XRD paper

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X-RAY CRYSTALLOGRAPHYRESEARCH PAPER BY

Manatad, Ma. Eda Roselle

Narciso, Karina Sophia

Ngo, Lorvin Lanze

Pilar, Ron Benedict

October 7, 2011

B I O C H E M I S T R Y 1 2 4

Physica l Biochemistry

O n e a m i n o a c i d d o e s n o t a p r o t e i n m a k e - l e t a l o n e a b e i n g ~ P r e s t o n C l o u d

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Table of Contents

X-ray Crystallography! 1Proteins and their Structures! 1X-ray Crystallography! 1

Methodology! 3Protein Crystallization Principle! 3Protein Crystallization Methods! 4Vapor Diffusion! 4Dialysis! 5Batch Experiment! 5Seeding! 6In situ Proteolysis! 6Protein Mounting! 8Instrumentation! 10X-ray Sources! 10Detectors! 10Analysis of Results! 11

Applications! 15Proteins on a Molecular Level! 15Function Confirmation! 15Drug Discovery and Design! 17Mutation and Manipulation Tracking! 18

Bibliography! 20

B i oc he m i s t ry 1 2 4 : P hy s i c a l B i oc he m i s t ry! X - r a y C r y s t a l l o g r ap h y

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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 structuresand the low-energy conformation is known as its native state. With only the amino acid

sequence, the protein can fold in a defined 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 specific and slight changes in its conformation will result in no activity.

! 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 specific

substrate to the enzymes active site. Other conformational changes can also be seen when a protein is subjected to acompletely 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 polarisa-

tion interferometry and Circular Dichroism. However, the major and the most common method that will also be dis-

cussed 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 first turned into well-ordered crystals. These crystals should be very uni-

form and should be large enough to provide a diffraction pattern when ex-posed to X-rays. The crystals act as an amplifier 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 diffrac-

tion can be described using the Bragg model of diffraction.

! From the diffraction pattern, a three-dimensional picture of theelectron 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 canbe found in databases. In order to compare the generated electron density map to a specific protein model, the R-factor

should be used. It is a measure of convergence between the intensities from the specific protein model and the observed

intensities from the electron density map. An R=0 marks a perfect fit and an R greater than 0.5 marks a bad fit.

Figure 2. Example of crystallized protein: Te-

tragonal Lysozyme crystals

B i oc he m i s t ry 1 2 4 : P hy s i c a l B i oc he m i s t ry! X - r a y C r y s t a l l o g r a p h y : M e t h o d o l o g y a n d A n a l y s i s

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Figure 1. 3D Protein Structure

of Myoglobin

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

! The most significant advantage of using X-ray diffraction in elucidating the protein structure is that it revealsthe 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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MethodologyTo determine the structure of proteins using X-ray crystallography, the protein

must first be in its solid state; the protein must be crystallized to sufficiently large

protein crystals, the most difficult step in the analysis.

! The methods of x-ray crystallography for protein structure determinationgenerally involves: (1) isolation and purification of the protein, (2) protein crys-

tallization, (3) crystal mounting, (4) diffraction pattern data collection, and (5)

data processing and analysis.

! The protein must first be extracted and isolated from a source, then, puri-fied. It is recommended that proteins are purified since crystallization would be

difficult 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.

Protein Crystallization Principle

Crystallization process occurs in two steps: nucleation and particle growth. In nu-

cleation, ions, atoms or molecules come together to form a nuclei or particle; the growth

on existing nuclei formed is the particle growth process.

! Crystallization occurs from a supersaturated protein solution. Holding the pH and temperature of the solutionconstant, protein crystallization is affected by precipitant and protein concentration. From the graph (figure above), the

nucleation range is called the labile zone and the particle growth range is the metastable zone. The transition from a sta-ble 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 crystal-

lography. 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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Figure 4. Workflow for Protein

Structure Determination

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

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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. PO 43- and NH4+ are seldom used as a precipitant for

protein.

! Salts change the ionic strength of the solution. The solubility of proteins as a function of ionic strength is shownbelow.

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 whichcauses the charges on the protein surface to interact better with the environment. The salting out range reduces solu-

bility 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 solu-

bility by lowering the dielectric constant of the solvent. PEG is usually used as a precipitant as it has been studied inten-

sively as a good precipitating agent for protein.

! The pH and temperature of a solution affects protein solubility. Generally, solubility is lowest at the isoelectricpoint 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 solu-

tion.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 high-

vacuum 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 sandwich-

ed by the cover on top and the coverslip on the bottom.


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Figure 7. Hanging Drop Method Set-up Figure 8. Sitt ing 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 similarbuffers 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 precipi-

tant 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 sufficient for nucleation is achieved.

Figure 12. Macrobatch experiment

Figure 13. Microbatch experiment


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Seeding

The ideal conditions for nucleation and particle growth differ, and from this, a logical approach is to separate optimiza-

tion 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 mi-

croseeding. 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 macromole-cules or other matter whose inclusion may have altered the crystal surface and caused it to stop growing. Thus, mac-

roseeding involves preparing solutions for nucleation and initial growth, for etching, and for growth to final size. Mi-

croseeding 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 efficiency 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 per-

cent boost in efficiency.


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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 bescreened 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 rea-

gents. Then, the condition that worked for crystallizing the protein should then be optimized to get sufficiently 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 screening and optimization of crystallization conditions. The mainadvantage is the small sample size a crystallization robot can handle reproducibly, but it needs some effort to set it up.


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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 (discussedlater). 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 sufficient cryoprotection for freezing protein crys-

tals. 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, finding the need to use

other cryoprotectants, such as glycerol or glucose. Once suitable cryoprotectant solutions have been identified, the be-

havior 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 sufficientresolution before data can be collected. Protein crystals are mounted on tiny nylon loops (0.05-1.0mm diameter) for dif-

fraction. 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 fished 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 fished out in the drop again and the loop plunged in liquid nitrogen;


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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 first configured and cooled to about 95K. The cryostream cools the protein crystalupon 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 diffrac-

tometer. The goniometer is motorized and moves through a range of 2-theta angle, the 2-theta angle in Braggs law. Priorto complete data collection, there is a need to screen for initial diffraction pattern, and then ascertain the crystal symme-

try, 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 reso-

lution 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 leftis the x-ray beam, the top left is the cryo jet (liq-

uid 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 flat faced cylinder is the detector eherethe 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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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 dif-

fracted 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 ten-billionth of a meter, or 10-10m. The perfect "rulers" to measure angstrom distances are x-rays. The x-rays used in crystal-

lography 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 theelectronsre-leased 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 field (turn the particles) and the electric field (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 tunethe 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 specific 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

flux, others measure the energy, position, and incidence time of each x-ray.Scintillation detectors work by converting x-

rays 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)], sin-

gle 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 flux rather than individual photons. A typical detector consists of a rectangular

gas cell with thin entrance and exit windows. Inside the detector, an electric field 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 cur-

rent amplifier. 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 elec-trons are attracted to the positive anode wire. Near the anode wire, the electrons are accelerated by the high electric field,

producing a cascade of electrons that result in a large electrical pulse, coupled to a low-noise preamplifier 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 con-

nects observed scattering with reflections 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 dif-

fraction pattern is an image of the reciprocal lattice, the reflection, 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 reflection

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 find 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 metalsand are located in their respective diffraction patterns and in the unit cell. Molecular replacement superimposes a knownB i oc he m i s t ry 1 2 4 : P hy s i c a l B i oc he m i s t ry! X - r a y C r y s t a l l o g r a p h y : M e t h o d o l o g y a n d A n a l y s i s

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homologous structure onto the diffraction data for the unknown structure. Lastly multiple wavelength anomalous dis-

persion (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 pro-

ducing the protein.

! Once an X-ray diffraction pattern has been transformed into an electron density map, fitting of models can nowbe done. The different quality parameters which can be associated with the models are resolution, refinement, b-factors

and model geometry. Resolution is related to the diffraction grating calculated in the diffraction pattern. A higher resolu-

tion of the data means higher resolution of the electron density maps, which in turn means higher accuracy of the posi-

tions of the atoms in the structure. This is illustrated in the following figure:

! Refinement is the measure of agreement between the crystallographic model and the original X-ray diffractiondata. The crystallographer calculates from the model the expected intensity of each reflection in the diffraction pattern,

and then compares these calculated "data" with the experimental data, which consist of measured positions and intensi-

ties. The R-factor is used to assess the progress of structure refinement, and the final 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 reflection in the diffraction pattern,and each |Fcalc| is the intensity of the same reflection 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 R-

factor greater than 0.5 implies that agreement between observed and calculated intensities is very poor, and many mod-

els 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 refinement methods. A desirable target R-factor for a

protein model refined with data to 2.5 is 0.2. Very rarely, small, well-ordered proteins may refine to R = 0.1, whereas


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

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small organic molecules commonly refine 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 vibratesaround the position specified in the model. Atoms at side-chain termini are expected to exhibit more freedom of move-

ment than main-chain atoms, and this movement amounts to spreading each atom over a small region of space. Diffrac-

tion 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 refinement. From the temperature factors com-

puted during refinement, 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 fit the data precisely.

! If the temperature factor Bj is purely a measure of thermal motion at atom j, then in the simplest case of purelyharmonic 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 isused to find problems in models during structure refinement. The conformational angles plotted are , the torsional

angle of the N-CA bond, defined by the atoms C-N-CA-C (C is the carbonyl carbon); and , the torsional angle of the

CA-C bond, defined by the atoms N-CA-C-N. In this figure, = = 180 (convergent stereo).

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

due. Each residue in the protein is represented with a dot or other mark on the plot.

! During the final stages of map fitting and crystallographic refinement, Ramachandran diagrams are a great aidin finding conformationally unrealistic regions of the model. Structure publications often include the diagram, with anexplanation 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 26. Molecular structure detailing phi and psi

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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 col-

ored by type: blue = positive, red = negative, yellow = polar, gray = nonpolar. Note that, in this very well-refined model, only glycines lie outside of allowed re-

gions (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 ge-

nomics and proteomics will lead to the discovery of structures and new fold, better understanding of specific diseases,

and, and new knowledge of representatives from each major protein family. For example, by understanding the struc-

tures of the macromolecules, one will be able to investigate the macromolecular machinery that controls vesicular trans-

port 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 Confirmation

The physiological role or functions of many macromolecules can be reconfirmed by using the data obtained from the x-

ray diffraction patterns produced from the protein crystals. In 2010, Ding, et. al., discovered a novel adaptor protein in-

volved 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 sig-

nal 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 pro-

B i oc he m i s t ry 1 2 4 : P hy s i c a l B i oc he m i s t ry! X - r a y C r y s t a l l o g r ap h y : A p p l i c a t i o n s

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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 beseen. 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 do-

main of PDCD10 represents a new fold. An eight-residue flexible 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 confirming the molecules intra-

cellular 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 con-

firms that fact that the gene could interact with the kinase and stabilize them to promote Golgi assembly and cell orienta-

tion. 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 flexible linker

are labeled and colored in orange, cyan, and gray, respectively. The hinge (Lys69-Lys70) is also labeled. (C) Two N-terminal domain firmly 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


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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 adhe-

sion 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 benefit from x-ray crystallography data because proteins are the mole-

cules 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 inflammation; thrombin, in thrombosis; renin, in hypertension; iNOS, for inflammation;

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 HumanImmunodeficiency 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 replica-

tion. 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 activ-

ity were able to bind to the N-terminal domain of CA (designated as CAN) and inhibit capsid assembly during viralmaturation.

! 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 con-

formation of Phe32 switch has a critical role in the normal capsid assembly. However, the affinity for CAP-1 to CAN is

significantly below the levels needed for therapeutic use. In relation to drug development, efforts are made to supply

inhibitors with improved affinities in relation to providing modifications that would improved the fit to the binding site.For example, the carbonyl oxygen of Ala31 is located within the hydrophobic cavity. Modifications 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 modifications. (add in-

strument 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 stud-

ied by means of x-ray crystallographic and biochemical analysis. The authors inactivated the psbTc gene by inactivatedby inserting a chloramphenicol-resistant cassette. The mutated PSII was extracted from the thylakoid membrane of the


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oxygenic organisms, purified 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 confirmed 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 representedby 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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B i oc he m i s t ry 1 2 4 : P hy s i c a l B i oc he m i s t ry! X - r a y C r y s t a l l o g r a p h y : R e f e r e n c e s
https://docs.google.com/viewer?a=v&q=cache:Kz9iDja8GWoJ:cat.middlebury.edu/~chem/chemistry/class/anal/ch311/PowerPointTalks/ProteinCrystallography.ppt+protein+x+ray+crystallography&hl=en&gl=ph&pid=bl&srcid=ADGEEShZmI6gMmZyFFeJCobfRfqsKnbr4hKmla-cjkU7K-OhuJkAHwphY_iU3ELjBxNt42VO41UGEo1z8af1goTc1VVui9iY3CO0tXFaaVlfYvODIMhTFR_qamKx_7zayPhulXgrQZ_5&sig=AHIEtbQERo9SIS6yv8oaLIhuXjDAxIhb1ghttps://docs.google.com/viewer?a=v&q=cache:Kz9iDja8GWoJ:cat.middlebury.edu/~chem/chemistry/class/anal/ch311/PowerPointTalks/ProteinCrystallography.ppt+protein+x+ray+crystallography&hl=en&gl=ph&pid=bl&srcid=ADGEEShZmI6gMmZyFFeJCobfRfqsKnbr4hKmla-cjkU7K-OhuJkAHwphY_iU3ELjBxNt42VO41UGEo1z8af1goTc1VVui9iY3CO0tXFaaVlfYvODIMhTFR_qamKx_7zayPhulXgrQZ_5&sig=AHIEtbQERo9SIS6yv8oaLIhuXjDAxIhb1ghttps://docs.google.com/viewer?a=v&q=cache:Kz9iDja8GWoJ:cat.middlebury.edu/~chem/chemistry/class/anal/ch311/PowerPointTalks/ProteinCrystallography.ppt+protein+x+ray+crystallography&hl=en&gl=ph&pid=bl&srcid=ADGEEShZmI6gMmZyFFeJCobfRfqsKnbr4hKmla-cjkU7K-OhuJkAHwphY_iU3ELjBxNt42VO41UGEo1z8af1goTc1VVui9iY3CO0tXFaaVlfYvODIMhTFR_qamKx_7zayPhulXgrQZ_5&sig=AHIEtbQERo9SIS6yv8oaLIhuXjDAxIhb1ghttp://www.proteincrystallography.org/http://www.bio.davidson.edu/Courses/Molbio/MolStudents/spring2003/Kogoy/protein.htmlhttp://www.bio.davidson.edu/Courses/Molbio/MolStudents/spring2003/Kogoy/protein.htmlhttp://www.lanl.gov/orgs/tt/pdf/techs/diffraction.pdfhttp://www.lanl.gov/orgs/tt/pdf/techs/diffraction.pdfhttp://www.lanl.gov/orgs/tt/pdf/techs/diffraction.pdfhttp://www.jic.ac.uk/staff/david-lawson/xtallog/summary.htmhttp://www.jic.ac.uk/staff/david-lawson/xtallog/summary.htmhttp://www.jic.ac.uk/staff/david-lawson/xtallog/summary.htmhttp://www.proteinstructures.com/Experimental/Experimental/electron-density.htmlhttp://www.proteinstructures.com/Experimental/Experimental/electron-density.htmlhttp://www.proteinstructures.com/Experimental/Experimental/electron-density.htmlhttp://pldserver1.biochem.queensu.ca/~rlc/work/teaching/definitions.shtmlhttp://pldserver1.biochem.queensu.ca/~rlc/work/teaching/definitions.shtmlhttp://spdbv.vital-it.ch/TheMolecularLevel/ModQual/http://spdbv.vital-it.ch/TheMolecularLevel/ModQual/http://www-structmed.cimr.cam.ac.uk/Course/Overview/Overview.htmlhttp://www-structmed.cimr.cam.ac.uk/Course/Overview/Overview.htmlhttp://www-structmed.cimr.cam.ac.uk/Course/Overview/Overview.htmlhttp://www-structmed.cimr.cam.ac.uk/Course/Overview/Overview.htmlhttps://docs.google.com/viewer?a=v&q=cache:Kz9iDja8GWoJ:cat.middlebury.edu/~chem/chemistry/class/anal/ch311/PowerPointTalks/ProteinCrystallography.ppt+protein+x+ray+crystallography&hl=en&gl=ph&pid=bl&srcid=ADGEEShZmI6gMmZyFFeJCobfRfqsKnbr4hKmla-cjkU7K-OhuJkAHwphY_iU3ELjBxNt42VO41UGEo1z8af1goTc1VVui9iY3CO0tXFaaVlfYvODIMhTFR_qamKx_7zayPhulXgrQZ_5&sig=AHIEtbQERo9SIS6yv8oaLIhuXjDAxIhb1ghttps://docs.google.com/viewer?a=v&q=cache:Kz9iDja8GWoJ:cat.middlebury.edu/~chem/chemistry/class/anal/ch311/PowerPointTalks/ProteinCrystallography.ppt+protein+x+ray+crystallography&hl=en&gl=ph&pid=bl&srcid=ADGEEShZmI6gMmZyFFeJCobfRfqsKnbr4hKmla-cjkU7K-OhuJkAHwphY_iU3ELjBxNt42VO41UGEo1z8af1goTc1VVui9iY3CO0tXFaaVlfYvODIMhTFR_qamKx_7zayPhulXgrQZ_5&sig=AHIEtbQERo9SIS6yv8oaLIhuXjDAxIhb1ghttps://docs.google.com/viewer?a=v&q=cache:Kz9iDja8GWoJ:cat.middlebury.edu/~chem/chemistry/class/anal/ch311/PowerPointTalks/ProteinCrystallography.ppt+protein+x+ray+crystallography&hl=en&gl=ph&pid=bl&srcid=ADGEEShZmI6gMmZyFFeJCobfRfqsKnbr4hKmla-cjkU7K-OhuJkAHwphY_iU3ELjBxNt42VO41UGEo1z8af1goTc1VVui9iY3CO0tXFaaVlfYvODIMhTFR_qamKx_7zayPhulXgrQZ_5&sig=AHIEtbQERo9SIS6yv8oaLIhuXjDAxIhb1ghttps://docs.google.com/viewer?a=v&q=cache:Kz9iDja8GWoJ:cat.middlebury.edu/~chem/chemistry/class/anal/ch311/PowerPointTalks/ProteinCrystallography.ppt+protein+x+ray+crystallography&hl=en&gl=ph&pid=bl&srcid=ADGEEShZmI6gMmZyFFeJCobfRfqsKnbr4hKmla-cjkU7K-OhuJkAHwphY_iU3ELjBxNt42VO41UGEo1z8af1goTc1VVui9iY3CO0tXFaaVlfYvODIMhTFR_qamKx_7zayPhulXgrQZ_5&sig=AHIEtbQERo9SIS6yv8oaLIhuXjDAxIhb1ghttps://docs.google.com/viewer?a=v&q=cache:Kz9iDja8GWoJ:cat.middlebury.edu/~chem/chemistry/class/anal/ch311/PowerPointTalks/ProteinCrystallography.ppt+protein+x+ray+crystallography&hl=en&gl=ph&pid=bl&srcid=ADGEEShZmI6gMmZyFFeJCobfRfqsKnbr4hKmla-cjkU7K-OhuJkAHwphY_iU3ELjBxNt42VO41UGEo1z8af1goTc1VVui9iY3CO0tXFaaVlfYvODIMhTFR_qamKx_7zayPhulXgrQZ_5&sig=AHIEtbQERo9SIS6yv8oaLIhuXjDAxIhb1ghttps://docs.google.com/viewer?a=v&q=cache:Kz9iDja8GWoJ:cat.middlebury.edu/~chem/chemistry/class/anal/ch311/PowerPointTalks/ProteinCrystallography.ppt+protein+x+ray+crystallography&hl=en&gl=ph&pid=bl&srcid=ADGEEShZmI6gMmZyFFeJCobfRfqsKnbr4hKmla-cjkU7K-OhuJkAHwphY_iU3ELjBxNt42VO41UGEo1z8af1goTc1VVui9iY3CO0tXFaaVlfYvODIMhTFR_qamKx_7zayPhulXgrQZ_5&sig=AHIEtbQERo9SIS6yv8oaLIhuXjDAxIhb1ghttps://docs.google.com/viewer?a=v&q=cache:Kz9iDja8GWoJ:cat.middlebury.edu/~chem/chemistry/class/anal/ch311/PowerPointTalks/ProteinCrystallography.ppt+protein+x+ray+crystallography&hl=en&gl=ph&pid=bl&srcid=ADGEEShZmI6gMmZyFFeJCobfRfqsKnbr4hKmla-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ttp://www.jic.ac.uk/staff/david-lawson/xtallog/summary.htmhttp://www.jic.ac.uk/staff/david-lawson/xtallog/summary.htmhttp://www.jic.ac.uk/staff/david-lawson/xtallog/summary.htmhttp://www.jic.ac.uk/staff/david-lawson/xtallog/summary.htmhttp://www.proteinstructures.com/Experimental/Experimental/electron-density.htmlhttp://www.proteinstructures.com/Experimental/Experimental/electron-density.htmlhttp://www.proteinstructures.com/Experimental/Experimental/electron-density.htmlhttp://www.proteinstructures.com/Experimental/Experimental/electron-density.htmlhttp://pldserver1.biochem.queensu.ca/~rlc/work/teaching/definitions.shtmlhttp://pldserver1.biochem.queensu.ca/~rlc/work/teaching/definitions.shtmlhttp://pldserver1.biochem.queensu.ca/~rlc/work/teaching/definitions.shtmlhttp://pldserver1.biochem.queensu.ca/~rlc/work/teaching/definitions.shtml

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XRD paper