B

Physical Biochemistry

X-RAY CRYSTALLOGRAPHY
RESEARCH PAPER BY

Manatad, Ma. Eda Roselle Narciso, Karina Sophia Ngo, Lorvin Lanze Pilar, Ron Benedict October 7, 2011

One amino acid does not a protein make - let alone a being ~ Preston Cloud

Table of Contents

X-ray Crystallography! Proteins and their Structures! X-ray Crystallography! Methodology! Protein Crystallization Principle! Protein Crystallization Methods! Vapor Diffusion! Dialysis! Batch Experiment! Seeding! In situ Proteolysis! Protein Mounting! Instrumentation! X-ray Sources! Detectors! Analysis of Results! Applications! Proteins on a Molecular Level! Function Conrmation! Drug Discovery and Design! Mutation and Manipulation Tracking! Bibliography!

1 1 1 3 3 4 4 5 5 6 6 8 10 10 10 11 15 15 15 17 18 20

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X-ray Crystallography

X-ray Crystallography
Proteins and their Structures Proteins are biochemical compounds that are made up of multiple polypeptide units. These polypeptide units are composed of linked amino acids bonded together by peptide bonds between the adjacent carboxyl and amino groups. ! The majority of these proteins fold into their unique three dimensional structures

and the low-energy conformation is known as its native state. With only the amino acid sequence, the protein can fold in a dened pathway to its native conformation although some proteins require molecular chaperones to assists in their folding. The structure of the protein is important in carrying out biochemical functions inside the biological system because it is highly specic and slight changes in its conformation will result in no activity. !
Figure 1. 3D Protein Structure of Myoglobin

Proteins do not have a rigid structure. Some of them may shift between different related structures while per-

forming their functions. These conformational changes can be seen mostly in enzymes wherein the binding of a specic substrate to the enzymes active site. Other conformational changes can also be seen when a protein is subjected to a completely different environment where it can undergo denaturation. ! In studying proteins, the higher level structures provide important data on how proteins perform their func-

tions. There are several experimental methods in determining protein structures like NMR spectroscopy, Dual polarisation interferometry and Circular Dichroism. However, the major and the most common method that will also be discussed in this paper is X-ray Crystallography which uses X-ray Diffraction. X-ray Crystallography In order to study protein structures using X-ray diffraction, the proteins must be rst turned into well-ordered crystals. These crystals should be very uniform and should be large enough to provide a diffraction pattern when exposed to X-rays. The crystals act as an amplier due to its ability to arrange huge numbers of molecules in the same orientation so that scattered waves add up in phase and raise the signal to a measurable level. The X-ray diffraction can be described using the Bragg model of diffraction. !
Figure 2. Example of crystallized protein: Tetragonal Lysozyme crystals

From the diffraction pattern, a three-dimensional picture of the

electron density can be produced using several computational methods that uses the Fourier Transform equation. ! The electron density map can provide the relative or mean posi-

tions of the atoms in the crystal and can be used in comparing the sample protein to other protein structures which can be found in databases. In order to compare the generated electron density map to a specic protein model, the R-factor should be used. It is a measure of convergence between the intensities from the specic protein model and the observed intensities from the electron density map. An R=0 marks a perfect t and an R greater than 0.5 marks a bad t.

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X-ray Crystallography: Methodology and Analysis

Figure 3 (Left to Right). (A) Electron density map; (B) Specic protein structure; (C) Fitted protein structure in the electron density map

The most signicant advantage of using X-ray diffraction in elucidating the protein structure is that it reveals

the three-dimensional position of all the atoms in the protein molecule. This structure is determined without using any additional information like the sequence, cofactor structure, etc. ! However, the major disadvantage of using this method is that it requires perfect crystals. This means that mole-

cules that cannot be converted into well-ordered crystals cannot be analysed using this method.

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X-ray Crystallography: Methodology and Analysis

Methodology
To determine the structure of proteins using X-ray crystallography, the protein must rst be in its solid state; the protein must be crystallized to sufciently large protein crystals, the most difcult step in the analysis. ! The methods of x-ray crystallography for protein structure determination

generally involves: (1) isolation and purication of the protein, (2) protein crystallization, (3) crystal mounting, (4) diffraction pattern data collection, and (5) data processing and analysis. ! The protein must rst be extracted and isolated from a source, then, puri-

ed. It is recommended that proteins are puried since crystallization would be difcult if contaminating proteins or macromolecules are present in the sample as it affects the homogeneity of the crystal formed, affecting diffraction patterns. In most cases, proteins must be better than 95% pure to produce a crystal. Purity can be evaluated through SDS-PAGE, isoelectric focusing, mass spectrometry, and other methods.
Figure 4. Workow for Protein Structure Determination

Protein Crystallization Principle Crystallization process occurs in two steps: nucleation and particle growth. In nucleation, ions, atoms or molecules come together to form a nuclei or particle; the growth

on existing nuclei formed is the particle growth process.

Figure 5. General concentration of protein vs. Concentration of precipitant phase diagram

Crystallization occurs from a supersaturated protein solution. Holding the pH and temperature of the solution

constant, protein crystallization is affected by precipitant and protein concentration. From the graph (gure above), the nucleation range is called the labile zone and the particle growth range is the metastable zone. The transition from a stable solution to a supersaturated solution can be achieved by increasing the concentration of either the protein of interest or the precipitant. If the solution is maintained in the labile zone for too long, then there will be rapid growth of too many small crystals, which is not ideal for protein crystallization since large protein crystals are needed for X-ray crystallography. The protein-precipitant solution must approach the nucleation zone very slowly so that the developing nuclei have enough time to grow.

To crystallize proteins, precipitants are used. The precipitant used must not denature the protein of interest.

Commonly used precipitants include salts, organic polymers, alcohols, and occasionally pure water. The ability of a salt to precipitate proteins is generally described by the Hofmeister series:
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PO43- > HPO42- = SO42- > citrate > acetate > Cl- > Br- > NO3- > ClO4- > SCN- (for anions) NH4+ > K+ > Na+ > Li+ (for cations) !
+

PO43- does not exist in the solution for a typical pH range for protein to crystallize, it exist as HPO42- and H2PO4-.

NH4 can lose H+ at high pH 8, which make the pH highly unstable. PO43- and NH4+ are seldom used as a precipitant for protein. ! below. Salts change the ionic strength of the solution. The solubility of proteins as a function of ionic strength is shown

Figure 6. Dependence of Solubility (S) on Ionic Strength by the Salt (I)

Solubility increases in the salting in range due to elevation of the dielectric constant of the solvent which

causes the charges on the protein surface to interact better with the environment. The salting out range reduces solubility as the charges of the precipitant compete for water molecules with the charges on the protein surface, in a way that it effectively lowers the overall hydration of the protein. Organic precipitants such as ethanol, methanol, propanol, MPD (2-methyl-2,4-pentanediol) or acetonitrile, and organic polymer such as PEGs (polyethyleneglycol) reduce protein solubility by lowering the dielectric constant of the solvent. PEG is usually used as a precipitant as it has been studied intensively as a good precipitating agent for protein. ! The pH and temperature of a solution affects protein solubility. Generally, solubility is lowest at the isoelectric

point of the protein since the protein carries a net charge of zero. Solubility of proteins in salt solutions tends to increase at low temperature; in PED or MPD solutions, protein solubility generally decreases with decreasing temperature. These can be accounted for the different interactions within the solutions and the free energy of the protein-precipitant solution. Protein Crystallization Methods Methods of protein crystallization include vapor diffusion, dialysis, batch experiments, seeding, and in situ proteolysis. Vapor Diffusion Vapor diffusion, which suits protein crystallization, can be the hanging drop, sitting drop or sandwich drop method. The hanging drop differs from the sitting drop method in the orientation of the protein solution drop but both require a closed system -- the system must be sealed off from the outside environment using an air-tight container or a highvacuum grease between glass surfaces. The drop lies on top of the cover (drop-side down) in the hanging drop method whereas the drop sits beside the well in the sitting drop method. The drop in the sandwich drop method is sandwiched by the cover on top and the coverslip on the bottom.
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Figure 7. Hanging Drop Method Set-up

Figure 8. Sitting Drop Method Set-up

Figure 9. Sandwich Drop Method Set-up

The drop contains the protein, buffers and the precipitant solution, while the well solution contains similar

buffers and precipitants in higher concentrations. The difference in precipitant concentrations between the drop and the well solution is the driving force that causes water to evaporate from the drop until the concentration of the precipitant in the drop equals that of the well solution. Through this, the drop becomes supersaturated for protein and the precipitant concentration in the drop increases to a level optimal for crystallization to occur. The volume of the well solution is way much greater than the drop, so dilution of the well solution by water vapor from the drop is negligible. When the system is in equilibrium, the optimum conditions are maintained until crystallization is complete, which takes days. Dialysis Dialysis utilizes diffusion and equilibration of small precipitant molecules through a semi-permeable membrane and slowly approach the concentration at which the protein crystallizes. Dialysis is uniquely suited to crystallizations at low ionic strength, and a convenient method for crystallization in the presence of volatile reagents such as alcohols.

Figure 10. Dialysis schematic Figure 11. Dialysis set-up

Batch Experiment In batch crystallization methods, all components are combined into a single solution, which is then left undisturbed. This simple method works well for hen egg white lysozyme, catalase, and cytochrome C554. In microbatch methods, a small (2-10 l) droplet containing both protein and precipitant is immersed in an inert oil to prevent droplet evaporation. The success of batch crystallization experiment requires that a supersaturation level sufcient for nucleation is achieved.

Figure 13. Microbatch experiment

Figure 12. Macrobatch experiment

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X-ray Crystallography: Methodology and Analysis

Seeding The ideal conditions for nucleation and particle growth differ, and from this, a logical approach is to separate optimization of these processes. This can be accomplished by seeding, a technique where crystals are transferred from nucleation conditions to those that will support only particle growth. There are two basic seeding methods: macroseeding and microseeding. In macroscopic seeding, a crystal is transferred from the mother liquor where nucleation and initial growth occur to a less supersaturated solution for continued growth. Prior to transfer, the crystal is placed in an unsaturated solution to etch its surface, a process called etching. This partial dissolution procedure removes misoriented macromolecules or other matter whose inclusion may have altered the crystal surface and caused it to stop growing. Thus, macroseeding involves preparing solutions for nucleation and initial growth, for etching, and for growth to nal size. Microseeding involves transferring nuclei to the growth medium. Crystal growth by microseeding can involve preparing stock seed solutions, which when added to the growth solution will produce only few large crystals. Seed solutions can be prepared by crushing crystals, then testing a set of serially diluted solutions to see which gives the desired number of crystals.

Figure 14. Macroseeding and Microseeding Set-up

In situ Proteolysis Due to the structures of some proteins having less tidy molecular structures than others, in which disordered amino acid chains dangle off the protein like split ends, a new crystallization method was developed, called In situ proteolysis. In order to boost the efciency of the crystallization process, Joachimiak and his colleagues at the MCSG (Midwest Center for Structural Genomics) and SGC (Structural Genomics Consortium) inserted a protease (chymotrypsin or trypsin) a certain type of enzyme that breaks down the bonds that connect a protein's amino acid. The protease preferentially bound to the proteins at the disordered regions, snipping off the loose ends. They then applied the usual crystallization procedure as discussed above. By using proteases to digest part of the protein sample, the scientists achieved a six percent boost in efciency.

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X-ray Crystallography: Methodology and Analysis

Figure 15. Ribbon representation of NE2398, a protein from the Nitrosomonas europaea bacterium. Dotted lines represent the parts of the protein digested with protease. Blue molecules represent other molecules in the crystal lattice. (Credit: Image courtesy of DOE/Argonne National Laboratory)

Crystallization conditions for different proteins vary from pH, concentration of precipitants, salts, buffers, tem-

perature, and protein concentration. A crystallization tray is needed if preliminary crystallization condition is to be screened for a protein. It typically contains 24 wells, cover slips, and a greasing agent to seal the wells. The protein is then subjected to widely varying pH, salts, and precipitants to screen initial crystallization conditions; this process is called sparse matrix method. Fortunately, screening kits are now available, allowing for easier screening of conditions for possible crystallization. An example of screening kit is the Hampton Research Crystal Screen, which contains 50 reagents. Then, the condition that worked for crystallizing the protein should then be optimized to get sufciently larger and more homogenous crystals, in which pH, concentration of salts, precipitants, and proteins, temperature are varied.

Figure 16. Crystallization tray for protein crystallization condition screening

! ! Robots are commonly used for automatic screeningand optimization of crystallization conditions. The main

advantage is the small sample size a crystallization robot can handle reproducibly, but it needs some effort to set it up.

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X-ray Crystallography: Methodology and Analysis

Figure 17. A Crystallization Robot for Automatic Screening and Optimization

Protein Mounting Most x-ray crystallographic data collection is done at low temperature (at about 100K), in order to minimize degradation of the crystal by free radicals generated by the x-ray beam, especially in intense synchrotron x-ray sources. To prevent crystals from cracking when frozen, cryoprotectants are used. In the presence of cryoprotectant, the protein and its thin layer of surrounding solution will form an amorphous glass in which the crystal suffers minimal damage, but retains maximum x-ray diffraction properties.
Table 1 Typical Cryoprotectants and Concentrations Required

Screening for suitable cryoprotectant for the protein crystals are needed prior to crystal mounting (discussed

later). The minimum amount of cryoprotectant required can be determined by pipetting 10 L drops of solution into liquid nitrogen. If the drops reliably freeze clear, then the solution has sufcient cryoprotection for freezing protein crystals. The choice of cryoprotectant will depend upon the crystallization solution composition; if protein crystallization conditions already contain a cryoprotectant (e g. PEG 400), it is often ideal to simply increase the concentration to the appropriate value. However, PEG has limited solubility for high salt concentration solutions, nding the need to use other cryoprotectants, such as glycerol or glucose. Once suitable cryoprotectant solutions have been identied, the behavior of protein crystals in these solutions should be observed which is often carried out at the same time as crystal mounting. The crystal should not disintegrate, or crack during cryo-soaking. ! It is necessary to mount a crystal in the x-ray beam of a diffractometer and determine if it diffracts to sufcient

resolution before data can be collected. Protein crystals are mounted on tiny nylon loops (0.05-1.0mm diameter) for diffraction. The loops are mounted on hollow rods that are mounted on magnetic caps that are conveniently stored under liquid nitrogen, of which the caps are easily placed on the goniometer head of the x-ray diffractometer. The loop size should be just slightly larger than the crystals. The crystal is shed out in the cover slip using the loop. The crystal is then soaked to cryoprotectant solution drop placed on spot plate. The crystal is then observed under a microscope to check for cracking or disintegration. The crystal is then shed out in the drop again and the loop plunged in liquid nitrogen;

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X-ray Crystallography: Methodology and Analysis

then check again for cracks or splits in the crystal. The crystal cap is then placed in cryovial and the cryovial immersed in liquid nitrogen prior to placing the cap in the diffractometer.

Figure 18. Mounting loops and cryovials

Figure 19. Mounting loops on Magnetic caps

Figure 20. Protein Crystal Mounted on a Tiny Loop

! ! The cryostream system is rst congured and cooled to about 95K. The cryostream cools the protein crystal

upon subjecting it to x-ray beam. The x-ray source is then energized: increasing the voltage to 50kV and current to 100mA. The loop is then placed on the goniometer (which literally means angle-measuring device) head of the diffractometer. The goniometer is motorized and moves through a range of 2-theta angle, the 2-theta angle in Braggs law. Prior to complete data collection, there is a need to screen for initial diffraction pattern, and then ascertain the crystal symmetry, the unit cell parameters, the crystal orientation and the resolution limit. Knowing this, a data collection strategy is derived to maximize both the resolution and completeness of the data set. The method is to rotate the crystal through a small angle, typically 1 degree, and record the x-ray diffraction pattern. If the diffraction pattern is very crowded, then the rotation angle should be reduced so that each spot can be resolved on the image. This is repeated until the crystal has moved through at least 30 degrees or to 180 degrees depending on the crystal symmetry; the lower the symmetry, the more data are required. A typical resolution data set may take up to 3 days using an ordinary x-ray source. For high resolution data collection, synchrotron x-ray sources are used where x-ray intensity is greater and therefore data collection times are shorter (can be as fast as 10mins). Having a complete data for diffraction pattern, every spots on each image are measured using computer softwares. Nitrogenase data sets contain around 300 images with over 5000 spots per image, so there is a need to conduct data analysis with computers

Figure 21. The crystal mounted on the loop placed in the diffractometer. The object on the left is the x-ray beam, the top left is the cryo jet (liquid nitrogen stream), the right is the beam stop, and bottom is where the crystal is mounted.

Figure 22. Another view of the crystal mounted on the loop

Figure 23. An x-ray crystallography instrument. The large at faced cylinder is the detector ehere the diffracted x-rays will be measured. When data is being collected, the detector swings around to be directly next to the crystal and x-ray beam

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X-ray Crystallography: Methodology and Analysis

Instrumentation

Figure 24. X-ray Crystallography set-up

The general process in x-ray crystallography is that the x-ray beam hits the protein crystal, where the beam will be diffracted on a detector, and a diffraction pattern will be obtained, which is correlated to the proteins structure. X-ray Sources In x-ray crystallography, the distances between atoms is measured in angstroms (). One angstrom equals one tenbillionth of a meter, or 10-10m. The perfect "rulers" to measure angstrom distances are x-rays. The x-rays used in crystallography are approximately 0.5 to 1.5 angstroms long, which is just the right size to measure the distance between atoms in a molecule. X-rays are generated by an x-ray tube, a vacuum tube that uses high voltage to accelerate theelectronsreleased by cathodeto high velocity. The high velocity electrons collide with a metal target, the anode, producing x-rays. The x-ray radiation most commonly used is that emitted by copper, whose characteristic wavelength for the radiation is 1.5418. ! Synchrotron x-ray sources are used nowadays in crystallography. Asynchrotronis a type of cyclic particle ac-

celerator wherein the magnetic eld (turn the particles) and the electric eld (accelerate the particles) are synchronized with the travelling particle beam, usually electrons. It is used to convert high energy electron energy to other form of electromagnetic radiation, usually used in X-ray crystallography. Using synchrotron in x-ray diffraction studies can tune the wavelength used, set the beam sizes, give a higher intensity of X-ray beams that it increases resolution and reduces time it takes to obtain results. Synchrotron X-ray diffraction can differentiate between oxidation states of an element, determine crystal structures from microcrystals, and study cation occupancies in materials where more than one element occupies or partially occupies a specic site in a sample/compound (zeolite chemistry). Detectors There are many kinds of x-ray detectors, some count single photons, some provides measurements of count rate or total ux, others measure the energy, position, and incidence time of each x-ray.Scintillation detectors work by converting xrays to optical photons in special materials and then detecting the light with a photomultiplier tube or a photodiode. The scintillator materials can be either organic scintillators, single crystals of thallium-activated sodium iodide [NaI(Tl)], single crystals of sodium-activated cesium iodide [CsI(Na)], or single crystals of bismuth germanate (BGO). CCD (charge
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coupled device) detectors are now used in a variety of ways for x-ray imaging. In most systems, a thin phosphor screen converts the incident x-rays into optical photons, which the CCD detects. Gas ionization detectors are commonly used as integrating detectors to measure beam ux rather than individual photons. A typical detector consists of a rectangular gas cell with thin entrance and exit windows. Inside the detector, an electric eld of about 100 V/cm is applied across two parallel plates. The electrons and ions are collected at the plates, and the current is measured with a low-noise current amplier. Gas proportional detectors consist of a small-diameter anode wire in an enclosed gas volume. They are usually used to count single photon events. When a photon interacts in the gas, some gas atoms are ionized, and the electrons are attracted to the positive anode wire. Near the anode wire, the electrons are accelerated by the high electric eld, producing a cascade of electrons that result in a large electrical pulse, coupled to a low-noise preamplier to give usable pulses giving the counts.

Table 2 Properties of common x-ray detectors

Analysis of Results With the protein now crystallized, X-ray diffraction can begin. The basis of diffraction is Braggs law. Braggs law connects observed scattering with reections from evenly spaced planes within the crystal. This law is commonly expressed as ; where d is the distance between the planes, is the angle of scattering and is the wavelength of the X-rays. A diffraction pattern is an image of the reciprocal lattice, the reection, of the crystal. A reciprocal lattice has vectors which are perpendicular to the real lattice plane, the lattice of the crystal, and the size of the unit cells found in the real lattice are inversely related to those of the reciprocal lattice. Therefore large unit cells are closely spaced in a reciprocal lattice and small unit cells result in a reciprocal lattice with large spaces. Every atom in a unit cell contributes to every reection in a diffraction pattern. For the diffraction pattern to be Fourier transformed into an electron density model, the phase angle of the electron density map must be found. To nd the phase angle several methods can be used. The primary method for determinig initial phases is the multiple isomorphous replacement; the crystals are soaked into heavy metals and are located in their respective diffraction patterns and in the unit cell. Molecular replacement superimposes a known
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homologous structure onto the diffraction data for the unknown structure. Lastly multiple wavelength anomalous dispersion (MAD) phasing can be used; MAD phasing is a new method for deriving initial phases by measuring diffraction data at several different wavelengths near the absorption edge of a heavy-atom. The anomalous signal that results from this can give very accurate phases. This is commonly done by replacing methionine with seleno- methionine when producing the protein. ! Once an X-ray diffraction pattern has been transformed into an electron density map, tting of models can now

be done. The different quality parameters which can be associated with the models are resolution, renement, b-factors and model geometry. Resolution is related to the diffraction grating calculated in the diffraction pattern. A higher resolution of the data means higher resolution of the electron density maps, which in turn means higher accuracy of the positions of the atoms in the structure. This is illustrated in the following gure:

Figure 25. Different resolutions of trp137 residue of Bacilus subtilis ferrochelatase

Renement is the measure of agreement between the crystallographic model and the original X-ray diffraction

data. The crystallographer calculates from the model the expected intensity of each reection in the diffraction pattern, and then compares these calculated "data" with the experimental data, which consist of measured positions and intensities. The R-factor is used to assess the progress of structure renement, and the nal R-factor is one measure of model quality. The R-factor is calculated as follows:

In this expression, each |Fobs| is derived from the measured intensity of a reection in the diffraction pattern,

and each |Fcalc| is the intensity of the same reection calculated from the current model. Values of R range from zero to about 0.6, the R-factor obtained when a set of measured intensities is compared with a set of random intensities. An Rfactor greater than 0.5 implies that agreement between observed and calculated intensities is very poor, and many models with R = 0.5 or greater will not respond to attempts at improvement unless more data are available. An early model with R near 0.4 is promising, and is likely to improve with various renement methods. A desirable target R-factor for a protein model rened with data to 2.5 is 0.2. Very rarely, small, well-ordered proteins may rene to R = 0.1, whereas

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small organic molecules commonly rene to better than R = 0.05. When R approaches about 0.15, it sometimes becomes possible to discern hydrogen atoms in electron-density maps. ! The B-factor or temperature factor can be thought of as a measure of how much an atom oscillates or vibrates

around the position specied in the model. Atoms at side-chain termini are expected to exhibit more freedom of movement than main-chain atoms, and this movement amounts to spreading each atom over a small region of space. Diffraction is affected by this variation in atomic position, so it is realistic to assign a temperature factor to each atom and to include the factor among parameters to optimize during least-squares renement. From the temperature factors computed during renement, we learn which atoms in the molecule have the most freedom of movement, and we gain some insight into the dynamics of our largely static model. In addition, adding the effects of motion to our model makes it more realistic and hence more likely to t the data precisely. ! If the temperature factor Bj is purely a measure of thermal motion at atom j, then in the simplest case of purely

harmonic thermal motion of equal magnitude in all directions, Bj is related to the magnitude of vibration as follows:

where Uj2 is the mean-square displacement of the atom from its rest position. ! Finally the Ramachandran plot shows the main-chain conformational angles in a polypeptide. This diagram is

used to nd problems in models during structure renement. The conformational angles plotted are , the torsional angle of the N-CA bond, dened by the atoms C-N-CA-C (C is the carbonyl carbon); and , the torsional angle of the CA-C bond, dened by the atoms N-CA-C-N. In this gure, = = 180 (convergent stereo).

Figure 26. Molecular structure detailing phi and psi

The pair of angles phi and psi of a single residue is greatly restricted by steric repulsion. The allowed pairs of

values are depicted on a Ramachandran diagram as irregular polygons that enclose backbone conformational angles that do not give steric repulsion (yellow, inner polygons) or give only modest repulsion (blue, outer polygons). Every point (phi, psi) on the diagram represents the conformational angles phi and psi on either side of the alpha carbon of one residue. Each residue in the protein is represented with a dot or other mark on the plot. ! During the nal stages of map tting and crystallographic renement, Ramachandran diagrams are a great aid

in nding conformationally unrealistic regions of the model. Structure publications often include the diagram, with an explanation of any residues that lie in high-energy ("forbidden") areas. Glycines, because they lack a side chain, usually account for most of the residues that lie outside allowed regions. If nonglycine residues exhibit forbidden conformational angles, there should be some explanation, such as structural constraints that overcome the energetic cost of an unusual backbone conformation.!

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Figure 27. Ramachandran diagram for cytochrome b5 (PDB 3b5c). Small squares represent glycine residues; small crosses represent all others. Residues are colored by type: blue = positive, red = negative, yellow = polar, gray = nonpolar. Note that, in this very well-rened model, only glycines lie outside of allowed regions (blue polygons).

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Applications
Proteins on a Molecular Level The main application for protein x-ray crystallography is for crystallographic structural studies. This technique aims to produce well-ordered protein crystals that are pure and large enough to diffract x-ray beams. ! X-ray crystallography is best applied in the world of biochemistry and medicine because it can be used in diag-

nostics and drug design and discovery because the three-dimensional (3D) structures of protein can be determined. The structure of the protein - how it is folded, which amino acids and their substituents are exposed and available for contact, the presence of cavities - is of great importance because it can clue us in the function of the said protein. Structural genomics and proteomics will lead to the discovery of structures and new fold, better understanding of specic diseases, and, and new knowledge of representatives from each major protein family. For example, by understanding the structures of the macromolecules, one will be able to investigate the macromolecular machinery that controls vesicular transport or the mechanisms of the carrier vesicle formation, cargo loading, accurate delivery, neurotransmitter release, and membrane recycling. This can be done by monitoring the impact of binding or substitution of different compounds on the biological molecules.

Figure 28. The possible information that may be obtained from knowing the 3D structure of a macromolecule

Function Conrmation The physiological role or functions of many macromolecules can be reconrmed by using the data obtained from the xray diffraction patterns produced from the protein crystals. In 2010, Ding, et. al., discovered a novel adaptor protein involved in human cerebral cavernous malformation. The Programmed Cell Death 10 (PDCD10) protein was previously found to have proliferative and apoptotic function. In another study, it was shown that PDCD10 is localized to the Golgi apparatus, forming a complex with proteins of the germinal cancer kinase III (GCKIII) family, which are involved in signal pathways of cell orientation and migration. Before this study, no structural investigation of the function of the gene has been done. In this study, the protein was was crystallized by means of the hanging-drop diffusion method along with the complexation of inositol-(1,3,4,5)-tetrakisphosphate (41P) to improve the diffraction patterns of the crystals proB i o c h e m i s t r y 1 2 4 : P h y s i c a l B i o c h e m i s t r y! X-ray Crystallography: Applications

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duced. The diffraction data was collected using an ADSC Quantum-270 charge-coupled device (CCD) detector. MOSFLM and SCALA from the CC-4 program were used to process, reduce, and scale the diffraction data. The structure was determined by the MAD method using SOLVE. ! Results showed that the molecule is an integrated dimer that involved a unique assembly that has yet to be

seen. Each PDCP10 monomer contains an N-terminal domain with a new fold involved in the tight dimer assembly and a C-terminal four-helix bundle domain. Further research using the Dali database showed that no other protein has been sought with a single or dimer N-terminal domain as with PDCD10. Thus, it can be concluded that the N-terminal domain of PDCD10 represents a new fold. An eight-residue exible linker connects the N-terminal and C-terminal domain that provides mobility to the C-terminal domain. This results in the conformational variability of PDCD10. A basic cleft on the top of the dimer interface binds to phosphatidylinositide and regulates the intracellular localization of PDCD10. This helps localize and stabilize its interaction with membrane-associated proteins, thus conrming the molecules intracellular localization and functional role. Two potential sites located on the two domains are critical for recruiting binding partners. A binding site (SITE I) of PDCD10 for GCKIII kinase has been localized on the N-terminal domain. This conrms that fact that the gene could interact with the kinase and stabilize them to promote Golgi assembly and cell orientation. A second binding site (SITE II) was found to potentially mediate paxillin binding in PDCD10 and could implicate PDCD10 in the cell adhesion.

Figure 29. (Upper) Structural characteristics and unique dimeric assembly of PDCD10. (A) Overall structure of PDCD10 complexed with 4IP. The two monomers of PDCD10 are colored in green and yellow, respectively. The 4IP molecule is shown as a ball-and-stick model. The disordered loop in monomer B is shown as a broken line. (B) The structural architecture of the PDCD10 monomer. The N-terminal domain, the C-terminal domain, and the exible linker are labeled and colored in orange, cyan, and gray, respectively. The hinge (Lys69-Lys70) is also labeled. (C) Two N-terminal domain rmly inter-clasp with each other to form a compact six-helix bundle, mediating a unique dimeric assembly. (Lower) Phosphatidylinositide binding. (A) A basic cleft on the top of the dimer interface contains a 4IP molecule. The 4IP molecule is represented as a sphere model. (B) Binding of the 4IP molecule. The residues interact with B i o c h e m i s t r y 1 2 4 : P h y s i c a l B i o c h e m i s t r y! X-ray Crystallography: Applications

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the 4IP molecule through a extended hydrogen-bond network. The 4IP molecule is shown as a ball-and-stick model. The hydrogen bonds are shown as broken lines. (Ding, Wang, Li, Hu, Zhang, & Wang, 2010)

Figure 30. Potential sites for recruiting different binding patterns. (A) Electrostatic potential analysis of PDCD10. Two hydrophobic patches (Site I and Site II), respectively located on the N- and C-terminal domain, for potential protein-protein interactions are boxed. (B) Homo-interactions between two Site I induce a compact packing in the 4-fold direction. The PDCD10 dimer and 4-fold-related symmetric dimer are colored in pink and cyan, respectively. The residues forming Site I are labeled and shown as stick models. (C) Superimposing the C-terminal domain of PDCD10 with the FAT domains of focal adhesion kinases indicates the conservation of Site II. The hydrophobic residues forming Site II and the basic residues surrounding Site II are labeled and shown as stick models. (Ding, Wang, Li, Hu, Zhang, & Wang, 2010)

Drug Discovery and Design The drug discovery and design process would benet from x-ray crystallography data because proteins are the molecules that carry out virtually all biological processes. Most drugs function by correcting the abnormalities of the proteins that causes diseases. X-ray crystallography enables the screening of potential pharmaceutical materials for binding to proteins or macromolecules. The effectiveness and suitability of the drug may be enhanced by optimizing ligand binding and identifying binding modes. Having knowledge and access to high-resolution atomic structures of a protein increases our awareness of different target sites that we can manipulate and the possible ways we can design a drug candidate in accordance to the proteins structure. Because of x-ray crystallography, more structural information is known regarding p38 kinase, which plays a role in inammation; thrombin, in thrombosis; renin, in hypertension; iNOS, for inammation; and EGF receptor tyrosine kinase, for cancer. ! In the study of Kelly, et. al., the structure of the antiviral assembly inhibitor CAP-1 complex with the Human
X-ray Crystallography: Applications

Immunodeciency Virus type 1 (HIV-1) CA protein was determined by X-ray crystallography and NMR spectroscopy
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(2007). The CA domain of the HIV-1 Gag polyprotein has an important role in the early and late phases of viral replication. Thus, this site is a possible antiviral target. During the early phase, the CA is a 231 amino acid domain within the 55 kDa Gag precursor polyprotein. During the late phase, the CA domain helps the in formation of 4000 copies of Gag into an immature virus cell. The processing of the domain by the viral protease forces the CA in conformational changes that promote its assemble in the capsid of the protein shell that is composed of 1500 CA molecules that encloses two copies of the viral genome and viral enzymes that are essential for its infectivity. It was found that compounds with antiviral activity were able to bind to the N-terminal domain of CA (designated as CAN) and inhibit capsid assembly during viral maturation. ! CAP-1, N-(3-chloro-4-methylphenyl)-N-{2-[({5-[(dimethylamino)-methyl]-2-furyl}-methyl)-sulfanyl]ethyl}-urea),

is the antiviral assembly inhibitor. When CAP-1 binds to the protein, the PHe32 is displaced from a buried position to open a deep hydrophobic cavity that serves as the ligand binding site. This makes it possible for the aromatic ring of CAP-1 to insert itself into the cavity with its urea groups forming hydrogen bonds with the backbone oxygen of Val59, and the dimethylammonium group of the inhibitor interacting with the side chains of Glu28 and Glu29. The change in the placement of Phe32 due to the binding CAP-1 is facilitated with a mid-chain conformation. This implies that the conformation of Phe32 switch has a critical role in the normal capsid assembly. However, the afnity for CAP-1 to CAN is signicantly below the levels needed for therapeutic use. In relation to drug development, efforts are made to supply inhibitors with improved afnities in relation to providing modications that would improved the t to the binding site. For example, the carbonyl oxygen of Ala31 is located within the hydrophobic cavity. Modications that enable hydrogen bonding with the buried oxygen atom would increase binding. Also, the backbone oxygen atom of Val59, and NH groups of Gly61 and His62 are located at the mouth of the cavity, thus, are available for binding modications. (add instrument used)

Figure 31. Structural changes induced in CAN when crystallized in the presence of CAP-1. (a) Ribbon diagram of CAN crystallized in presence (darker colors) or absence of CAP-1. Phe32 is shown explicitly. N and C termini, secondary structural elements, and the cyclophilin A binding site are labeled. (b) Close up stereo view of the structural changes in the presence of CAP-1. (c) Surface representation of CA crystallized in the absence (left) and presence (right) of CAP-1. Phe32 is shown explicitly in the open and closed conformations. (Kelly, et al., 2007)

Mutation and Manipulation Tracking Mutations in the biomolecule may also be determined by means of x-ray crystallographic studies. In a study by Henmi and his collegues last 2008, a mutant photosystem II (PSII) of a T. vulcanus whose psbTc gene was inactivated was studied by means of x-ray crystallographic and biochemical analysis. The authors inactivated the psbTc gene by inactivated by inserting a chloramphenicol-resistant cassette. The mutated PSII was extracted from the thylakoid membrane of the
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oxygenic organisms, puried using an anion exchange column, and crystallized using the hanging drop method. The crystals were subjected to x-ray beams and the diffraction data was processed with HKL-2000 and the electron density maps were calculated. The crystals were found to be highly isomorphous with the wild type crystals, meaning that they have the same space group and unit-cell dimensions and the types and the positions of atoms except in the replacement in one structure. The electron density maps showed that the psbTc transmembrane helix was still present in the mutant,
yet the C-terminal loop of the gene was lost. Subsequent biochemical analysis conrmed that the C-terminal loop functions to maintain the stability of the PSII dimer and the activity of oxygen evolution.

Figure 32. Electron density map of the mutant PSII crystal (a) and a difference fourier map of wild-type-minus-mutant PSII (b) around the transmembrane helix and its C-terminal loop of PsbTc. The electron density map (a) at a resolution of 3.8 Armstrong is represented by 2.0sigma. The difference fourier map (b) is computed at a resolution of 6.0 armstrong and displayed at 2.0 sigma, where positive signls are represented by black mesh and negative signals are represented by a light-gray mesh. Psbtc is depicted by cartoon with side chain, and other subunits are depicted as ribbons. (Henmi, Iwai, Ikeuchi, Kawakami, Shen, & Kamiya, 2008)

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Bibliography
Campbell, Roberts. Protein Crystallography Lecture De:initions. Queens University. Retrieved from: http://pldserver1.biochem.queensu.ca/~rlc/work/tea ching/de:initions.shtml. October 6, 2011. Ding, J., Wang, X., Li, D.-F., Hu, Y., Zhang, Y., & Wang, D.-C. (2010). Crystal structure of human programmed cell death 10 complexed with inositol-(1,3,4,5)- tetrakisphosphate: A novel adaptor protein involved in human cerebral cavernous malformation. Biochemical and Biophysical Research Communications , 399, 587- 592. Henmi, T., Iwai, M., Ikeuchi, M., Kawakami, K., Shen, J.-R., & Kamiya, N. (2008). X-ray crystallographic and biochemical character- izations of a mutant photosys- tem II complex from Thermosynechococcus vulcanus with the psbTc gene inactivated by an insertion muta- tion. Journal of Synchotron Radiation , 304-307. Karadaghi, Salam Al. Electron, density,resolution, pro- tein crystallography. 2011. Retrieved from: http://www.proteinstructures.com/Experimental/Exp erimental/electron-density.html. October 6, 2011. Kelly, B., Kyere, S., Kinde, I., Tang, C., Howard, B., Rob- inson, H., et al. (2007). Structure of the Antiviral As- sembly Inhibitor CAP-1 Complex with the HIV-1 CA Protein. Jounal of Molecular Biology , 373, 355-366. Lawson, D. (n.d.). A Brief Introduction to Protein Crys- tallography. Retrieved September 10, 2011, from John Innes Centre (JIC): http://www.jic.ac.uk/staff/david-lawson/xtallog/sum mary.htm Licensable Technolgies. (2006, December). Powder Diffraction X-ray Crystallography. Retrieved October 2, 2011, from LANL: http://www.lanl.gov/orgs/tt/pdf/techs/diffraction.pd f Protein Crystallization. (2003). Retrieved September 10, 2011, from http://www.bio.davidson.edu/Courses/Molbio/MolS tudents/spring2003/Kogoy/protein.html d=bl&srcid=ADGEEShZmI6gMmZyFFeJCobfRfqsKnbr4 hKmla-cjkU7K-OhuJkAHwphY_iU3ELjBxNt42VO41UGE o1z8af1goTc1VVui9iY3CO0tXFaaVlfYvODIMhTFR_qam Kx_7zayPhulXgrQZ_5&sig=AHIEtbQERo9SIS6yv8oaLIh uXjDAxIhb1g. October 6,2011. Read, R. J. (2008, April 22). Overview of macromolecu- lar X-ray crystallography. Retrieved September 10, 2011, from Structural Medicine: http://www-structmed.cimr.cam.ac.uk/Course/Overvi ew/Overview.html Rhodes, Gales. Quality of Macromolecular Models. January 1, 2000. Retrieved from: http://spdbv.vital-it.ch/TheMolecularLevel/ModQual/ . October 6,2011.

Protein Crystallography. (2006). Retrieved September 10, 2011, from Protein Crystallography: http://www.proteincrystallography.org/ Protein X-ray Crystallography. Retrieved from: https://docs.google.com/viewer?a=v&q=cache:Kz9iDj a8GWoJ:cat.middlebury.edu/~chem/chemistry/class/ anal/ch311/PowerPointTalks/ProteinCrystallography. ppt+protein+x+ray+crystallography&hl=en&gl=ph&pi
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