title: Membrane protein structure determination by SAD, SIR, or SIRAS phasing in serial femtosecond crystallography using an iododetergent.
abstract: The 3D structure determination of biological macromolecules by X-ray crystallography suffers from a phase problem: to perform Fourier transformation to calculate real space density maps, both intensities and phases of structure factors are necessary; however, measured diffraction patterns give only intensities. Although serial femtosecond crystallography (SFX) using X-ray free electron lasers (XFELs) has been steadily developed since 2009, experimental phasing still remains challenging. Here, using 7.0-keV (1.771 Å) X-ray pulses from the SPring-8 Angstrom Compact Free Electron Laser (SACLA), iodine single-wavelength anomalous diffraction (SAD), single isomorphous replacement (SIR), and single isomorphous replacement with anomalous scattering (SIRAS) phasing were performed in an SFX regime for a model membrane protein bacteriorhodopsin (bR). The crystals grown in bicelles were derivatized with an iodine-labeled detergent heavy-atom additive 13a (HAD13a), which contains the magic triangle, I3C head group with three iodine atoms. The alkyl tail was essential for binding of the detergent to the surface of bR. Strong anomalous and isomorphous difference signals from HAD13a enabled successful phasing using reflections up to 2.1-Å resolution from only 3,000 and 4,000 indexed images from native and derivative crystals, respectively. When more images were merged, structure solution was possible with data truncated at 3.3-Å resolution, which is the lowest resolution among the reported cases of SFX phasing. Moreover, preliminary SFX experiment showed that HAD13a successfully derivatized the G protein-coupled A2a adenosine receptor crystallized in lipidic cubic phases. These results pave the way for de novo structure determination of membrane proteins, which often diffract poorly, even with the brightest XFEL beams.
authors: Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo 113-0033, Japan.
authors: Takanori
authors: T
authors: Nakane
authors: Department of Chemistry, Graduate School of Science, Osaka University, Osaka 563-0043, Japan.
authors: Shinya
authors: S
authors: Hanashima
authors: Institute for Protein Research, Osaka University, Osaka 565-0871, Japan.|RIKEN SPring-8 Center, Hyogo 679-5148, Japan.
authors: Mamoru
authors: M
authors: Suzuki
authors: Department of Chemistry, Graduate School of Science, Osaka University, Osaka 563-0043, Japan.
authors: Haruka
authors: H
authors: Saiki
authors: Department of Applied Chemistry, Graduate School of Engineering, Osaka University, Osaka 565-0871, Japan.
authors: Taichi
authors: T
authors: Hayashi
authors: Department of Chemistry, Graduate School of Science, Osaka University, Osaka 563-0043, Japan.|Lipid Active Structure Project, Japan Science and Technology Agency, Exploratory Research for Advanced Technology, Osaka 560-0043, Japan.
authors: Keisuke
authors: K
authors: Kakinouchi
authors: Department of Chemistry, Graduate School of Science, Osaka University, Osaka 563-0043, Japan.|Lipid Active Structure Project, Japan Science and Technology Agency, Exploratory Research for Advanced Technology, Osaka 560-0043, Japan.
authors: Shigeru
authors: S
authors: Sugiyama
authors: Department of Chemistry, Graduate School of Science, Osaka University, Osaka 563-0043, Japan.|Lipid Active Structure Project, Japan Science and Technology Agency, Exploratory Research for Advanced Technology, Osaka 560-0043, Japan.
authors: Satoshi
authors: S
authors: Kawatake
authors: Department of Chemistry, Graduate School of Science, Osaka University, Osaka 563-0043, Japan.|Lipid Active Structure Project, Japan Science and Technology Agency, Exploratory Research for Advanced Technology, Osaka 560-0043, Japan.
authors: Shigeru
authors: S
authors: Matsuoka
authors: Department of Chemistry, Graduate School of Science, Osaka University, Osaka 563-0043, Japan.
authors: Nobuaki
authors: N
authors: Matsumori
authors: RIKEN SPring-8 Center, Hyogo 679-5148, Japan.
authors: Eriko
authors: E
authors: Nango
authors: RIKEN SPring-8 Center, Hyogo 679-5148, Japan.
authors: Jun
authors: J
authors: Kobayashi
authors: Department of Cell Biology, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan.
authors: Tatsuro
authors: T
authors: Shimamura
authors: Department of Cell Biology, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan.
authors: Kanako
authors: K
authors: Kimura
authors: Department of Cell Biology, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan.
authors: Chihiro
authors: C
authors: Mori
authors: RIKEN SPring-8 Center, Hyogo 679-5148, Japan.
authors: Naoki
authors: N
authors: Kunishima
authors: RIKEN SPring-8 Center, Hyogo 679-5148, Japan.
authors: Michihiro
authors: M
authors: Sugahara
authors: Department of Applied Chemistry, Graduate School of Engineering, Osaka University, Osaka 565-0871, Japan.
authors: Yoko
authors: Y
authors: Takakyu
authors: RIKEN SPring-8 Center, Hyogo 679-5148, Japan.|Department of Cell Biology and Anatomy, Graduate School of Medicine, The University of Tokyo, Tokyo 113-0033, Japan.
authors: Shigeyuki
authors: S
authors: Inoue
authors: RIKEN SPring-8 Center, Hyogo 679-5148, Japan.|Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kyoto 611-0011, Japan.
authors: Tetsuya
authors: T
authors: Masuda
authors: Division of Structural and Synthetic Biology, RIKEN Center for Life Science Technologies, Yokohama 230-0045, Japan.
authors: Toshiaki
authors: T
authors: Hosaka
authors: Japan Synchrotron Radiation Research Institute, Hyogo 679-5198, Japan.
authors: Kensuke
authors: K
authors: Tono
authors: Japan Synchrotron Radiation Research Institute, Hyogo 679-5198, Japan.
authors: Yasumasa
authors: Y
authors: Joti
authors: Japan Synchrotron Radiation Research Institute, Hyogo 679-5198, Japan.
authors: Takashi
authors: T
authors: Kameshima
authors: RIKEN SPring-8 Center, Hyogo 679-5148, Japan.
authors: Takaki
authors: T
authors: Hatsui
authors: RIKEN SPring-8 Center, Hyogo 679-5148, Japan.
authors: Makina
authors: 0000-0002-2472-1684
authors: M
authors: Yabashi
authors: Department of Applied Chemistry, Graduate School of Engineering, Osaka University, Osaka 565-0871, Japan.
authors: Tsuyoshi
authors: T
authors: Inoue
authors: Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo 113-0033, Japan.
authors: Osamu
authors: O
authors: Nureki
authors: RIKEN SPring-8 Center, Hyogo 679-5148, Japan.|Department of Cell Biology, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan.
authors: So
authors: S
authors: Iwata
authors: Department of Chemistry, Graduate School of Science, Osaka University, Osaka 563-0043, Japan; murata@chem.sci.osaka-u.ac.jp mizohata@chem.eng.osaka-u.ac.jp.|Lipid Active Structure Project, Japan Science and Technology Agency, Exploratory Research for Advanced Technology, Osaka 560-0043, Japan.
authors: Michio
authors: M
authors: Murata
authors: Department of Applied Chemistry, Graduate School of Engineering, Osaka University, Osaka 565-0871, Japan; murata@chem.sci.osaka-u.ac.jp mizohata@chem.eng.osaka-u.ac.jp.
authors: Eiichi
authors: E
authors: Mizohata
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism*; D003460:Crystallization; D003461:Crystallography / Q000379:methods; D003902:Detergents / Q000737:chemistry; D004583:Electrons; D006217:Halobacterium; D007834:Lasers; D008565:Membrane Proteins / Q000737:chemistry*; D011487:Protein Conformation; D014283:Triiodobenzoic Acids / Q000737:chemistry
chemical_list: D001426:Bacterial Proteins; D003902:Detergents; D008565:Membrane Proteins; D014283:Triiodobenzoic Acids
keywords: G protein-coupled receptor; de novo phasing; detergent; heavy atom; phase problem