Structural changes in a cryo-cooled protein crystal owing to radiation damage

The high intensity of third-generation X-ray sources, along with the develo pment of cryo-cooling of protein crystals at. temperatures around 100 K, ha ve made it possible to extend the diffraction limit of crystals and to redu ce their size. However, even with cryo-cooled crystals, radiation damage be comes a limiting factor. So far, the radiation damage has manifested itself in the form of a loss of overall diffracted intensity and an increase in t he temperature factor. The structure of a protein (myrosinase) after exposu re to different doses of X-rays in the region of 20 x 10(15) photons mm(-2) has been studied. The changes in the. structure owing to radiation damage were analysed using Fourier difference maps and occupancy refinement for th e first time. Damage tvas obvious in the form of breakage of disulfide bond s, decarboxylation of aspartate and glutamate residues, a loss of hydroxyl groups from tyrosine and of the methylthio group of methionine. The suscept ibility to radiation damage of individual groups of the same kind varies wi thin the protein. The quality of the model resulting from structure determi nation might be compromised owing to the presence of radiolysis in the crys tal after an excessive radiation dose. Radiation-induced structural changes may interfere with the interpretation of ligand-binding studies or MAD dat a. The experiments reported here suggest that there is an intrinsic limit t o the amount of data which can be extracted from a sample of a given, size.