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Toshiya Senda

High Energy Accelerator Research Organization (KEK), Japan

Title: The native (Sulfur) SAD method in Photon Factory

Biography

Biography: Toshiya Senda

Abstract

Crystallography has been a major method to determine 3D structures of biological macromolecules at atomic resolution. While a new method with cryo-EM is becoming another major technique for the 3D structure analysis, crystallography still has some advantages. Recently, many crystal structures of biological macromolecules are determined by MAD/SAD method with seleno-methionine proteins (SeMet-proteins). Since selenium has an X-ray absorption edge near 1Å, it is convenient to utilize in the MAD/SAD method. While this technique is useful, crystallographers need to prepare SeMet-proteins. If we can develop a method to solve the phase problem without using SeMet-proteins, it would be highly useful for crystallographers. So, we have tried to develop the native SAD (or sulfur SAD) method, in which anomalous signals from sulfur atoms in the native protein are utilized. However, there are some problems in the native SAD method. First, sulfur gives only weak anomalous signals with X-ray typically utilized in protein crystallography (X-ray wavelength of around 1Å). To increase the anomalous signals, we need to use a longer wavelength X-ray than usual. However, since a longer wavelength X-ray shows significant absorption by air, solvent, protein etc., data quality is degraded by the absorption. The native SAD method, therefore, requires a specific system for high quality data collection. To achieve routine utilization of the native SAD method, we have developed a beamline (BL-1A) dedicated for the native SAD method. In BL-1A, we can utilize a long wavelength X-ray (1.9 – 3.5 Å). Furthermore, the goniometer and X-ray detector are installed inside a He chamber to prevent the absorption problem. Our system enables us to solve crystal structures of proteins by the native SAD method. In the presentation, we will present several examples of crystal structure determination with native SAD. Also, we will mention our unique method for crystal freezing to collect high quality diffraction data required in native SAD experiments.

References:

  1. Liebschner, D., Yamada, Y., Matsugaki, N., Senda, M. & Senda, T. (2016). On the influence of crystal size and wavelength on native SAD phasing. Acta Crystallogr. D72, 728–741.
  2. Hiraki, M., Matsugaki, N, Yamada, Y. & Senda, T. (2016) Development of sample exchange robot PAM-HC for beamline BL-1A at the photon factory. API Conf. Proc. 1741, 030029.
  3. Nagae, M., Liebschner, D., Yamada, Y., Morita-Matsumoto, K., Matsugaki, N., Senda, T., Fujita, M., Kinoshita, T., Yamaguchi, Y. (2017) Crystallographic analysis of murine p24g2 Golgi dynamics domain. Proteins, 85, 764-770.  doi: 10.1002/prot.25242
  4. Nagae, M., Mishra, S. K., Neyazaki, M., Oi, R., Ikeda, A., Matsugaki, N., Akashi, S., Manya, H., Mizuno, M., Yagi, H., Kato, K., Senda T., Endo, T., Nogi, T. & Yamaguchi Y. (2017) Genes to Cells, doi 10.1111/gtc.12480 [Epub ahead of print]
  5. Senda, M., Hayashi, T., Hatakeyama, M., Takeuchi, K., Sasaki, A. T. & Senda, T. (2016) Use of multiple cryoprotectants to improve diffraction quality from protein crystals. Crystal Growth & Design 16, 1565-1571.  Doi: 10.1021/acs.cgd.5b01692