Data collection in protein crystallography: capillary effects and background corrections

In protein crystallography, observed diffraction intensities must be corrected for background radiation due to scatter from air and scatter and absorption by capillary., crystal and mother liquor. A systematic study shows that a major contribution to background intensity is air scatter arising from the air intercepted by the direct X-ray beam as 'seen' by the receiving-counter aperture. As a result there is a first-order dependence of background on the 20 angle. The second-order variations in this function are principally due to absorption of the direct beam or air-scattered radiation by the capillary and to diffraction by the glass in the direct beam. To reduce data collection time and crystal exposure, individual background measurements may be approximated by interpolation from empirical background curves or, alternatively, by collecting background intensities for short times and fitting these data with a multidimensional function. If isotropic interpolation is used, i.e., if background is considered to be a function of 2θ alone, systematic errors of up to about 30% can be introduced into the interpolated backgrounds. Methods of accounting for the anisotropy in the background are derived and shown to reduce this error to I-2%.