Biophys J, June 2000, p. 3178-3185, Vol. 78, No. 6

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

Robert Glaeser,^* Marc Facciotti,^§ Peter Walian, Shahab Rouhani,^§ James Holton,^* Alastair MacDowell,^¶ Richard Celestre,^¶ Daniela Cambie,^¶ and Howard Padmore^¶

 ^*Department of Molecular and Cell Biology;  Life Sciences Division and  Physical Biosciences Division, Lawrence Berkeley National Laboratory;  ^§Graduate Group in Biophysics; and  ^¶Experimental Systems Group, Advanced Light Source, Lawrence Berkeley National Laboratory, University of California, Berkeley, California 94720 USA

The 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.

Biophys J, June 2000, p. 3178-3185, Vol. 78, No. 6
© 2000 by the Biophysical Society   0006-3495/00/06/3178/08  $2.00