S-SAD phasing at RIKEN structural genomics beamline of SPring-8

PharmAxess, Inc.
â—‹Raita Hirose Yuki Nakamura Tomokazu Hasegawa Akihito Yamano Kensaku Hamada

S-SAD is a phasing method in macromolecular crystallography which uses anomalous signal from sulfur atoms. The method has an advantage over other phasing methods such as MAD and MIR when neither a Se-Met nor a heavy atom is present in the crystal. A disadvantage of SAD, however, is that it is impossible to obtain a unimodal phase probability distribution in principle. In order to resolve the phase ambiguity, it is necessary to combine the SAD approach with density modification and other crystallographic techniques.
Generally, the anomalous signal of sulfur atoms in a protein crystal is so weak that it can readily be buried in background noises and systematic errors. We believe that those noises and errors come mainly from scattering by liquid around the crystal, characteristics of the X-ray detector itself and so on. Therefore, some techniques and modifications to reduce background noises and increasing accuracy of diffraction data are employed to improve success rate of S-SAD phasing.

We examined S-SAD experiments using insulin crystals at BL26B2 in SPring-8, and succeeded in automatic phasing and model building under some specific conditions. For successful datasets, the amount of liquid around the crystal was minimized by mounting crystals on MicroMountsTM, a novel crystal mounting device consisted with a nib-shaped Kapton film. Moreover, scattering and absorption by the black paper shading IP's from visible light were greatly reduced by replacing it with a poly-carbonate sheet.

This research was supported by "Program for Strategic Use of Advanced Large-scale Research Facilities of the Ministry of Education, Culture, Sports, Science and Technology."