Data collection in protein crystallography: experimental methods for reducing background radiation

Abstract

The primary source of background intensity when diffraction intensities are measured by diffractometry is air scatter from the volume of air irradiated by the direct X-ray beam which scatters incident radiation into the photon-detection system. In protein crystallography where crystals are generally enclosed in glass capillaries, scattering of the direct beam from the glass is the second most significant source of background intensity. Glass scattering leads to a broad diffuse ring centered at spacings of 3"0 A (20 = 20-32°), whereas air scattering continuously diminishes in intensity between 20= 0 ° and 45 ° [Krieger, Chambers, Christoph, Stroud & Trus, (1974), Acta Cryst. A 30, 740-748]. Compared to these sources of background radiation, other sources are usually insignificant. Two experimental methods are described which reduce the air-scattered component of backgrounds by as much as a factor of 100. Use of these methods increased the peak-to-background ratio by a factor of three-ten times in the case of typical diffractometry of protein crystals. This can be translated into time saving during data collection, and into an increase in the range of measurable intensities. DATA COLLECTIONIN PROTEIN CRYSTALLOGRAPHY

The accuracy of refined protein structures: comparison of two independently refined models of bovine trypsin

“…Crystallographic data for the current structure were collected to a resolution of 1.35/~, (20 = 69.6 °, Cu Ka radiation; 48 492 independent hkl reflections) on a fullcircle diffractometer modified to reduce background levels (Krieger, Chambers, Christoph, Stroud & Trus, 1974;Krieger & Stroud, 1976) and data were corrected as described by Stroud et al (1974).…”

A limited-range step-scan method for collecting X-ray diffraction data

“…Our experience with the limited-range, step-scan method suggests that reliable intensity data can be collected at a rate approximately twice that for the fullscan method, but certain precautions should be observed. In particular, if backgrounds are not measured for each reflection, care must be exercised in checking whether the background can be assumed to be a function only of 0 or whether it should be fit by a function similar to that used for the width parameter or by some other function (Krieger, Chambers, Christoph, Stroud & Trus, 1974;Krieger & Stroud, 1976).…”

On the evaluation of root-mean-square errors in atomic coordinates in protein crystallography

“…437 previous figures. Such a background intensity distribution in the region 0.1 < Isl < 0.21 A -~ seems to be caused primarily by solvent scattering, as air and glass scatter at different angles (Krieger & Stroud, 1976).…”