High-resolution x-ray diffraction investigation of radiation damage in hen egg white lysozyme crystals

High-resolution X-ray diffraction was used to examine small changes in diffracted intensity from hen egg white lysozyme crystals due to radiation damage with the goal of showing the usefulness of this technique to detect defects in protein crystals. A rotating anode generator with four-circle diffractometer was equipped with two graded, parabolic mirrors to permit these measurements in a modest, laboratory environment. An metric, two-reflection silicon monochromator showed the best compromise between reducing wavelength dispersion and decreasing flux. Triple axis reciprocal space maps of reflections from different lysozyme crystals and different X-ray reflections within the same crystal show significant, measurable differences, which means that this technique can indeed be used in the protein crystallography community. A discussion of whether protein crystals diffract kinematically or dynamically is given. As a first investigation, changes in diffracted intensity near a Bragg peak from two lysozyme crystals were monitored during an X-ray irradiation study. Interestingly, the widths of the peaks originally decreased, implying a brief increase in structural quality. At long irradiation times, the peak intensity decreased as the peak widths increased, which would be expected for a system with increasing disorder and defects. Most of the diversion of diffracted intensity appeared as an anisotropic mosaic spread. If the transition between Huang and Stokes-Wilson regimes represents a characteristic size (i.e., a defect size) causing the scattering mechanism to change as it does for semiconductor materials, then this characteristic size is shown to be on the order of microns, and to decrease over the irradiation study. Possible mechanisms for radiation-induced protein crystal defects are discussed.