W0202

SAD Phasing Using Sulfur Anomalous Scattering with Chromium Radiation. C. Yang, J.W. Pflugrath, C.N. Stence, D.A. Courville, J.D. Ferrara*, Rigaku/MSC, Inc. 9009 New Trails Dr., The Woodlands, TX 77381.

Anomalous scattering with soft X-ray radiation opens new possibilities in phasing for macromolecular crystallography. Anomalous scattering from sulfur atoms collected on an in-house chromium radiation (2.29 Å) source was used to phase the X-ray diffraction data of thaumatin (22 kDa), trypsin (24 kDa) and glucose isomerase (40 kDa) crystals. The contribution to the anomalous term, Δƒ”=1.14 °, from sulfur for CrK_α radiation is double compared to that for CuK_α radiation, Δƒ”=0.56°. The direct methods program SHELXD successfully found sulfur positions using data sets with the resolution limited to 3.5 Å. The statistical phasing program SHARP was used to produce the electron density maps using the sulfur anomalous signal alone at a low resolution (~3.5 Å). An interpretable electron density map for each structure was obtained solely from these phases derived from single-wavelength anomalous dispersion (SAD) data from CrK_α radiation. Much less data, that is lower redundancy, is required for this sulfur SAD phasing procedure compared to the highly redundant data reported in the sulfur SAD phasing procedure with CuK_α radiation. CrK_α radiation can also improve the strength of anomalous scattering of many other intrinsic elements in macromolecules, like Ca²⁺, Zn²⁺, and P, because of the doubled value of Δƒ”. Furthermore, the anomalous scattering of selenium is increased substantially when CrK_α radiation is used, because its Δƒ” is increased to 2.28° from 1.14° with CuK_α radiation.

In order to measure small Bijvoet differences accurately, several devices were developed for the experiment, including an Osmic Confocal MaxFlux optic optimized for CrK_α radiation, a helium path and a beam stop. In the cases studied here, radiation damage to the samples and reduction of anomalous signal were observed in some long data sets. Therefore, an adequate collection strategy to maximize the completeness in a short scan range was used in subsequent data collections. The results show the anomalous signal of sulfur atoms can be quickly collected. Since the absorption of solvent and the loop with CrK_α radiation may not be negligible any more, the orientation of the crystal and exposure time were accounted for in order to minimize the effects of radiation damage and absorption. This experimental study shows using CrK_α radiation can provide sufficient phasing power from sulfur anomalous signals for routinely phasing protein data in-house.