X-ray crystallography is a powerful technique for visualizing the structure of
It is a tool used for identifying the atomic and molecular structure of a
In crystallography the crystalline atoms cause a beam of incident X-rays to
diffract into many specific directions.
Then crystallographer can produce a three-dimensional picture of the density
of electrons within the crystal.
From this electron density, the mean positions of the atoms in the crystal can
X-ray crystallography can locate every atom in a zeolite, an aluminosilicate.
3. X-Ray Crystallography
What is X-Ray Crystallography?
– A form of very high resolution microscopy.
– Enables us to visualize protein structures at the atomic level
– Enhances our understanding of protein function.
What is the principle behind X-Ray Crystallography?
– It is based on the fact that X-rays are diffracted by crystals
5. Steps in Structure Determination
1. Protein purification.
2. Protein crystallization.
3. Data collection.
4. Structure Solution (Phasing)
5. Structure determination (Model building and refinement)
6. Step1:Protein Purification
What is Protein Purification?
is a series of processes intended to isolate one or a few proteins from a complex
mixture, usually cells, tissues or whole organisms.
Why Protein Purification?
Characterization of the function.
Interactions of the protein.
minimum of 5 to 10 milligrams pure soluble
protein are required with better than 95% purity
7. Step2:Protein crystallization
X-ray scattering from a single unit would be unimaginably weak.
A crystal arranges a huge number of molecules in the same orientation.
Scattered waves add up in phase and increase Signal to a level which can be
This is often the rate-limiting step in straightforward structure determinations,
especially for membrane proteins
The source of the X-rays is often a synchrotron.
The typical size for a crystal for data collection may be 0.3 x 0.3 x 0.1 mm.
The crystals are bombarded with X-rays which are scattered from the planes
of the crystal lattice.
The scattered X-rays are captured as a diffraction pattern on a detector such
as film or an electronic device.
Rotate crystal through 1 degree and Record XRD pattern
If XRD pattern is very crowded, reduce the degree of rotation
Repeat until 30 degrees were obtained
Sometimes 180 degrees depending on crystal symmetry
Lower the symmetry= More data are required
For high resolution, use Synchrotron
14. Step4:Structure Solution (Phasing)
Methods for solving the phase problem
Molecular Replacement (MR)
Multiple/Single Isomorphous replacement (MIR/SIR)
Multiple/Single wavelength Anomalous Diffraction(MAD/SAD)
Principle using Fourier Transform (FT) :
FT of the diffraction data gives us a representation of the contents of the crystal.
15. Step5: Structure determination (Fitting):
Fitting of protein sequence in the electron density.
Electron density – Not self explanatory
Can be automated, if resolution is close to 2Å or better.
What can be interpreted is largely defined by resolution.
18. Process of resolution of molecular and
crystal structures by X-ray diffraction
For crystals composed of large molecules, such as
proteins and enzymes, the phase problem can be solved
successfully with three main methods, depending of the
(i) introducing atoms in the structure with high
scattering power. This methodology, known as MIR
(Multiple Isomorphous Replacement) is therefore based
on the Patterson method.
(ii) introducing atoms that scatter X-rays anomalously,
also known as MAD (Multiwavelength Anomalous
(iii) by means of the method known as MR (Molecular
Replacement), which uses the previously known
structure of a similar protein.
19. Applications of X-ray Crystallography
Scientists also determined the X-ray crystallographic structure of HIV protease, a viral
enzyme critical in HIV’s life cycle, in 1989.
Pharmaceutical scientists hoped that by blocking this enzyme, they could prevent the
virus from spreading in the body.
By feeding the structural information into a computer modeling program, they could use
the model structure as a reference to determine the types of molecules that might block
To create an effective painkiller in case of arthritis that doesn’t cause ulcers, scientists
realized they needed to develop new medicines that shut down COX-2 but not COX-1.
Through structural biology, they could see exactly why Celebrex plugs up COX-2 but not
Get whole 3D structure by analysis of good crystallized material.
Produces a single model that is easy to visualize and interpret.
More mathematically direct image construction
Quality indicators available (resolution, Rfactor)
Large molecules can be determined.