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Biophys J. 2000 Jun;78(6):3178-85.

Characterization of conditions required for X-Ray diffraction experiments with protein microcrystals.

Author information

1
Department of Molecular and Cell Biology, Lawrence Berkeley National Laboratory, University of California-Berkeley 94720, USA. rmglaeser@lbl.gov

Abstract

The x-ray exposure at which significant radiation damage occurs has been quantified for frozen crystals of bacteriorhodopsin. The maximum exposure to approximately 11-keV x-rays that can be tolerated for high-resolution diffraction experiments is found to be approximately 10(10) photons/microm(2), 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 10(9) photons/s-microm(2). Crystals of bacteriorhodopsin that contain approximately 10(11) unit cells are found to be large enough to give approximately 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.

PMID:
10827994
PMCID:
PMC1300899
DOI:
10.1016/S0006-3495(00)76854-8
[Indexed for MEDLINE]
Free PMC Article

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