Evaluation of the experimental conditions for lower energy SAD phasing at the Photon Factory

Photon Factory, High Energy Accelerator Research Organization
â—‹Yusuke Yamada Noriyuki Igarashi Tadashi Satoh Naohiro Matsugaki Soichi Wakatsuki

Recent advancements in the X-ray data-acquisition techniques and phasing algorithms have enabled structure determination using weak anomalous signals from light elements such as sulfur that are naturally present in almost all proteins. This is rapidly becoming a useful technique for high-throughput protein crystallography because it does not require preparation of heavy atom derivatives or Se-Met substituted proteins. This is also a useful technique for the structure determination of membrane proteins of higher organism, because it is quite difficult to produce these proteins in bacteria and Se-Met incorporated variant can not be produced. In the Photon Factory a new insertion device beamline, BL-17A, has been built and the user operation will commence in this October. Because BL-17A is designed with the emphasis on diffraction experiments with microcrystals and lower energy, it is expected to be a useful facility for the structure determination using anomalous signals from light elements. For the successful phasing, it is important to carry out diffraction experiments under an optimal condition. For example, at a lower energy of X-rays, light elements exhibit higher anomalous signals but an absorption effect becomes higher, which not only enhances radiation damages but also deteriorates the data quality. There should be an optimal energy which maximizes the signal-to-noise ratio of anomalous signals. To find out such optimal experimental condition, we carried out our case studies with the crystals of a yeast protein Emp46p as a standard sample and under various experimental conditions, such as X-ray energy, resolution, processing software and so on. We will report the relationship between these experimental conditions and resulting statistics of the SAD phasing.