Techniques for Structure Determination

of Proteins-X ray Crystallography

Dr. Aamir Shehzad

Senior Scientist,
Drug Discovery and Structural Biology Lab,
Health Biotechnology Division (HBD),
NIBGE
Structural Biology and Nobel Prizes
2017 in Chemistry was awarded jointly to J.
Dubochet, J. Frank and R. Henderson "for
developing cryo-electron microscopy for the high-
resolution structure determination of biomolecules
in solution."

Sense diverse signals outside the

cell (light, odor, hormone)
800 GPCRs, Targets of 50% drugs
X-ray crystallography of proteins:
Three-dimensional structure
Function

Proteins:
5 nm.
Large complexes (ribosomes 30 nm)
Covalent bonds in proteins is ∼0.15 nm

Why X-rays?
The visible light (wavelengths 400–700 nm)

X-rays (wavelengths 0.01 – 10 nm)

 The first protein structures (myoglobin
and haemoglobin) were solved ∼60
years ago

There are over 181,000 structures determined through X-ray crystallography

Development of molecular biology in the 1980s,

computing power and synchrotron light sources in the 1990s.
Synchrotron
Why crystallize proteins?
A single protein molecule - scattering signal below the detection capabilities

Protein-destroyed before a useful signal is recorded.

Protein crystals -amplify the scattering signal and mitigate the damaging effects
Crystallization:

An ordered precipitation of molecules.

Protein crystallisation - developed as a method

to demonstrate protein purity.

Requirements for protein crystallization:

i) Purity
ii) Monodispersity: Uniform size. Reduce conformational heterogeneity.
iii) Stability: Low flexibility

Affinity chromatography and gel

filtration
Crystallisation: Two-step process
Crystallization of proteins proceeds in two distinct but inseparable steps, nucleation and
growth.

i) Nucleation: Supersaturated solution to microscopic clusters (critical nuclei).

Molecules pass from a wholly disordered state to an ordered one. Internal rearrangement
ultimately yields small, ordered assemblies known as critical nuclei. A critical nucleus is an
ordered cluster of molecules that is of sufficient size (has a surface to volume ratio) such
that it acquires new molecules at a rate greater than that of losing molecules.

ii) Growth: Nuclei into a three-dimensional crystal.

Supersaturation: Some quantity of the

macromolecule in excess of the solubility limit
is present in solution.

Undersaturated solution modified to reduce the

ability of the medium to solubilize the
macromolecule.

The concentration of the precipitating agent is

just a few percent less than that which yields an
amorphous precipitate.
Methods for protein crystallization:

i) Vapor-diffusion

Concentration of the
protein is below its
solubility limit, or
alternatively at its
solubility maximum

Gradually alter
conditions so that the
solubility of the protein
in the sample is
significantly reduced,
thereby rendering the
Hanging-drop vapor-diffusion method solution
supersaturated
Sitting-drop vapor-diffusion method
Microdialysis method
Microdialysis method
Microbatch
Constituents of the reservoir solution/mother liquor (precipitants):
(i) Salts
(ii) Organic solvents
(iii) Long-chain polymers
(iv) Low-molecular-weight polymers and nonvolatile organic compounds (MW < 1000)

Deprive the protein of solvating water and promote protein-protein association

Volume-exclusion effects
(Less solvent available Maintain the
space for the other protein in a
macromolecules). homogeneous
Reduce the Induce separation of state
dielectric of the proteins from solution. (can be
medium. There is a possibility that physiologically or
(Lower dielectric PEG may also act as an biochemically
Dehydrating constant of adhesive intermediary relevant small
proteins medium, lower between protein molecules,
through ability to dissolve molecules. chemical
competition for charged PEG is extremely protectants)
water molecules) hydrophilic and binds
molecules water molecules.
Seeding:
Seeds to produce protein crystals.

Seeds can be obtained from low quality crystals.

i) Macroseeding
ii) Microseeding

Macroseeding
 Crushing

Serial dilution

Doping/Transfer through Cat’s whisker

Equilibration

Microseeding
 1 455 479 649 1048

N P450 FMN FAD C

Crystallisation Screening:
Commercial crystallisation kits

Screens are biased.

Optimisation may be required to obtain

well-diffracting crystals.

Protein concentrations: 5–50 mg/ml.

Heavy precipitates - protein concentration is too high.

Clear drops - protein concentration is too low

Crystallisation Robots:
screening hundreds of conditions using
small amounts of protein, 50–300 nl, per drop.

Two minutes to dispense protein and

crystallisation solution for the entire plate

Initial hits are optimised on a larger scale

Protein–ligand complexes:

i) Co-crystallization
~20-fold molar excess of the ligand to the protein.

ii) Soaking
Crystals contain 50% solvent on average (25 to 90%).
Remaining volume protein

Proteins in crystals are structurally similar to that found in solution.

Diffusion of conventional chemical compounds, which may be ions, ligands, substrates,

coenzymes, inhibitors, or drugs, may be freely diffused into and out of the crystals.
 Soaking of a large crystal of the protein canavalin with the blue dye xylene cyanol

Protein–protein complexes:
Co-expressed and purified.

Pre-purified proteins - mixed and the complex purified by size exclusion chromatography.
Crystals:
Intermediaries in the crystallographic process.

X-ray diffraction patterns as the raw data which allow the direct visualization of the
macromolecules or their complexes
Synchrotron X-ray tube
Radiation damage:
X-rays- ionising radiation.
Synchrotron: 1012 photons per second
Beam cross-sections: 20–50 µm.
Covalent bonds break: destruction of the protein
Disorder within the sample and loss of diffraction strength.
Higher resolution reflections fade quickly and disappear.
Cryocooling:
Dry nitrogen gas at 100K to slow radiation damage
Cryo-cooling diminishes the damage effects resulting from diffusion of certain active
radicals throughout the crystal.
Creates problems in because of ice formation

Cryoprotection:
Cryoprotectants are small molecules such as organic solvents (glycerol, PEG 400 ) that
inhibit the formation of ice in protein crystals
Cryoprotectant replaces water
Cryoprotectants are used in the 20–30% range.
Mounting and centring the sample:
Placed on the goniometer either manually or through a robot
Maintained at a cryogenic temperature
If the crystal is stationary during exposure, only a few reflections are diffracting. More
reflections come into diffraction condition if the crystal is rotated
 Bragg’s Law
William Henry Bragg and William Lawrence Bragg observed that crystals diffracted X-rays in an
ordered manner

The interaction of the incident X-ray beam with electrons.

Constructive interference if the phase shift of waves reflected by different planes was a
multiple of 2π.
Resolution:
The ability to distinguish between neighbouring features in an electron density map.
Reflections towards the centre of the detector: low resolution information.
Reflections towards the perimeter of the detector: high resolution data.
Unit cell:
The smallest volume repeats identically in three dimensions to form the crystal.
Characterised by three distances (Å) a, b, c and three angles α, β, γ (°).
Indexing (iMosflm, XDS and DIALS are excellent for indexing)
Space group:
Unique ways molecules can be arranged symmetrically within a unit cell and still obey the
translational symmetry in all three directions.
If the space group of the crystal cannot be determined correctly, the crystal structure
cannot be solved
65 macromolecular space groups
Asymmetric unit:
The smallest portion of a crystal structure to which symmetry operations can be applied in
order to generate the complete unit cell (the crystal repeating unit).
Proteins can pack with multiple copies within the asymmetric unit.
A complete data set should contain all reflections within the asymmetric unit of the
reciprocal space for the particular symmetry of the crystal.
X-ray detectors record the intensity
Mathematical function that relates the electron density to the diffracted waves is the
Fourier transform.
Three parameters needed for the Fourier transform: wavelength, amplitude and the phase
(the angle at which the wave peaks)
Thus, Fourier synthesis to calculate the electron density map depends upon the structure
factor (amplitude and the phase) of each measured reflection.

Joseph Fourier
Phase solution:

Electron-density map
Model building and refinement:
Model building is completed when the electron density is interpreted to the best of the
user’s knowledge.
Unexpected electron density ‘blobs’.
During refinement, the observed data will be compared to the model.
Refmac, Phenix.refine, Buster, SHELXL, Coot.
Each model deposited at the PDB is an interpretation of its electron density.

Asn95
Trp96

Lys94

Lys97
COOT

Electron-density map
Validation
To ensure that the model makes chemical, physical and biological sense.
Molprobity (http://molprobity.biochem.duke.edu)

All-atom close contact analysis (clash score)

Model geometry (angles etc)
Quality of rotamers, hydrogen binding pattern.
Deposition and publication:
The PDBe validation service (https://validate-rcsb-1.wwpdb.org) runs many deposition
checks before the model is deposited and freely available.
The PDB deposition generates a validation report required for the reviewing stage
in many journals.

The Protein Data Bank (PDB)––the single global

repository of experimentally determined 3D
structures of biological macromolecules and their
complexes––was established in 1971

The X-ray scattering power of the H atom is very

low and, therefore, H atoms are normally omitted
in modeling macromolecular crystal structures