Introduction of X-ray Crystallography

Time : 2005/10/03
10:20~11:10pm

Department of Chemistry, NTU

Andrew H.-J. Wang

Institute of Biological Chemistry

Academia Sinica
Four steps to apply X-ray method
{ Crystal
{ X-ray diffraction
{ Electron density map
{ Molecular structure (atomic level)
Basic diffraction physics
{ Bragg’s law Path difference between wave 1 and 2 :

2dsinθ = nλ
2dsinθ

n=integer, in phase, constructive interference

d : distance between planes
λ: wavelength of incident beam
θ : diffraction angle

incident beam reflected beam In phase

θ θ
d
dsinθ dsinθ Out of phase
Crystallization Method
{ Vapor Diffusion Method

1µl of protein solution

1µl of mother liquid

A siliconized cover slide

24-well crystallization plate

easy to perform,
require only a small
amount of sample
500µl of mother liquid
crystals growth around
Hanging drop Sitting drop 60~70 % concentration of
mother liquid
Different crystal forms-Polymorphism

The crystals of Bacillus stearothermophilus 6-phosphoglucose isomerase B

under various crystallization conditions
Unit cell
{ The smallest cell that can give all the
symmetries
{ The diffraction signal is contributed
from the scatters in the unit cell space

1
Diffraction signal, Intensity I∝ 2
V
Space groups

System Lattice Space group

Triclinic P P1
C
Monoclinic P,C P2,P21,C2
Orthorhombic P,C,I,F C222,P222,P212121,P21212,P2221,C2221,
F222,I222,I212121
β α B
Tetragonal P,I P4,P41,P42,P43,I4,I41
γ P422,P4212,P4122,P41212,P4222,P42212,
P4322,P43212,I422,I4122
A Trigonal P (or R) P3,P31,P32,R3
P312,P321,P3121,P3112,P3221,P3212,R32
7 crystal systems
Hexagonal P P6,P61,P62,P63,P64,P65
14 Bravais lattices
P622,P6122,P6222,P6322,P6422,P6522
65 (230) space groups Cubic P,I,F P23,F23,I23,P213,I213
P432,P4132,P4232,P4332,F432,F4132,I432,I4132
Crystal mounting
{ Crystal Mounting (Cryo)

Cryoprotectants

Glycerol, Glucose, Sucrose, Xylitol,

(2R ,3R)-(-)-butane-2,3-diol, Erythritol
MPD, PEG8000, PEG6000, PEG3000, PEG400
MPEG 500, Ethylene glycerol, Jeffamine
Isopropanol, Pump oil, Paratone-N, Li2SO4 ……
 Electron density maps- Fourier maps

Institute of Biological Chemistry, Academia Sinica

X-ray diffraction data
{ The diagram of X-ray diffractometer

Data collection time :

In-house X-ray : one day to one week ; Synchrotron X-ray : 30 min. to 3~4 hrs
In-house X-ray

Goniometer and Phi axis

X-ray tube detector

X-ray data collection
{ Resolution makes difference
2 tan θ D
reflected X-ray tan 2θ = =
1 − tan 2 θ R
D
D 2θ
nλ
incident X-ray θ R d=
θ
θ
origin 2 sin θ
crystal

R = crystal-to-distance distance
D = The distance between spot
and origin (direct beam center)
10.0 Å 3.5 Å 2.0 Å
 The average resolution of macro-
molecular crystals
2~3 Å

Resolution makes difference

There is no question that a model of
phenylalanine (the 6-ring structure) can be
correctly placed into the 1.2 Å data. This still
can be done with confidence in the 2 Å case,
but at 3 Å we already observe a deviation of
the centroid of the ring from the correct model.

Resolution limitation
Upper limit of q = 90o
2d sin θ = n λ
The maximum d = λ/2

In-house X-ray : 1.54 Å (CuKα)

Synchrotron Radiation source : 0.5~2.0 Å
Institute of Biological Chemistry, Academia Sinica
One dataset is not enough
{ The structure factor, F
F (hkl ) = A(hkl ) + iB (hkl )
Amplitude : C

C
α B

A
wavelength : λ

I h1k1l1

∑ F (hkl ) = ∑ A(hkl ) + ∑ iB(hkl )

j j j
I
A(hkl ) = ∑ f i cos 2π (hx j + ky j + lz j )
h0k0l0
fj

B(hkl ) = ∑ f i sin 2π (hx j + ky j + lz j )

j

j
One dataset is not enough
{ Fourier transform
∑ F (hkl ) = ∑ f cos 2π (hx
j j
i j + ky j + lz j ) + ∑ f i sin 2π (hx j + ky j + lz j )
j

ρ ( xyz ) = ∑ ∑ ∑ F (hkl ) exp[− 2πi (hx + ky + lz )]

1
Vc h k l
{ Real measurement : Intensity

I ∝ F2 Phase angle (α) is unknown, α =0~360

Phase problem
Phasing method
{ MR (Molecular Replacement)

Published PDB file

experimental data
from unknown
An artificial unit cell, P1 macromolecular
crystal
An artificial structure factor, F

Rotation and translation search

Phasing method
{ MIR (Multiple Isomorphous Replacement)
The phase

Fp : protien
FH: heavy atom
One heavy atom Two heavy atom
FPH : protein +heavy atom

Need at least two different heavy atom derivates

The variants between crystals are hardly controlled
Phasing method
{ MAD (Multiwavelength Anomalous Dispersion)

Use Se-Met instead of S-Met

Adjust the wavelength to the absorption edge of Se

One kind of heavy atom (One crystal) is enough

Prevent the variants of crystals

I = Itheoretical + system error

Easy to build the structure

Need synchrotron source!!!

Absorption edge for Se-MAD:

0.9797, 0.9795, 0.9801 Å
 Facilities for Structural Genomics
High Throughput Protein X-ray Crystallography Core

5F
5F
4F 2F

4F, 5F, Institute of Biological Chemistry 2F, Institute of Molecular Biology

Synchrotron Radiation Protein Crystallography Facilities

BL17B2, BL13B, BL13C, NSRRC, Taiwan BL12B2, SPring-8, Japan MBC beamline 4.2.2, ALS, USA

Taiwan 3 GeV Synchrotron (2010?)

 Current Status of ALS MBC beamline

Advanced Light Source (ALS), Berkeley, U.S.A

MBC beamline 4.2.2

• Bruker Proteum 30-cm Round Lens-

Coupled CCD System

• BLU-ICE Interface
• Automated Crystal Screening System
 New Synchrotron Protein Beamlines for NRPGM
SR
Vertical Focusing Mirror (13A)

Double Crystal Monochromator (13B)

Toroidal Mirror (13B)

BL13A Collimating Mirror (13B)

Curved Crystal Monochromator (13A)

BL13C Vertical Focusing Mirror (13C)

BL13B

Curved Crystal Monochromator (13C)

Experimental Station
Hard X-ray Source at NSRRC
17
10
2
(Brilliance (ph./s/0.1%BW/mm /mr)

Se K-Edge
2

16 SPring-8 BM
10

NSRRC SM6 ( l =6cm, B=3.2T, N=28)

15
10
NSRRC W20
NSLS BM
14
10
NSRRC BM

13
10

12
10
0 5000 10000 15000 20000 25000 30000

Energy (eV)
 Data Quality from Q-315 (S-SAD)

Atom Peak Heights

Spring-8 BL 45XU NSRRC BL17B2
CYS 30 5.60 12.55
CYS 64 4.97 10.77
Cys 80 CYS 80 4.96 10.35
MET 105 4.64 14.22
Cys 76
Cys 64 CYS 76 4.22 10.76
CYS 94 4.05 11.73
Cys 94 CYS 115 3.81 12.07
CYS 6 3.54 10.15
CYS 127 < 3.00 8.34
CL 201 (1) 5.05 8.09
CL 202 3.21 7.75
Met 105 CL 203 3.43 7.60
CL 204 3.30 11.24
Met 12 CL 205 3.72 7.69
Cys 115 CL 206 3.00 7.41
Cys 30

Anomalous Difference Signal for 15 S atoms

Cys 127 @8 keV ~ 1.4%
Cys 6 @10 keV ~ 0.9%
@12.4 keV ~0.6%
Model building
{ Identify the secondary structure

Cα model Final model

Structural refinement
{ Agreement of Fobservation and Fcalculation

Fo (hkl ) ∝ I I=Itheoretical + error

ρ ( x, y, z ) = ∑∑∑ Fo (hkl ) cos[2π (hx + ky + lz ) − φ (hkl )]

2
V h k l
Compare &
minimize Molecular model, N atoms

iφ j = 2π (hx j + ky j + lz j )
F (hkl )calc = ∑ f i exp(iφ j )
N

fi, atomic scattering factors

j =1
Structural refinement
{ Slow cooling strategy
 Cross-validation : free R

R=
∑ F −F
o c

∑F o

The diffraction data are divided into two sets – a large

working set (typically comprising 90% of the data) and a
complementary test set (comprising the remaining 10%).

The diffraction data in the working set are used in the

normal crystallographic refinement process, whereas the
test set data are not.

The cross-validated free R-value computed using test set

data provides a more objective guide during model building
and refinement process.
Institute of Biological Chemistry, Academia Sinica
Institute of Biological Chemistry, Academia Sinica
 Motions in proteins

N
C

C Blue <30
Green 30~50
Yellow 50~70
N Red >70

Institute of Biological Chemistry, Academia Sinica

Structural validation: PDB!
{ Ramachandran plot
X-ray software
{ Data collection
CrystalClear (Control Software of Rigaku/MSC X-ray machine)
Other control software (Blu-Ice……) at beamline station
{ Data processing
HKL (US$1500), HKL2000 (US$7000)
MOSFLM
DPS (GUI interface of MOSFLM)
D*Trek (Unix/Linux version of CrystalClear, temporary license)
XDS
X-ray software
Phasing Model Building Refinement Validation Others

SOLVE RESOLVE

CNS

XtalView

CCP4 suite

SHELXCDE SHELX

Accelrys

Uppsala software
Some current exciting topics of structural biology

1. Membrane proteins (7)

2. Receptor extracellular domain(s) (4)

3. Macromolecular assembly (4)

4. Biologically important single enzymes (18)

5. Important protein-protein and protein-DNA complexes (26)

Membrane Protein of Known 3D Structure (UCI)

Unique Proteins in Database = 96

Number of Coordinate Files in Database = 177
http://blanco.biomol.uci.edu/membrane_proteins_xtal.html
 1. Membrane proteins
1
Methane monooxygenase
2 + +
A bacterial Na /H antiporter
3 + -
A bacterial homologue of Na /Cl -
4
A bacterial V-type Na+-
(2.8 Å), Nature (3.45 Å), Nature dept transporter (1.65 Å) ATPase (2.1 Å)

A bacterial F-type Na+- MsbA (4.2 Å), a bacterial A rat voltage-dept K+-channel
ATPase (2.4 Å), Science ABC transporter, Science (2.9 Å)

5 6 7
Antibody fragments are useful for crystallization

Essential crystal contacts (white

arrows) of the dimeric yeast
Structural model of the dimeric yeast QCR and QCR–Fv fragment complex are
schematic representation of the Q cycle within a mediated by the Fv fragment
functional unit. (encircled in red).

Hunte, C. FEBS letters, review, 2001.

Crystal Structure of NalP: a β-barrel Membrane Protein

Crystal structure of NalP . (A) Side view of NalP shows a 12-stranded β-barrel (blue
ribbon representation) with a shear number of 14. The hydrophobic membrane-
embedded region is flanked by aromatic residues (yellow ball-and-stick). (B)
Schematic representation of the mixed character of the α-helix and its interactions with
the β-barrel wall with the α-helical residues depicted on a helical wheel. Colour coding:
positively charged residues in blue, negatively charged residues in red, hydrophilic
residues in green and nonpolar residues in orange. (C) Top view of the NalP β-barrel.
Oomen, C. J. et al., EMBO, 2004.
Views of the Kv1.2–β2 subunit complex.

Four subunits of the channel (including the

T1 domain, voltage sensor, and pore) are
colored uniquely. Each subunit of the β
subunit tetramer is colored according to the
channel subunit it contacts. The NADP+
cofactor bound to each β subunit is drawn
as black sticks. TM indicates the integral
membrane component of the complex.
Long, S. B. et al., Science, 2005.
The Complex II Crystal Structures in the Mitochondrial Respiratory Chain

The relative size and shape of mitochondrial Complex II–IV, as well as a schematic of
Complex I, are shown. The transfer of electrons is shown by red lines. Mitochondrial
Complex II couples the Krebs cycle with the electron transfer chain comprised of
Complex I, Complex III (Iwata et al., 1998), and Complex IV (Tsukihara et al., 1996). UQ
indicates ubiquinone, which serves as a mobile carrier to transport electrons from
Complex I or II to Complex III.
Sun, F. et al., Cell, 2005.
 2. Macromolecular assembly
1 A bacterial type III secretion 2 Nitrogenase complex (2.1 Å) (one of
the nucleotide-hydrolyzing proteins),
system (1.8 Å), Nature
Science

3 The IBDV subviral particle

(3.0 Å) 4
The mitochondrial respiratory
complex II (2.4 Å), Cell
VP2 particles and crystal
 3. Biologically significant single enzymes
Drug discovery-related
1SIV gp120 (4.0 Å), Nature
2 Zn-binding domain of HCV
3 Aromatic prenyltransferase (1.6 Å)
(synthesis of natural anti-oxidants, anti-
replicase (2.5 Å), Nature microbial, anti-viral, anti-cancer …),
Nature

4 5 6 7
Human complement Enzyme for biosynthesis of MfpA (2.0 Å), a bacterial Human formylglycine-
components C3 (3.3 Å) and fosfomycin (1.8 Å), a clinically protein that mimics B-form generating enzyme
C3c (2.4 Å), Nature important antibiotic, Nature DNA, Science (1.55 Å), Cell
 Coronavirus particles are
irregularly-shaped, ~60-220nm in
diameter,
with an outer envelope bearing
distinctive, 'club-shaped' peplomers
(~20nm long x 10nm at wide distal
end).

This 'crown-like' appearance (Latin,

corona) gives the family its name.

The centre of the particle appears

Kathryn V. Holmes amorphous in negatively stained
N Engl J Med 348 (20): 1949 EM preps, the nucleocapsid being
May 15, 2003 www.nejm.org in a loosely wound rather
disordered state.
 Dimer structure of SARS 3CLpro
Wild type C145A
product

185-306
Helical Domain
102-184
1-101
Domain II
Domain I

His41 Cys145
Substrate
Catalytic dyad
Dimer structure of SARS 3CLpro with a potent inhibitor
 Report

Acknowledgments

ƒ DR. C.-C. Chou (Assistant Research Specialist, GRC)

ƒ Mr. K.-F. Huang (NTU Ph.D. student)
ƒ Mr. R.-T. Guo (TIGP Ph.D. student)

ƒFor their slides

Academia Sinica