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

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 Å (2θ = 20-32°), whereas air scattering continuously diminishes in intensity between 2θ = 0° and 45° [Krieger, Chambers, Christoph, Stroud & Trus, (1974), Acta Cryst. A30, 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.