Dose dependence of radiation damage for protein crystals studied at various X‐ray energies

Radiation damage to protein crystals is the most serious problem in obtaining accurate structures from protein crystallography. In order to examine the photon energy dependence of radiation damage, 12 to 15 data sets from each of nine tetragonal lysozyme crystals were collected at nine different X‐ray energies (6.5, 7.1, 8.3, 9.9, 12.4, 16.5, 20.0, 24.8 and 33.0 keV) using beamline BL41XU at SPring‐8. All results were compared on the basis of absorbed dose, expressed in Gray (Gy). Crystallographic statistics, such as the values of lattice constants, R merge and I /σ( I ), for each data set degraded at all nine energies as the exposure time for each crystal increased. In all data sets, radiation damage was observed after the absorbed dose exceeded 10 6  Gy. However, from the point of view of crystallographic statistics normalized to the absorbed dose, no clear dependence on photon energy was observed in these results. Structural refinement showed that the average B ‐factor for the last data set was larger than that for the first data set at all energies tested. However, no energy dependence of radiation damage on B ‐factor was found. Furthermore, disruption of disulfide bonds due to radiation damage was observed in electron density maps even at the highest photon energy (33 keV) used in this study. Therefore, these results suggest that radiation damage in the energy range investigated could be evaluated based on absorbed dose without energy dependence, and that it is important to minimize the absorbed dose in a crystal sample for obtaining an accurate protein structure.