Characterization of Conditions Required for X-Ray Diffraction Experiments with Protein Microcrystals

AbstractThe x-ray exposure at which significant radiation damage occurs has been quantified for frozen crystals of bacteriorhodopsin. The maximum exposure to ∼11-keV x-rays that can be tolerated for high-resolution diffraction experiments is found to be ∼1010 photons/μm2, very close to the value predicted from limits that were measured earlier for electron diffraction exposures. Sample heating, which would further reduce the x-ray exposure that could be tolerated, is not expected to be significant unless the x-ray flux density is well above 109 photons/s-μm2. Crystals of bacteriorhodopsin that contain ∼1011 unit cells are found to be large enough to give ∼100 high-resolution diffraction patterns, each covering one degree of rotation. These measurements are used to develop simple rules of thumb for the minimum crystal size that can be used to record x-ray diffraction data from protein microcrystals. For work with very small microcrystals to be realized in practice, however, it is desirable that there be a significant reduction in the level of background scattering. Background reduction can readily be achieved by improved microcollimation of the x-ray beam, and additional gains can be realized by the use of helium rather than nitrogen in the cold gas stream that is used to keep the protein crystals frozen