.01.3

Crystallography without Crystals: Breaking the Crystallization Paradigm

1 1 2 3
Dilano Saldin , Hin-Cheuck Poon , Henry Chapman , John Spence
1 2
University of Wisconsin-Milwaukee, Milwaukee, WI, United States, Center for Free Electron
3
Laser Science, Hamburg, Germany, Arizona State University, Tempe, AZ, United States

The extreme brilliance of the x-ray free electron laser (XFEL) has provoked speculation about
1
the possibility of protein structure determination from single molecules . However, even in the
proposed diffract and destroy experiment, the number of scattered photons per detector pixel
2
from a typical protein is estimated to be a number much less than unity . Methods have been
2,3,4
proposed for structure determination even under such circumstances . We propose a
method for structure determination of uncrystallized proteins, by directly recovering the
diffraction pattern of a single molecule from that of multiple randomly positioned and randomly
oriented ones through their correlated scattering, and subsequent iterative phasing of this
5
diffraction pattern to recover the molecular electron density . We suggest a possible
6
application to structure determinations of membrane proteins in situ . Possible applications for
the proposed single-molecule XFEL experiments will also be discussed, as well as ideas for
3,7
extracting time-resolved information .

References

[1] R. Neutze, et al., Nature 406, 752 (2000).

[2] R. Fung et al., Nature Physics 5, 64 (2009).

[3] D. K. Saldin et al., J. Phys: Condens. Matter 21, 134014 (2009).

[4] N.-T. D. Loh and V. Elser, Phys. Rev. E 80, 026705 (2009).

[5] D. K. Saldin et al., New J. Phys., in press

[6] D. K. Saldin et al., submitted to Phys. Rev. B.

[7] J. C. H. Spence et al., Abstracts of the Meeting of the Microscopical Society of America
(2010).
04.01.4

Stucture of a bacterial ribonuclease P holoenzyme in complex with tRNA

1 1 1 1 1,2
Nicholas Reiter , Amy Osterman , Alfredo Torres-Larios , Kerren Swinger , Tao Pan ,
1
Alfonso Mondragón
1 2
Northwestern, Evanston, IL, United States, University of Chicago, Chicago, IL, United States

Maturation of transfer RNA (tRNA) requires processing at both its 3’ and 5’ ends.
Ribonuclease (RNase) P is the ribozyme responsible for 5’ -end tRNA processing and is found
in all three domains of life. We report the 3.85 Å resolution crystal structure of Thermotoga
Phe
maritima RNase P holoenzyme in complex with tRNA . The entire 154 kDa complex,
consisting of a large catalytic RNA (P RNA), a small protein cofactor, and mature tRNA, is
revealed in the structure. The structure shows the presence of tertiary RNA-RNA and RNA-
protein interactions that mediate substrate recognition and catalysis. Specific contacts at the
RNase P/tRNA interface explain the structural basis for substrate recognition. The structure
identifies the active site location and shows that it is composed of phosphate backbone
moieties, a universally conserved nucleotide, and metal ions. Additional experiments position
the leader sequence and show the interactions with the protein and the RNA component.
Overall, the structure is consistent with existing biochemical and biophysical data. The active
site structure and conserved RNase P/tRNA contacts suggest a universal mechanism of
catalysis by RNase P.
04.01.5

Structural Studies of the Complement-Like Innate Immune Response in anopheles

gambiae.

presented.

Richard Baxter1, Stefanie Steinert2, Yoga Chelliah1, Gloria Volohonsky2, Elena

Levashina2, Johann Deisenhofer1
1
University of Texas Southwestern Medical Center, Dallas, Texas, United States
Minor Outlying Islands, 2Université de Strasbourg, Strasbourg, France

Structural studies of the complement-like innate immune response in Anopheles

gambiaeMalaria, the world's most devastating parasitic disease, is cause by
apicomplexan parasites of the genus Plasmodium. The major vector for malaria in
Africa is the mosquito Anopheles gambiae. Significant progress has been made in
the past decade in demonstrating that A. gambiae possesses a robust innate
immune response to infection by Plasmodium parasites that may be a potential
source of novel vector control strategies. The complement-like protein thioester-
containing protein 1 (TEP1) labels P. berghei ookinetes for lytic destruction in the
basal lamina of the midgut epithelium. The three dimensional structure of TEP1 is
homologous to human complement factor C3. Further studies however, demonstrate
that the mechanism of TEP1 activation is distinct from vertebrate complement
factors, involving a pair of leucine-rich repeat proteins, LRIM1 and APL1. Recent
results of crystallographic and solution x-ray scattering studies regarding LRIM1 and
APL1 shall be
04.01.6

Morphological and Structural Changes of Lignocellulosic Biomass during Dilute Acid

Pretreatment using SANS
1 1 1 1 2
Sai Venkatesh Pingali , Volker Urban , William Heller , Hugh O'Neill , Marcus Foston , Dean
1 2 1
Myles , Arthur Ragauskas , Barbara Evans
1 2
Oak Ridge National Laboratory, Oak Ridge, TN, United States, Georgia Institute of
Technology, Atlanta, GA, United States

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04.01.7

Structure of the Natural Killer (NK) cell activating receptor NKp30 and identification of
its ligand binding site by utilization of a NKp30 blocking antibody and specific NKp30
peptides which map the protein surface
1 2 1
M. Gordon Joyce , Marco Colonna , Peter D. Sun
1 2
NIAID/NIH, Rockville, MD, United States, Washington University School of Medicine, St.
Louis, MO, United States

NK cells are a group of innate immune cells which are critical for the detection and destruction
of virally infected or cancerous cells on a daily basis in humans. These lymphocytes are
activated without any prior immune priming and elicit their effects using a group of activating
molecules (NKp30, NKp46 and NKp44). NKp30 is known to be the dominant receptor
responsible for the destruction of a number of tumor cell types. The precise mechanism of
how NKp30 interacts with cancerous cells initiating NK cell killing is an intriguing question and
to date, a number of NKp30 interacting molecules have been proposed. These molecules are
quite diverse ranging from Heparan sulphate to a Plasmodium falciparum molecule and most
recently a B7 Immunoglobulin-like homolog protein. However, the precise mechanism of how
NKp30 recognizes these molecules and initiates NK cell killing is not known.

To address this question we have determined the crystal structure of human NKp30. Multiple
protein constructs were expressed to find a protein construct which could be crystallized.
Crystals diffracted to 1.85 angstrom and although the closest known structure has a seq id of
22%; utilizing Balbes and EPMR, the structure was solved by Molecular Replacement. NKp30
has an I-type Ig-like structure, with a large dimer interface (previously unknown) and it is
structurally quite different from both NKp44 and NKp46. The closest homologue to NKp30 is
Programmed Death Ligand-1 and a structure of this protein in complex with its ligand,
Programmed Death-1 is known. Comparing our structure NKp30 with this complex indicates
that NKp30 ligand binding may utilize one face of the protein, specifically sheets C, F and G.

Cell killing assays using NK cells and cancer cells allowed us to identify an antibody which
strongly blocks NKp30 activation and prevents cancer cell killing. We also designed a group
of biotinylated peptides (each 15 aa in size) which map the entire surface of NKp30. Binding
studies with the NKp30 peptides and blocking antibody allowed us to identify Sheet F as a
critical portion for NKp30 activation. A NKp30 polyclonal antibody and a scrambled peptide
were used as controls.

Together, the structure determination of NKp30, comparison of NKp30 to its homologues and
the identification of a specific NKp30 region critical for NK cell killing is the first step in
understanding how NKp30 binds diverse ligands resulting in the destruction of cancerous
cells.
04.02.1

DNA-directed colloidal crystal formation: assembly and reorganization

1 2 2 1 2
Byeongdu Lee , Robert Macfarlane , Haley Hill , Soenke Seifert , Chad Mirkin
1 2
Argonne National Laboratory, Argonne, IL, United States, Northwestern University,
Evanston, IL, United States

While it was shown more than a decade ago that DNA oligonucleotides can be attached to Au
nanoparticles to direct the formation of larger assemblies, the conceptually simple yet
powerful idea that functionalized nanoparticles might serve as basic building blocks that can
be rationally assembled through programmable base-pairing interactions into highly ordered
macroscopic materials remains poorly developed, and the approach has mainly resulted in
polymerization having no long range order, or periodicity between particles within the
assembled material. Recently, it is demonstrated that DNA can be used to control the
crystallization of nanoparticle–oligonucleotide conjugates to the extent that different DNA
sequences guide the assembly of the same type of inorganic nanoparticle into different
1,2
crystalline states . Several factors that found critical to govern the process will be discussed.
Crystallization mechanism studied by in-situ isothermal and nonisothermal SAXS experiment
will be also presented to show that the crystals grow via a three step process: an initial
“random binding” phase resulting in disordered DNA-AuNP aggregates, followed by localized
reorganization and subsequent growth of crystalline domain size, where the resulting crystals
are well-ordered at all subsequent stages of growth.
04.02.2

In situ SAXS studies of silver nanoparticles in synthetic lung fluid

1 1 1 2
Andrew Allen , Vincent Hackley , Robert MacCuspie , Jan Ilavsky
1 2
Materials Science and Engineering Lab., NIST, Gaithersburg, MD, United States, Advanced
Photon Source, Argonne Natl. Lab., Argonne, IL, United States

Silver nanoparticles (AgNPs) have emerged as the most commonly identified nanoscale
material in consumer products, principally because of their broad-spectrum biocidal
properties. It remains unclear if nanoscale silver presents a new form of silver, or is simply a
new vector for solubilized Ag ions. Consequently, research on the environmental, health, and
safety (EHS) impact and risk of AgNPs has gained substantial momentum in recent years.
Within this context, the dispersion stabilization of AgNPs in synthetic lung fluid has been
studied to interrogate the effects on colloidal stability of the principal constituents in the fluid.
The colloidal stability of 20 nm citrate-AgNPs dispersed in the presence of each constituent of
the synthetic lung fluid (individually, the complete fluid, and without additives) was observed
during the titration of an increasing sodium chloride concentration into the solution. In situ
small-angle X-ray scattering (SAXS) measurements using a capillary flow cell were combined
with other complementary measurement techniques (dynamic light scattering, ultraviolet-
visible absorption spectroscopy, and atomic force microscopy). It was observed that AgNPs
continue to adsorb bovine serum albumin (BSA) protein from the synthetic lung fluid solution
as the sodium chloride concentration increases, until a maximum BSA coating is achieved
prior to reaching the physiological sodium chloride concentration of 154 mmol/L. BSA was
determined to be the constituent of the synthetic lung fluid required to provide colloidal
stability at high salt loadings, though phospholipid constituents also exert a subtle effect.
Since AgNPs are a distinctly different class of nanoparticles from the carbon nanotubes and
titania nanoparticles initially reported to be dispersible using this fluid, the work demonstrates
the broad applicability of synthetic lung fluid in providing stable dispersions for engineered
nanoparticles. Colloidal stability is inherently entangled with the surface functionalization of
nanoparticles. Since the surface that a nanoparticle presents to a living cell will impact its
biological fate and toxicity profile, understanding and controlling the nanoparticle surface in
dispersion protocols is a key element of correctly interpreting data in nano-EHS studies. This
AgNP study demonstrates how SAXS can play a critical role in understanding the EHS
consequences of nanoparticle interactions with soft matter and biological systems.
04.02.3

New opportunities for Anomalous Small-Angle X-Ray Scattering to characterize

Charged Soft Matter Systems

Michael Sztucki, Emanuela Di Cola, Theyencheri Narayanan

European Synchrotron Radiation Facility, Grenoble, France

Better understanding of charged soft matter systems is of direct relevance to many areas of
biological sciences. In general, electrostatic forces are more difficult to handle in modeling
and simulation owing to their long-range character. Examples include charge stabilized
colloids, polyectrolytes, proteins, surfactant micelles, membranes, etc. One of the key
parameters in the complete understanding of such systems is the spatial distribution of
counterions. In this contest, quantitative Anomalous Small-Angle X-ray Scattering (ASAXS)
offers a unique method for the structural characterization of charged systems. The spatial
distribution of counterions can be deduced with high precision by tuning the energy in the
vicinity of the absorption edge of the counterions. This information is not readily accessible by
conventional scattering techniques as the contributions of the counterions and the macroions
superimpose and thus cannot be distinguished.

This presentation will give an overview of recent results from different charged systems: e.g.
flexible hydrophobic polyelectrolytes in semi-dilute regime with rubidium as counterions (K-
edge, 15.2keV) and micellar surfactant systems studied near the bromine K-edge
(13.474keV). In the case of polyelectrolytes, the ASAXS effect is rather weak. Nevertheless,
the normalized intensities can be decomposed into three components: the energy
independent normal SAXS, a cross-term involving the amplitudes of normal SAXS and the
resonant scattering of the counterions and the elusive resonant scattering term due to
counterions. This latter term allows determining directly the spatial distribution of counterions.
On the other hand, the strong ASAXS effect in case of the micellar systems allows direct
modeling of the charge distribution.

The low concentration of anomalous species as well as the stability and radiation sensitivity of
soft matter systems pose high demands on the instrumental setup for ASAXS. This
contribution will also review the experimental requirements and recent technical advances in
ASAXS instrumentation at the high brilliance SAXS beamline (ID2), ESRF.
04.02.4

Scattering and Cryo-TEM studies of oleic acid based emulsions for investigating self-
assembly during human digestion
1 2 2
Heiner Santner , Stefan Salentinig , Otto Glatter
1 2
Anton Paar GmbH, Graz, Austria, Department of Chemistry, University of Graz, Graz,
Austria

This contribution presents self-assembly structures in biological relevant emulsified oleic acid
- monoolein (OA-MO) mixtures at different pH values. Small angle x-ray scattering (SAXS),
performed with the SAXSess system, as well as Cryo-TEM and dynamic light scattering
(DLS) were used to investigate structures and follow structure transition.

The solubilization of OA in MO based cubosomes decreases the interfacial curvature of the

liquid crystalline phase to more negative values. Structure transitions from bicontinuous
cubosomes, to hexosomes, emulsified Fd3m and EME occur with increasing OA
concentration. Similar effects were recently reported for the solubilization of tetradecane in
monolinolein based emulsions [1].

pH variation between 2 and 8 in a OA-MO system shows that the internal particle structure
strongly depends on the pH of the aqueous phase. At high enough OA concentration,
transformations from structure less emulsions to emulsified microemulsion (EME), emulsified
Fd3m, hexosomes, bicontinuous cubosomes and vesicles can be observed as a function of
pH. Interestingly, the liquid crystalline structure to vesicle transition always occurs at intestinal
pH values. The hydrodynamic radius of the particles decreases from around 120nm for
internally structured particles to around 60nm for vesicles [2]. All transitions with pH are
reversible.

An apparent pKa for OA in MO is evaluated from the change of structure with pH. This value is
within in the physiological pH range of the intestine (between pH 5.5 and 7.5). For pure OA a
higher pKa value between 8 and 8.5 was found [3].

[1] A. Yaghmur, et al., Langmuir 21, 569 (2005).

[2] S. Salentinig, et al., J. of Coll. and Interf. Sci. 326, 211 (2008).

[3] D.P. Cistola, et al., Biochemistry, 27, 1881 (1988).

04.02.5

Evaluation of the Microstructure of Semicrystalline Solid Dispersions

Qing Zhu, Lynne Taylor, Michael Harris

Purdue University, West Lafayette, United States

As a result of an increase in the number of emerging therapies with dissolution limited

bioavailability, formulation strategies such as solid dispersions[1] that enhance the rate of
solubilization are of interest.[2-5] In this study, the microstructure of solid dispersions
prepared with polyethylene glycol (PEG) and several model compounds with different
physicochemical properties were evaluated using a variety of experimental techniques. Solid
dispersions were prepared by fusion and evaluated using small angle X-ray scattering
(SAXS), powder X-ray diffraction (PXRD) etc. SAXS results indicated that, aceclofenac and
chlorpropamide solid dispersions favored the interlamellar aggregation of the drug in the PEG
matrix. Optical microscopy did not show any evidence of interspherulitic accumulation for any
of the model compounds. Haloperidol was highly crystalline in the dispersions, whereas
evidence of amorphous material was found for the other model compounds. Results indicated
that both the crystallization tendency of the drug and its solubility in amorphous regions of
PEG played important roles in determining the aggregation mode and size range of the drug
within the dispersion.

References

1. Sekiguchi, K.; Obi, N. Chem. Pharm. Bull. 1961, 9, 866-872.

2. Chiou, W. L.; Riegelman, S. J. Pharm. Sci. 1971, 60, 1281-1302.

3. Serajuddin, A. T. M. J. Pharm. Sci. 1999, 88, 1058-1066.

4. Leuner, C.; Dressman, J. Eur. J. Pharm. Biopharm. 2000, 50, 47-60.

5. Craig, D. Q. M. Int. J. Pharm. 2002, 231, 131-144.

04.02.6

Review of Application of Ultra-Small-Angle X-ray Scattering (USAXS) to Polymers and

Colloids
1 2 1 1
Jan Ilavsky , Fan Zhang , Gabrielle Long , Pete Jemian
1 2
Argonne National Laboratory, Argonne, IL, United States, NIST, Gaithersburg, MD, United
States

Ultra-small-angle X-ray scattering (USAXS) is capable of probing, in one single measurement,

structural inhomogeneities in the size range of 1 to 1000 nm. Recent developments of X-ray
sources and optics make USAXS increasingly relevant to polymer and colloids research. In
this review, we examine the current unique capabilities of USAXS and how these are
matched to the needs of these fields. We will present selected examples of USAXS
applications in areas of polymer nanocomposites, polymer gels and solutions, polymer
blends, polymer micelles and microemulsions, and colloidal sciences. As case example, we
will review published study of a unique colloidal stabilization mechanism, known as
1
nanoparticle “haloing”. This mechanism has been predicted theoretically and inferred
experimentally in microsphere-nanoparticle mixtures that possess high charge and size
asymmetry. The term “halo” implies the existence of a non-zero separation distance between
the highly charged nanoparticles and the negligibly charged microspheres that they surround.
Direct characterization of this system by means other than USAXS is challenging due to large
size difference between the two components of the system - approximately micron sized
spheres and ~50 nanometer particles forming the “halo”. However, using the USAXS, it was
possible to, quantitatively, characterize not only the separation distance of the “halo”, but also
number of particles in the “halo” and ratio of particles in the “halo” and surrounding
suspension. In conclusion, we predict more USAXS studies on polymeric and colloidal
systems, especially those with large-scale structure or hierarchical microstructures.
1
F. Zhang, G.G. Long, P.R. Jemian, J. Ilavsky, V.T. Milam, and J.A. Lewis, Langmuir, 2008,
24 (13), p. 6504-6508

Acknowledgement: Use of the Advanced Photon Source at Argonne National Laboratory was
supported by the U. S. Department of Energy, Office of Science, Office of Basic Energy
Sciences, under Contract No. DE-AC02-06CH11357.
04.02.7

In Situ Studies by Small-Angle X-ray Scattering of Kerogens under High Pressure CO2
1 1 1 2 3
Randall Winans , Darren Locke , Soenke Seifert , Tony Clemens , Joseph Calo
1 2
Argonne National Laboratory, Argonne, IL, United States, CRL Energy Limited, Lower Hutt,
3
New Zealand, Brown University, Providence, RI, United States

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04.02.8

Quantification of Structural Topology in Branched Polyolefins

Ramnath Ramachandran, Gregory Beaucage, Durgesh Rai

University of Cincinnati, Cincinnati, OH, United States

1, 2
A novel scaling method to quantify the topology of complex structures will be presented.
Specifically, the application of the method to branched polyolefin systems will be discussed.
3 4
The approach is useful in estimating the short-chain and long-chain branch contents in
these polymers. Results from this approach are compared and related to data obtained from
4
common techniques such as NMR and rheological measurements. Additionally, the method
provides a unique measure of the average long-chain branch length and the number of inner
segments in polyolefins. Inner segments reflect the degree of hyperbranching (branch-on-
branch) in the polymer systems.

To quantify the topology in polyolefin systems, the scaling method is applied to small-angle
3-4
neutron scattering (SANS) data from dilute solutions of polymer in deuterated good solvent.
Characteristic mass-fractal dimensions describing the topology (tortuosity and connectivity) of
branched polyolefins can be obtained. Further, quantities such as mole-fraction long-chain
branch content (Φ br), number of long-chain branches per chain (nbr), average branch length
(zbr) and number of inner segments per chain (ni) are estimated, the former two quantities
4
being unique to this approach. Such extensive topological information can be useful in better
understanding the effects of various catalyst systems and processing conditions on the
molecular structure of polyolefin resins. The scaling method can also be adapted to systems
other than polyolefins. For example, biomolecules, nano-aggregates cyclic and star polymers.

1. Beaucage, G., Determination of branch fraction and minimum dimension of mass-

fractal aggregates. Physical Review E 2004, 70 (3).

2. Kulkarni, A. S.; Beaucage, G., Quantification of branching in disordered materials.

Journal of Polymer Science Part B-Polymer Physics 2006, 44 (10), 1395-1405.

3. Ramachandran, R.; Beaucage, G.; Kulkarni, A. S.; McFaddin, D.; Merrick-Mack, J.;
Galiatsatos, V., Persistence Length of Short-Chain Branched Polyethylene. Macromolecules
2008, 41 (24), 9802-9806.

4. Ramachandran, R.; Beaucage, G.; Kulkarni, A. S.; McFaddin, D.; Merrick-Mack, J.;
Galiatsatos, V., Branch Content of Metallocene Polyethylene. Macromolecules 2009, 42
07.24.1

Phase transitions in perovskites - further uses of symmetry

Christopher J. Howard

University of Newcastle, Callaghan, Australia

In collaborative work with H.T. Stokes and others, the author has made extensive use of
computer program ISOTROPY to establish hierarchies of possible structures based on
various proposed distortions. Such hierarchies and their application (to the elucidation
structures in SrZrO3, for example) were reviewed five years ago [1]. The exploration of
complex distortions using ISODISPLACE is proving useful in more recent work [2].

The typical experimental study follows the evolution with temperature or pressure of lattice
parameters (these are particularly precise if measured using HRPD at the ISIS neutron
facility) and internal coordinates. It is often advantageous to construct symmetry-adapted
strains from the lattice parameters, and symmetry-adapted (normal mode) coordinates from
the internal coordinates. These symmetry-adapted quantities transform according to different
irreps of the parent space group. Certain of these quantities represent the order parameter(s)
for the transitions, and the connections between, say, the strains and order parameters can
be checked against the predictions of Landau theory. Illustrative examples to be described
may include further analysis of data from SrZrO3, results from a complex perovskite in the
(Ca,Sr)TiO3 solid solution, and the onset of Jahn-Teller distortion monitored by examination
of the relevant mode amplitude.

The influence of strain on phase transitions is nicely illustrated through recent measurements
of the 'plateau effect' on the system (Pr,La)AlO3 [3]. Low levels of the La dopant have no
effect on the transition temperature (strain field around La dopant atoms do not overlap), but
doping to around 2% La starts to reduce this temperature. This result leads to an estimate of
the scale of the strain fields around impurity atoms in perovskites.

[1] C J Howard & H T Stokes, Acta Cryst. A61, 93-111 (2005)

[2] C J Howard & M A Carpenter. Acta Cryst. B66, 40-50 (2010)

[3] M A Carpenter, R E A McKnight, C J Howard, Q Zhou, B J Kennedy & K S Knight, Phys.

Rev. B 80, 214101 (2009).
07.24.2

Phonons, phase transitions, and symmetry-mode analysis: examples from perovskites

and perovskite-related materials.

Ian Swainson

National Research Council of Canada, Chalk River, ON, Canada

Applying symmetry analysis to phonon modes can be done in a simple manner: one approach
is by inspection of a simple structure-type in direct-space, looking at the basic mechanics of
the structure. Frequently, a few candidate distortions can be revealed, which can then be
mapped onto a modulation vector in k-space. Symmetry programs, such as ISOTROPY, can
then be interrogated to find the basis functions of irreducible representations that correspond
to these phonon modes. I will give an example of the layered, A2BX4, organic-inorganic
perovskite-related structures. Specifically, I will discuss the propylammonium
tetrachlorometallates in which there is a complex series of transitions to tilt structures, and to
both commensurately and incommensurately modulated structures, in which the layers are
buckled. Using such methods it is possible to show that the inorganic sublattice solely
determines the possible distortions in these structures, while the final choice is made by the
energetics of the overall system. The same codes can be very useful in the analysis of real
phonon spectra: an example will be shown of unusual zone boundary scattering from the
relaxor perovskite, PMN, Pb(Mg1/3Nb2/3)O3. The relevant phonon branches can be assigned,
based on compatibility, observations of the lifting of degeneracy, and by comparing the
structure factor calculations from the basis functions of candidate irreducible representations
to measured intensity variations. From this, one can identify incipient zone boundary soft
modes, and identify the nature of the fluctuations.
07.24.3

Phase transitions in framework materials: insights from diffraction, NMR and theory

John Evans

Durham University, Durham, United Kingdom

Phase transitions are ubiquitous in functional materials and often directly related to the
emergence of specific properties. Examples range from paraelectric to ferroelectric
transitions in perovskites, through structural phase transitions in cuprate and pnictide
superconductors, to volume-reducing phase transitions in negative thermal expansion
materials.

In this contribution I'll discuss two examples where understanding phase transitions and their
structural consequences is crucial for understanding the material's properties, and will show
how insight from ISODISPLACE symmetry mode analysis provides insight.[1] The first study
relates to displacive phase transitions in (MoO2)2P2O7 where combining information from
31
powder diffraction, multi-dimensional solid state P NMR and DFT calculations is crucial in
unravelling the structural complexity.[2] In the second example I'll discuss the phase
transitions and structural/magnetic properties of a new family of transition metal
oxychalcogenides related to the pnictide superconductors.[3]

[1] e.g. Stokes, H.T., Campbell, B.J., Hatch, D.M., http://stokes.byu.edu/isodisplace.html.

[2] Lister, S.E., A. Soleilhavoup, R.L. Withers, P. Hodgkinson, and J.S.O. Evans, Structures
and Phase Transitions in (MoO2)2P2O7. Inorganic Chemistry, 2010. 49(5): p. 2290-2301.

[3] McCabe, E.E., Free, D.G., Evans, J.S.O., unpublished results.

07.24.4

The fundamental role of secondary modes in perovskite phase transitions

1 1 1 2
Ross Angel , Di Wang , Yonggang Yu , Michael Carpenter
1 2
Virginia Tech, Blacksburg, VA, United States, University of Cambridge, Cambridge, United
Kingdom

The description of perovskite structures and the phase transitions between them in terms of
the tilts of essentially rigid octahedra is long established, and has provided a consistent
framework within which to analyse perovskite structures that do not involve electronic effects
such as Jahn-Teller distortions or ferroelectric displacements. The formal description of these
tilts in terms of two 3-dimensional order parameters associated with the M- and R- points of
the Brillouin zone of the cubic parent phase has allowed the character of the transitions to be
constrained through Landau theory, the detailed coupling between the order parameters and
the spontaneous strain to be determined, and the resulting evolution of the elastic tensor
coefficients to be predicted.

However, if the octahedra were truly rigid as assumed in this analysis, then volume reduction
under high pressure could only be achieved by increasing octahedral tilts, and thus phase
transitions to lower-symmetry structures. However, in 3:3 perovskites such as YAlO3, LaGaO3
or LaAlO3, pressure results in decreased tilts and transitions to structures with higher
symmetry. We have used DFT calculations to determine the structural evolution of
orthorhombic YAlO3 over a pressure range (and with a precision) not available to
experimental measurement. The structure undergoes a Pnma to Imma phase transition at
~140 GPa at 0K, a transition normally attributed solely to the tilts associated with the M-point
becoming zero, and the amplitude of the corresponding M3+ mode becoming zero. However,
this does not fully describe the structural evolution of the Pnma phase, and in particular it
does not describe the simultaneous compression and reduction in distortion of the AlO6
octahedra. Analysis with the Isodisplace software shows that, in addition, the amplitudes of
the X5+and R5+ symmetry-adapted modes evolve significantly in the Pnma phase, while the
M2+ mode remains essentially zero. As expected from the crystal-chemical argument outlined
above, our analysis therefore shows that these additional modes must be considered in any
complete analysis of the physics of perovskite phase transitions.
07.24.5

Phase transition mechanisms in clinopyroxenes under non-ambient conditions

1 3 4 2
Matteo Alvaro , Fabrizio Nestola , Fernando Cámara , Chiara M. Domeneghetti , Vittorio
2 1
Tazzoli , Ross J. Angel
1 2 3
Virginia Tech, Blacksburg, VA, United States, Universita' di Pavia, Pavia, Italy, Universita'
4
di Padova, Padova, Italy, C.N.R. - Istituto di Geoscienze e Georisorse, Pavia, Italy

Clinopyroxenes are chain silicate minerals, in which there are two chains, made up by corner-
sharing SiO4 - tetrahedra running parallel to the c – axis. The two chains are crosslinked by
2+ 3+ 2+
M1 (containing Mg, Fe , Fe , and Al) and M2 (containing Ca, Fe , Mg and Na ) polyhedra.
There are three polymorphs (HT-C2/c, P21/c, HP-C2/c) stable at different pressure and
temperature conditions. At ambient conditions the space group is normally P21/c, which
transforms to HT-C2/c and HP-C2/c at high-temperature and high-pressure conditions
respectively. The HP and HT C2/c structures have the same Wyckoff positions as one
another but different geometries. Single crystal structure determinations under non-ambient
conditions clearly show that the most significant changes affecting the whole structure
geometry at the transition from P to C are those involving the kinking angle (angle between
shared oxygens of the same tetrahedral chain), together with the volume of the M2
polyhedron. In particular the kinking angle plays a crucial role in the entire structural evolution
as well as the phase transition mechanisms. As the two HP and HT polymorphs differ in their
topology the transition mechanism is also expected to be different.

By choosing a specific chemical composition we have been able to study the transition to both
the HP and HT-C2/c polymorphs. At ambient conditions in the P21/c phase, both the
tetrahedral chains are extremely kinked. Far from the transition point, with increasing P the
structure becomes more “compressed”, as expected: the two distinct chains become more
kinked with a decrease of volume of the M polyhedra. By contrast, at high-T the structure
starts “expanding”; the chains extend and the M2 polyhedra expand. At the high-P transition
there is an abrupt change of the rotation of the A chain and the two tetrahedral chains
become equivalent (with a kinking angle that is smaller than those of the P21/c structure), and
the M2 polyhedral volume undergoes a sudden decrease as expected for the first-order
character of the transition. By contrast, the high-T transition is continuous, and within the
P21/c phase there is a progressive change in rotation of both A and B chains until they
become equal at the transition temperature.
07.24.6

Triple Structural Transition below Room Temperature in the Antifilarial Drug

Diethylcarbamazine Citrate
1 1 1 2
Javier Ellena , Cecilia C. P. da Silva , Felipe T. Martins Martins , Sara B. Honorato , Núbia
3 1
Boechat Boechat , Alejandro P. Ayala
1
Instituto de Física de São Carlos, Universidade de São Paulo, São Carlos, São Paulo, Brazil,
2
Departamento de Física, Universidade Federal do Ceará, Fortaleza, Ceará, Brazil,
3
Fundação Oswaldo Cruz - FioCruz, Instituto de Tecnologia em Fármacos–FarManguinhos,
Rio de Janeiro, Rio de Janeiro, Brazil

A very unusual triple structural transition pattern below room temperature was observed for
the antifilarial drug diethylcarbamazine citrate. Besides the first thermal, crystallographic and
vibrational investigations of this first-line drug used in clinical treatment for lymphatic filariasis,
a noteworthy behavior with three structural transformations in function of temperature was
demonstrated by differential scanning calorimetry, Raman spectroscopy and single-crystal X-
ray diffractometry. Our X-ray data on single crystals allow for a complete featuring and
understanding of all transitions, since the four structures associated with the three solid-solid
phase transformations were accurately determined. Two of three structural transitions show
an order-disorder mechanism and temperature hysteresis with exothermic peaks at 224 K
(T1’) and 213 K (T2’) upon cooling and endothermic ones at 248 K (T1) and 226 K (T2) upon
heating. The other transition occurs at 108 K (T3) and it is temperature-rate sensitive.
Molecular displacements onto the (010) plane and conformational changes of the
diethylcarbamazine backbone as a consequence of the C—H•••N hydrogen bonding
formation/cleavage between drug molecules explains the mechanism of the transitions at
T1’/T2. However, such changes are observed only on alternate columns of the drug
intercalated by citrate chains, which leads to a doubling of the lattice period along the a axis
of the 235 K structure with respect to the 150 K and 293 K structures. At T2’/T1, these
structural alterations are completed in the crystal. At T3, there is a rotation on the axis of the
N—C bond between the carbamoyl moiety and an ethyl group of one crystallographically
independent diethylcarbamazine molecule besides molecular shifts and other conformational
alterations. The impact of this study is based on the fascinating finding in which the versatile
capability of structural adaptation dependent on the thermal history was observed for a
relatively simple organic salt, diethylcarbamazine citrate.
T-261

Diluted kagomé antiferromagnet in a single-crystal of segnitite studied by neutron

diffraction
1 1 2
Huibo Cao Cao , Bryan C. Chakoumakos Chakoumakos , Brian C. Sales Sales , Stuart J.
3
Mills Mills
1
Neutron Scattering Science Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831,
2
Oak Ridge, TN, United States, Materials Science and Technology Division, Oak Ridge
3
National Laboratory, Oak Ridge, TN 37831, Oak Ridge, TN, United States, University of
British Columbia, Department of Earth and Ocean Sciences, Vancouver, Canada, Vancouver,
BC, Canada

Iron kagomé lattices in the alunite-jarosite family provide a model kagomé Heisenberg
antiferromagnet, which is a highly geometrically frustrated system [1-2]. Segnitite,
PbFe3(AsO4)2(OH)5(H2O), belongs to the alunite supergroup [3], and naturally formed single
crystals offer a chance to perform neutron studies on this highly frustrated system. Single
crystal neutron diffraction was undertaken on the HB-3A four-circle diffractometer at HFIR of
ORNL. A 5% aluminum dilution in the iron kagomé layers was determined by both single
crystal X-ray and neutron diffraction. SQUID magnetometer identifies an antiferromagnetic
transition at T = 50 K, which lies well in the range of the antiferromagentic iron kagomé family
[1]. But in the specific heat measurement, we found that its entropy of transition is only 8% of
the total entropy, which also is much less than for pure iron kagomé lattices [1]. Preliminary
neutron diffraction was carried out to search for the (1 1 3/2) reflection and other reflections
with a propagation vector k = (0 0 3/2), where q-scans with a counting time of up to 60 s did
not show any antiferromagnetic signal. Further study will be undertaken to fully develop a new
magnetic model in this diluted kagomé antiferromagnet.

This research is supported by UT Battelle, LLC under Contract No. DE-AC05-00OR22725 for
the U.S. Department of Energy, Office of Science.

[1] D. Grohol et al. Nature Mater. 4, 323 (2005), Phys. Rev. B 67, 064401 (2003).

[2] J. Frunzke et al. J. Mater. Chem. 11, 179-185 (2001).

[3] S. J. Mills et al. Eur. J. Mineral. 21, 1073-1080 (2009).

07.25.1

VECTOR APPROACH IN TWO-DIMENSIONAL X-RAY DIFFRACTION

Bob He

Bruker AXS, Madison, WI, United States

Two-dimensional diffraction pattern contains information in a large solid angle. In the two-
2
dimensional X-ray diffraction (XRD ) geometry, the 2D image can be described by the
diffraction intensity distribution in both 2θ and directions. Unit diffraction vector is used in the
data analysis of the 2D diffraction pattern. The unit diffraction vector for all the pixels in the 2D
pattern can be calculated in the laboratory coordinates. The data analysis requires the unit
diffraction vector expressed in the sample coordinates, which can be obtained by vector
transformation. The unit vector expression can be used for many applications. For stress
analysis, the fundamental equation is given by scalar product of the strain tensor ½ ij with the
{hkl }
unit vector {h1 , h2 , h3} : ¾( , , , ) ¾ ij hi h j
{hkl }
where ¿( , , , ) is the measured strain from 2D pattern. For texture analysis, the pole figure
angles ( , ) are given by pole mapping equations:

h1 0 if h2 0
sin 1 h3 cos 1
h12 À h22 , cos 1

h12 h22 0 if h2 0

The diffraction unit vector is also used in polarization correction, absorption correction and
effective volume calculation for crystal size evaluation by -profile analysis.

Ref: Bob He, Two-dimensional X-ray Diffraction, John Wiley & Sons, 2009
07.25.2

Symmetry detection via symmetry-mode analysis

1 1 2 2
Branton J. Campbell , Sean Kerman , John S. O. Evans , Francesca Perselli , Harold T.
1
Stokes
1 2
Dept. of Physics & Astronomy, Brigham Young University, Provo, UT, United States, Dept.
of Chemistry, University of Durham, Durham, United Kingdom

For any crystal structure that can be viewed as a low-symmetry distortion of some higher-
symmetry parent structure, one can represent the details of the distorted structure in terms of
symmetry-adapted distortion modes of the parent structure rather than the traditional list of
atomic xyz coordinates. While both descriptions are entirely equivalent, and share the same
number of structural degrees of freedom, the symmetry-motivated basis has the advantage
that only a handful of the available degrees of freedom tend to be active (i.e. have non-
negligible values). Once these active modes are identified, even the Rietveld refinement of a
highly complex distortion can become simple. Consider a subtle low-symmetry distortion of
an otherwise high-symmetry parent structure that gives rise to a large supercell and a low
point symmetry. If the peak splittings are small and the superlattice intensities weak,
structural complexity can increase dramatically without a comparable increase in the
information content of an experimental powder-diffraction pattern. In such a case, we will
demonstrate that symmetry-mode analysis can reliably determine the real space-group
symmetry and simplify the refinement of the distorted phase.
07.25.3

Synchrotron powder X-ray diffraction studies of the alkali metal phenolates to probe
Kolbe-Schmitt reaction mechanism and intermediates
1 2 2
Matthew Suchomel , Hyunsoo Park , Maren Pink
1 2
Argonne National Laboratory, Argonne, IL, United States, Indiana University, Bloomington,
IN, United States

Time-resolved powder X-ray diffraction is a powerful tool to investigate the reaction

mechanisms, phase transformations and crystallization of solid-state processes. It can
provide valuable insights into dynamic processes in extended structures such as zeolites,
metal-organic frameworks and other porous crystalline solids to aid the rational design of new
materials.

In the current study, in-situ investigations of the carbon insertion mechanism in the Kolbe-
Schmitt reaction were performed using powder XRD methods. This important reaction is
widely used in industry for the? ÁÂÃÄ·ÅÆÇÃÈ? ÃÉ? ÁÇÊËÌÈÆÍK? É¡ÂÆÇÎÇ ¡‒ ? \‹ ? fi⁄\‒«\¦¡· ¦\ Z
⁄›•¡ ¡‒? ⁄¡?¡‚\¦ ?«¡¦⁄\‹ «?›¢? ⁄¡?j› ¡Lr¦⁄« ?‒¡\¦ ›‹? ? ? ·‹¦ ¡\‒M??s›?⁄¡ fi?¢·‒ ⁄¡‒
¡‹ ¢„? ⁄¡? ‒¡\¦ ›‹? ¡ \ ? \‹ ? ‹ ¡‒«¡ \ ¡ K time-resolved powder XRD measurements
were performed on synchrotron powder diffraction beamlines at the Advanced Photon Source
(1-BM and 11-BM). Time-resolved powder XRD techniques (1-BM) were used to observe the
reaction between alkali metal phenolate and CO2 gas. The experiments were performed as a
function of time, temperatures and CO2 pressures in order to probe the effects of reaction
parameters on the reaction products. Crystalline reaction intermediates isolated during the in-
situ experiments were further characterized in detail via high-resolution powder XRD (11-BM).

In this presentation, we will discuss the possible reaction pathways of the solid-state Kolbe-
Schmitt reaction and propose new crystal structures of the crystalline intermediate phases.
The work will also highlight the potential of performing rapid in-situ powder X-ray diffraction
measurements at high-intensity synchrotron sources like the Advanced Photon Source (APS)
and identify resources available for similar experiments by potential users within the
crystallography community.
07.25.4

Pressure-induced switching in magnetic framework materials.

Gregory Halder, Karena Chapman, Peter Chupas, John Schlueter

Argonne National Laboratory, Argonne, IL, United States

The design and characterization of molecular materials with targeted functionalities, such as
magnetism and/or nanoporosity, is part of a major international push aimed at developing
systems with technologically important applications (e.g., molecular sensing and storage). As
such, the accurate elucidation of their often complex structure-function relationships presents
a crucial step in their advancement. For molecular magnetism, these approaches are
commonly focused on variations of temperature and/or magnetic field, while comparatively
little attention has been given to how these materials behave as a function of pressure. Here,
we report magneto-structural investigations of magnetic molecular materials using
synchrotron-based structural studies (powder diffraction and pair distribution function) and
magnetic susceptibility measurements at high pressures. These studies have revealed a
range of interesting phenomena such as orbital reorientations, spin crossover, phase
transitions, and extreme compressibility.
07.25.5

Extending the Power of PowSnB

1,2 2,3 2,3
Hongliang Xu , Charles M. Weeks , Robert H. Blessing
1 2
SUNY College at Buffalo, Buffalo, NY, United States, Hauptman-Woodward Institute,
3
Buffalo, NY, United States, SUNY at Buffalo, Buffalo, NY, United States

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07.25.6

Ab initio study of the structure of porous metal-organic frameworks and covalent-

organic frameworks from powder X-ray diffraction using charge flipping

Fernando Uribe-Romo, Felipe Gandara, Michael O'Keeffe, Omar Yaghi

Center for Reticular Chemistry at the California NanoSystems Institute, Department of

Chemistry and Biochemistry, University of California Los Angeles, Los Angeles, California,
United States

Metal-organic frameworks (MOFs) and covalent-organic frameworks (COFs) are crystalline

extended materials that are constructed from rigid organic linkers connected either by
inorganic metal clusters (MOFs) or completely organic building blocks (COFs), respectively.
In the pursuit of the synthesis of functional porous crystalline materials, the preparation of
single crystals for a complete structural assignment can be challenging; thus it is common
that the only available source of structural information must be obtained from microcrystalline
powder samples. Here we describe a series of MOFs and COFs that have been structurally
characterized by analysis of their powder X-ray diffraction (PXRD) patterns. Electron density
maps of the synthesized materials were calculated ab initio (no chemical or symmetry
information) using the charge-flipping algorithm from laboratory PXRD data. Atomic resolution
data (dmin = 1.3 Å) enabled the solution of the crystal structure of several MOFs whereas low-
resolution data (dmin = 2.0 Å) yielded the assignment of the pore system (cavities) of COFs.
For the MOF systems, we identified the atomic positions of the metal and organic units as
well as the presence of guest molecules within the pore cavities. Despite the intrinsic low
quality of the diffraction of COFs, we identified regions of electron density corresponding to
the framework and volumes with very low electron density attributed to the void space of the
pores. Indeed this pore system is in agreement with the proposed structural.
07.26.1

Practical Methods of Modeling Disordered Solvent Molecules Using SHELXTL and

PLATON.
1 1 2
Charles Campana , Bruce Noll , Holger Ott
1 2
Bruker AXS Inc., Madison, Wisconsin, United States, Bruker AXS GmbH, Karlsruhe,
Germany

Some of the most difficult problems in obtaining chemically reasonable and

crystallographically accurate structures involve the use of appropriate techniques to model
and account for disordered solvent molecules and counter ions within the crystal lattice.
Through careful application of some of the advanced features of the SHELXTL and PLATON
software packages, we have been able to significantly improve the quality of the final
crystallographic results obtained for a variety of chemically important systems, often with low
resolution data. We will present several examples to illustrate some these methods.
07.26.2

Solvent: The Necessary Evil

Tom Emge

Rutgers University, Piscataway, NJ, United States

This brief survey reports on the deleterious effects of highly disordered solvent in a variety of
crystal structures. The improvements of derived results and R indices based upon different
schemes for modeling the disordered solvent are detailed. Structures analyzed include those
with lattices containing large pores, such as MOFs and cavitands, and those containing
disordered solvents or counterions of high molecular weight, such as bromoform,
triphenyltritellurium and trisphenyltelluridomercurate.
07.26.3

Crystallographic Determination of Metallic Oxide Fullerenes: The Interplay Between

Crystallographic and Computational Results
1 4 2 3
Brandon Mercado , Christine Beavers , Steven Stevenson , Joseph Poblet , Marilyn
1 1
Olmstead , Alan Balch
1 2
University of California - Davis, Davis, CA, United States, University of Southern Mississippi,
3
Hattiesburg, MS, United States, Universitat Rovira i Virgili, Tarragona, Spain, United States,
4
Advanced Light Source, Lawrence Berkeley National Lab, Berkeley, CA, United States

Fullerenes have a variety of potential applications that require a better understanding of the
interior environment of empty cage and endohedral metallofullerenes (EMFs).
Crystallographic data of EMFs offer definitive answers to questions concerning fullerene cage
symmetry, inter/intramolecular interactions. X-ray structure
determination of EMF single crystals can present numerous
problems due to issues such as large thermal motion and
small crystal size. Our lab’s approach to solving these
problems has included the application of engineered co-
crystal systems, synchrotron radiation, and advanced
refinement techniques. Computational optimizations of
fullerene structures can also provide answers concerning the
chemical nature of a fullerene, molecular orbital energy
diagrams, and electrostatic potential maps. These results
may or may not corroborate crystallographic data. Several
new EMF structures will be presented here, specifically
metallic oxide fullerenes: Sc4(ð3-O)n@C80 (n = 2, 3 see
Figure1. Sc4(3-O)3@C80; the tetrahedral Figure 1) and Sc2(ð2-O)@C82. Special consideration will be
array of four scandium atoms with three of
the faces capped with oxygen atoms is the
given to the comparison of crystallographic and
largest moiety to be encapsulated in an computational analyses.
fullerene to date.
07.26.4

Solution and Refinement with the cctbx and smtbx

Luc Bourhis, Oleg Dolomanov, Richard Gildea, Judith Howard, Horst Puschmann

Durham University, Durham, United Kingdom

The Computational Crystallography Toolbox (cctbx) opened a new era in crystallographic

computing by providing a free, open and comprehensive implementation of the fundamentals
of crystallography (symmetries, Fourier, scattering, etc). As the foundation of the
macromolecular suite PHENIX, it has a certain connotation which is undeserved since the
algorithms and data structures it features are correct for any crystal structure.

Thus we endeavoured to use it for small molecule work, as part of the EPSRC grant "Age
Concern". This lead to the creation of a companion library, the Small Molecule Toolbox
(smtbx). It shares the same philosophy as the cctbx: it is designed to make the writing of short
scripts easy as well as to make it possible to build or to integrate it into large programs. One
example of the latter is the program Olex 2, also developed under the same EPSRC grant,
through which the practising crystallographer is given access to most smtbx features. It
provides tools covering the whole workflow of small molecule work: e.g. charge flipping and
map symmetry search for the solution stage, full matrix refinement for the refinement stage
and solvent disorder modelling similar to the SQUEEZE procedure in PLATON. In this talk,
we will give an overview of the capabilities of the smtbx/cctbx for small molecule work,
focusing on those key computational details which have been the treasures of the classic
programs CRYSTALS or SHELX.
07.26.5

A New Solvent Masking Procedure

Richard Gildea, Luc Bourhis, Oleg Dolomanov, Judith Howard, Horst Puschmann

Durham University, Durham, United Kingdom

Disordered solvent molecules or counterions are most commonly modelled with two or
more overlapping fragments, often requiring the extensive use of restraints and/or constraints
to keep the model chemically reasonable. When appropriate, a somewhat more elegant
alternative may be to model atoms as continuously disordered along some special figure,
such as a line, a ring or the surface of a sphere, as featured by the program CRYSTALS [1].

However, there are often cases where extended disorder, unknown solvent composition, or
incompatibility of the symmetry of the solvent molecule with its site, is such that neither
approach is appropriate. Van der Sluis and Spek [2] suggested a method whereby the
contribution to the calculated structure factors of the disordered solvent area is calculated via
a Fourier transform of that area. This solvent contribution can then either be added to that
calculated from the ordered part of the structure, or alternatively subtracted from the observed
data before further cycles of refinement. This method has been made widely popular by the
SQUEEZE routine available through the program PLATON [3].

We present a new implementation of the above method based on the cctbx (Computational
Crystallographic Toolbox) [4], and available through the software Olex2 [5]. Our
implementation is compatible with our own smtbx-based refinement program [6], and other
refinement programs accepting a hkl file as input.

1. L. Schröder, D. J. Watkin, A. Cousson, R. I. Cooper and W. Paulus, J. Appl. Cryst. (2004).

37, 545-550.

2. P. van der Sluis and A. L. Spek, Acta Cryst. (1990). A46, 194-201.

3. A.L.Spek, J. Appl. Cryst. (2003). 36, 7-13.

4. http://cctbx.sourceforge.net

5. O. V. Dolomanov, L. J. Bourhis, R. J. Gildea, J. A. K. Howard and H. Puschmann, J. Appl.

Cryst. (2009). 42, 339-341.

6. L. J. Bourhis, R. J. Gildea, O. V. Dolomanov, J. A. K. Howard and H. Puschmann, IUCr

Comp. Comm. Newsletter (2009). 10, 18-31.
07.26.6

Truth is a Meadow – Solvent Molecules in Crystal Structures

Peter Mueller

MIT, Cambridge, MA, United States

“Truth is”, according to German author Stefan Heym, “a meadow where everyone picks the
flower he likes best”. Science is the search for knowledge (lat. scientia: knowledge), not truth;
yet scientists strive to get as close to The Truth as possible, and the three-dimensional
molecular models afforded by modern crystallographic methods certainly look “true”, however
flawed they may be. On the example of a particularly challenging solvent disorder this
presentation will offer a few flowers from the aforementioned meadow for the audience to
examine.
07.26.7

Host-Guest Hydrogen Bonding in Clathrate Hydrates

Konstantin Udachin, John Ripmeester

National Research Council, Ottawa, Canada

Clathrate hydrates with low melting points (often below –20C) are difficult subjects for single
crystal data collection. A high level of guest molecule disorder inside the high symmetry
cages causes difficulty for structure determination of such crystals as well. Recent advances
in single crystal X-ray diffraction have allowed this technique to be used as a valuable tool for
the analysis of hydrate structure and composition. With detailed analysis of guest and water
molecules disorder, not only the guest positions are clearly defined, but also it becomes
possible to find interactions between guest and water molecules.

For the first time, single crystal x-ray crystallography is used to detect
the presence of guest host hydrogen bonding in structure I, II and structure H clathrate
hydrates. Clathrates studied are the tert-butylamine (tBA) sII clathrate with H2S and Xe help
gases, the pinacolone + H2S binary sH clathrate, 1,3-Dioxolane hydrate, chlorine, and
bromine hydrates.

X-ray structural analysis shows that the tBA nitrogen atom has a distance of 2.64 Å from the
closest large cage oxygen atom. This water molecule is pulled inwards toward the tBA guest
(cage center) and the structure of the large cage is substantially distorted in comparison to
the ideal cage structure. The pinacolone oxygen atom is determined to have a distance of
2.96 Å from the closest large cage oxygen atom. This distance is compatible with pinacolone
– water hydrogen bonding.