Phasing with Sulphur Anomalous Diffraction

Phase information is necessary for solving macromolecular crystal structures. This information can be obtained largely ab initio for small molecules that diffract to very high resolution, but protein crystals tend to diffract to moderate resolution, and the phase information has to be obtained independently. One method is by harnessing the inherent anomalous diffraction from the sulphur present in 90% of proteins, in cysteine or methionine residues. The anomalous diffraction effect due to S is very weak at the wavelengths commonly employed in diffraction experiments, but Station 10.1 was designed to access longer wavelengths, up to 2.5 Å, easily. At a wavelength of 2.0 Å, the anomalous effect from S is doubled, and becomes useful for phasing.

Using longer wavelengths also increases the absorption in the sample, preferentially at the S sites. If these are in disulphide bridges between cysteine residues, they tend to be oxidised by the ionising X-ray radiation. Therefore, the level of exposure is deliberately lowered to make the sample last longer. Because the effect is weak, this usually necessitates averaging over a relatively large number of repeat measurements (suppressing the noise and enhancing the weak signal). The radiation damage incurred by shining X-rays on the sample limits the number of repeats that are possible from one sample.

station 10.1