GEN10000563

X-ray crystallography

8/1/03. By Richard Twyman

Firing X-rays at protein crystals to find out their 3D stucture.

When x-rays strike a protein crystal they are scattered (diffracted) by individual atoms. The way in which individual x-rays are diffracted depends upon the types of atom in the molecule and the way they are arranged.

Using sophisticated mathematical techniques, an x-ray diffraction pattern can be used to work out the relative positions of different atoms in a molecule. X-ray crystallography can thus be used to determine three-dimensional protein structures.

Key principles

When light strikes the edge of a solid object it spreads out by a process called diffraction. Individual waves of light then show interference (they either reinforce each other or cancel each other out, in much the same way as ripples on a pond surface). This results in a series of dark and light bands on the other side of the object.

X-rays behave in a similar manner when they strike the atoms in a protein molecule, i.e. they are diffracted by them.

If many molecules are oriented precisely, as they are in a crystal, then x-rays striking particular atoms will all be diffracted in the same way. A diffraction pattern is generated by the interference of individual x-rays.

The diffraction pattern represents the way atoms are arranged in the molecule and can be used to determine the structure of the protein.

How does it work?

A very pure sample of protein is required and the protein must be prepared as a crystal. This is the most difficult part of the procedure and can take several months to achieve. Once a crystal is available it is mounted on a rotating holder and exposed to a source of x-rays. The x-rays are diffracted by the crystal and generate a precise pattern of dots on a detector, such as photographic film. The dots are called reflections.

Sophisticated mathematical techniques are then used to analyse the diffraction pattern. The way in which x-rays are diffracted is related to the number of electrons in an atom, so a map of electron density in the protein can be built up. This in turn can be converted into a structural model.

How is it used?

Like nuclear magnetic resonance (NMR) spectroscopy, the main application of X-ray crystallography in biology is to determine the structure of proteins. X-ray crystallography produces very accurate structural models and can even show how proteins interact with other molecules.

A protein structure solved by X-ray crystallography is regarded as the gold standard to which all other structures are compared.