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Substrate binding proteins (SBP) bind to specific ligands in the periplasmic region and bind to membrane proteins to participate in transport or signal transduction. Typical SBPs consist of two α/β domains and recognize the substrate by hinge motion between two domains. Conversely, short length Rhodothermus marinus SBP (named as RmSBP) exists around the methyl-accepting chemotaxis protein. We previously determined the crystal structure of RmSBP consisting of a single α/β domain, but the substrate recognition mechanism is still unclear. To better understand the short length RmSBP, we performed comparative structure analysis, computational substrate docking, and X-ray crystallographic study. RmSBP shares a high level of similarity in α/β domain with other SBP proteins, but it has a distinct topology in the C-term region. The substrate binding model suggested that conformational change in the peripheral region of RmSBP was required to recognize the substrate. We determined the crystal structures of RmSBP at pH 5.5, 6.0, and 7.5. RmSBP showed structural flexibility of the β1-α2 loop, β5-β6 loop, and extended C-term domain based on the electron density map and temperature B-factor analysis. These results provide information that will further the understanding of the function of the short length SBP.

Serial crystallography (SX) is an innovative technology in structural biology that enables the visualization of molecular dynamics of macromolecules at room temperature. SX experiments always require a considerable amount of effort to deliver a crystal sample to the X-ray interaction point continuously and reliably. Here, a sample delivery method using a capillary and a delivery medium is introduced. The crystals embedded in the delivery medium can pass through the capillary tube, which is aligned with the X-ray beam, at very low flow rates without requiring elaborate delivery techniques and drastically reducing sample consumption. This simple but highly efficient sample delivery method can allow researchers to deliver crystals precisely to X-rays in SX experiments.

Serial crystallography (SX) allows crystal structures to be observed at room temperature through the steady delivery of crystals to the X-ray interaction point. Viscous delivery media are advantageous because they afford efficient sample delivery from an injector or syringe at a low flow rate. Hydrophobic delivery media, such as lipidic cubic phase (LCP) or grease, provide a very stable injection stream and are widely used. The development of new hydrophobic delivery materials can expand opportunities for future SX studies with various samples. Here, I introduce fat-based shortening as a delivery medium for SX experiments. This material is commercially available at low cost and is straightforward to handle because its phase (i.e., solid or liquid) can be controlled by temperature. Shortening was extruded from a syringe needle in a very stable injection stream even below 200 nl/min. X-ray exposed shortening produced several background scattering rings, which have similar or lower intensities than those of LCP and contribute negligibly to data processing. Serial millisecond crystallography was performed using two shortening delivery media, and the room temperature crystal structures of lysozyme and glucose isomerase were successfully determined at resolutions of 1.5–2.0 Å. Therefore, shortening can be used as a sample delivery medium in SX experiments.

HES (Hard X-ray EndStation)-2 beamline is located at the hard X-ray experimental hall at PAL-XFEL. The main objective of HES-2 beamline is to deliver a hard X-ray FEL beam to target materials in such a manner that a coherent diffraction study is possible. This endstation is supposed to provide brilliant hard x-rays and to measure the diffraction patterns with forward scattering geometry. In particular, the instruments are designed for serial femtosecond X-ray crystallography (SFX) and coherent diffraction imaging (CDI). In this poster, we introduce HES-2 beamline at PAL-XFEL in terms of two perspectives: beamline instrumentation and sample environment. In the instrumentation part, the current status of HES-2 beamline is described in details. This includes beamline layout, x-ray optics, beam diagnositics and the upcoming commissioning plan for HES-2 beamline. In the sample environment part, we aim to present scientific goals based on the sample environments for CXI and SFX respectively. Finally, we discuss the feasible demo-experiments, which is expected to be done in 2016., Proceedings of the 7th Int. Particle Accelerator Conf., IPAC2016, Busan, Korea