XRD

X-ray Crystallography
Manatad, Narciso, Ngo, Pilar October 7, 2011 Biochemistry 124; Physical Biochemistry
Friday, October 7, 2011
Proteins
Biochemical compounds made up of polypeptides Fold into unique 3D structure
Low energy conformation: native Amino acid sequence: sufcient for the protein to nd specic pathway Molecular chaperones
Protein Structure
Not rigid - can shift between related structures
Binding of substrate in enzymes Subject to different environment
Important in bioactivity: highly specic reactions Slight changes or denaturation in the

conformation will result in no activity
Protein Structure Determination
Higher level structures - provide info about the

protein functions XRD
Experimental methods: NMR, Circular Dichroism, XRD

Major and most common method in determining protein structures Requires protein crystals
Protein Crystals
Well-ordered, uniform, and
highly pure
Large enough to provide

diffraction pattern
Crystals - signal amplier:
ability to arrange huge number of molecules in the same orientation (raise signal to a measurable level)
4 major steps in protein structure
determination
Step 1: Grow perfect crystals (Protein crystallization) Step 2: Collect x-ray diffraction pattern (XRD) Step 3: Covert to diffraction pattern to electron density map Step 4: Convert electron density map to 3D molecular structure
Diffraction Pattern Electron Density Map Protein Structure Fitted Protein Structure
Advantages and Disadvantages
Advantage
Reveals 3D position of all atoms in the protein molecule Does not require additional info like AA sequence
Disadvantage
Needs well-ordered crystals
Highly puried Crystal size: large
Workow of X-ray Crystallography

(1) Isolation and purication of the protein (2) Protein crystallization (3) Crystal mounting (4) Diffraction pattern data collection (5) Data processing and analysis
Isolation and Purication

Proteins must be better than 95% pure to
produce a crystal
Purity evaluated through SDS-PAGE, isoelectric
focusing, mass spectrometry, and other methods
Crystallization
Nucleation
Ions, atoms, or molecules come together to form a nuclei or particle
Particle Growth
Growth on existing nuclei formed
General Concentration of Protein vs. Concentration of Precipitant Phase Diagram

Organic - ethanol, methanol, propanol, MPD (2methyl-2,4-pentanediol), acetonitrile (polyethyleneglycol)
Precipitants
Organic polymer such as PEGs

Reduce protein solubility by lowering the dielectric constant of the solvent
Salts
The ability of a salt to precipitate proteins is generally described by the Hofmeister series PO43- > HPO42- = SO42- > citrate > acetate > Cl- > Br- > NO3- . ClO4- > SCN- (for anions) NH4+ > K+ > Na+ > Li+ (for ca@ons)
Crystallization Methods
Vapor diffusion Dialysis Batch experiments Seeding In situ proteolysis
Vapor Diffusion
Hanging Drop
Sitting Drop
Sandwich Drop
Macrobatch Experiment
Microbatch Experiment Dialysis

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
Crystallization conditions vary from pH, Screening for crystallization condition
Screening kit
concentration of precipitants, salts, buffers, temperature, and protein concentration
Optimization
pH, concentration of salts, precipitants, and proteins, temperature are varied
X-ray crystallographic done at low temperature

(at about 100K)
Minimize degradation of the crystal by free radicals generated by the x-ray beam, especially in intense synchotron x-ray sources
Cryoprotectants
Prevent crystals from cracking when frozen 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
Typical Cryoprotectants and Concentrations Required
Cryostream system is X-ray source is
congured and cooled to about 95K energized: increasing the voltage to 50kV and current to 100mA goniometer (angle measuring device) head of the diffractometer
Loop placed on the
Need to screen for initial diffraction

Ascertain the crystal symmetry, the unit cell parameters, the crystal orientation and the resolution limit
Method
Rotate the crystal through a small angle, typically 1 degree, and record the x-ray diffraction pattern If the diffraction pattern is very crowded, the rotation angle is reduced so tht each spot can be resolved on the image Repeated until the crystal has moved through at least 30 or to 180 depending on the crystal symmetry; the lower the symmetry, the more data are required.
A typical resolution data

Up to 3 days using an ordinary x-ray source
Synchotron x-ray sources (x-ray intensity is greater)

Data collection times are shorter (can be as fast as 10 mins)
Every spots on each image are measured using

computer softwares
Nitrogenase data sets contain around 300 images

with over 5000 spots per image
Instrumentation
X-ray
0.5 to 1.5 long Generated by an x-ray tube
Vacuum tube that uses high voltage to accelerate the electrons released by cathode to high velocity High velocity electons collide with a metal target, the anode, producing x-rays Most commonly used - emitted by copper (characteristic wavelength for the radiation is 1.5418)
Synchotron x-ray
X-ray
Synchotron - type of cycle particle accelerator the magnetic eld (turn the particles) and the electric eld (accelerate the particles) are synchronized with the travelling particle beam, usually electrons Convert high energy electron energy to other forms of electromagnetic radiation, usually used in x-ray crystallography Tune the wavelength, set the beam sizes, gives a higher intensity Increases resolution and reduces time it takes to obtain results
Many kinds of x-ray detectors

Count single photons
Detectors
Provides measurements of count rate or total ux Measure the energy, position, and incidence time of each xray
Scintillation detectors converts x-rays to optical

photons and detecting the light with a photomultiplier tube or a photodiode
CCD (Charge Coupled Device) detectors -- a thin

phosphor screen converts the incident x-rays into optical photons, which the CCD detects
Detectors
Gas ionization detectors -- as integrating detectors Gas proportional detectors
Consist of a small-diameter anode wire in an enclosed gas volume 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
to measure beam ux rather than individual photons
- Begin as spherulite - Extremely thin needles clustered around a single nucleation site - Appearfuzzy - Single needles - Nucleation rate is too high which is why they are too many and too thin - Extremely thin needles growing from a single nucleation center
- Plate (2D) grow from a single nucleation site and overlay each other, which is far from optimal - 3D crystal - Fully formed - Not essentially useful unless the diffraction is checked
After Your Crystal

Diffraction of protein crystal Heavy atom derivative of protein X-ray diffraction of derivative Fourier Transform of X-ray diffraction &
derivative
Create electron density models

The Phase Problem

Phase - position of a waves maximum relative to
an origin
To solve the phase angle:

Multiple isomorphous replacement Molecular wavelength anomalous dispersion (MAD) phasing
Limitations (Aside from Phase Problem)
Crystallizing Protein
Fragile Requires a crystal with shortest side 0.2mm
Flaws of Crystallization
Disorder in Unit Cell Vibrations of molecules Distortion in Crystallization
Solve with cryogenic freezing (-196C) to

increase resolution and lower vibration
Real Lattice and Reciprocal Lattice
Real Lattice - the regular spacing of the origins of

the individual unit cells
Reciprocal Lattice
Reections that result from diffraction The relationships of size of the reciprocal lattice are inversely related to those of the real lattice Large unit cells result in a very closely spaced reciprocal and small unit cells result in a reciprocal lattice with large intervals
Resolution
Resolution gives the size of the smallest molecule
you can see or resolve lattice
Is the minimum interplanar spacing of the real Braggs law
Different Resolutions of Residue Trp147 of Bacilus subtilis ferrochelatase

Convergence: The R-Factor
Where F is related to the intensity the reection it describes
r 0.6 0.5 0.4 0.2 0.05 0 Very bad Bad Recoverable Good for protein Good for small organic molecules Perfect t
Temperature-factor or Debye-Waller factor

where Ui2 is the mean square displacement of atom i. This produces a weigh@ng factor on the contribu@on of atom i to the Fourier transform by:
B-factor
Indicates the true static or dynamic mobility of an

atom
Indicates where there are error in model building

Ramachandran Plot
Visualize backbone dihedral angles against of
amino acid residues in protein structure modest repulsion (blue, outer polygons)
No steric repulsion (yellow, inner polygons) or Residues are colored by type:

blue = positive red = negative yellow = polar gray = non-polar
Ramachandran diagram for cytochrome b5 (PDB 3b5c). Small squares represent glycine residues; small crosses represent all others
Main Application
Crystallographic structural studies Best applied in biochemistry and medicine

Function conrmation, diagnostics, drug design
Function conrmation
Understand structures/function
How is it folded What are exposed for interaction Impact of binding and manipulation
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 (2010)
PDCD10, Novel Adaptor Protein
Functions
Involved in human cerebral cavernous malformation Proliferative, apoptopic Found in Golgi apparatus to complex with germinal center kinase III
Structural investigation?
Integrated dimer:
Domains: N & C-terminal 8-residue exible linker provide mobility to C-terminal domain
Basic cleft binds to phosphatidylinositide and regulated localization of molecule
Two potential sites on the two domains for recruiting binding partners
Drug Discovery and Design

Proteins virtually carry out all biological process Drugs/medicine
Correct abnormalities that cause the diseases Screen for binding Effectiveness, stability Enhance by optimizing ligand binding and identifying binding modes
Drug Discovery and Design

High-resolution atomic structures of proteins
More structural information Increased awareness of target sites
of the StructureCAP-1 Antiviral Assembly HIV-1 Inhibitor Complex with the CA Protein (2007)
Inammation: p38 kinase, iNOS Thrombosis: Thrombin Hypertension: Renin Cancer: EGF receptor tyrosine kinase
CAP-1 with HIV-1 CA Protein (2007)
Structure of the antiviral assembly inhibitor

replication
CAP-1 complex with HIV-1 CA (capsid) protein
Important role in early and late phase in viral Antiviral activity

Bind to N terminal domain of CA Inhibit capsid assembly
CAP-1 binds, Phe32 displaced, Enter aromatic ring of CAP-1

Urea to Val59, Dimethylammonium group with Glu28 and Glu29
Modications to t binding site by enabling more Hbonding Ala31,Val59, Gly61, His62
Diagnostics
Changes in structure or functional groups Mutations X-ray Crystallographic and Biochemical
Characterizations of a Mutant Photosystem II Complex from Thermosynechococcus vulcanus with the psbTc gene inactivated by an insertion mutation (2008)
Inactivated psbTc gene of PSII

A mutant photosystem II (PSII) of a T. vulvanus
whose psbTc gene was inactivated
Insertion of a chloramphenicol-resistant casette Results

Highly isomorphous with wild type crystals psbTc transmembrane helix was still present C-terminal loop was lost
Maintain stability of the PSII dimer and activity of oxygen evolution
Disappearance of electron density of the C-terminal loop

XRD

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XRD

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X-ray Crystallography

Friday, October 7, 2011

Friday, October 7, 2011

Important in bioactivity: highly specic reactions Slight changes or denaturation in the

Friday, October 7, 2011

Protein Structure Determination

Higher level structures - provide info about the

Experimental methods: NMR, Circular Dichroism, XRD

Friday, October 7, 2011

Large enough to provide

Crystals - signal amplier:

Friday, October 7, 2011

Friday, October 7, 2011

Friday, October 7, 2011

Advantages and Disadvantages

Friday, October 7, 2011

Highly puried Crystal size: large

Workow of X-ray Crystallography

Friday, October 7, 2011

Isolation and Purication

Purity evaluated through SDS-PAGE, isoelectric

focusing, mass spectrometry, and other methods

Friday, October 7, 2011

Friday, October 7, 2011

General Concentration of Protein vs. Concentration of Precipitant Phase Diagram

Organic - ethanol, methanol, propanol, MPD (2methyl-2,4-pentanediol), acetonitrile (polyethyleneglycol)

Organic polymer such as PEGs

Microbatch Experiment Dialysis

concentration of precipitants, salts, buffers, temperature, and protein concentration

Friday, October 7, 2011

Friday, October 7, 2011

X-ray crystallographic done at low temperature

Friday, October 7, 2011

Typical Cryoprotectants and Concentrations Required

Friday, October 7, 2011

Friday, October 7, 2011

Cryostream system is X-ray source is

Loop placed on the

Friday, October 7, 2011

Need to screen for initial diffraction

Friday, October 7, 2011

Friday, October 7, 2011

A typical resolution data

Synchotron x-ray sources (x-ray intensity is greater)

Every spots on each image are measured using

Nitrogenase data sets contain around 300 images

Friday, October 7, 2011

Friday, October 7, 2011

Many kinds of x-ray detectors

Scintillation detectors converts x-rays to optical

CCD (Charge Coupled Device) detectors -- a thin

to measure beam ux rather than individual photons

Friday, October 7, 2011

After Your Crystal

Create electron density models

The Phase Problem

To solve the phase angle:

Friday, October 7, 2011

Limitations (Aside from Phase Problem)

Solve with cryogenic freezing (-196C) to

Real Lattice and Reciprocal Lattice

Real Lattice - the regular spacing of the origins of

Friday, October 7, 2011