Membrane protein structure determination by SAD, SIR, or SIRAS phasing in serial femtosecond crystallography using an iododetergent

Takanori Nakane, Shinya Hanashima, Mamoru Suzuki, Haruka Saiki, Taichi Hayashi, Keisuke Kakinouchi, Shigeru Sugiyama, Satoshi Kawatake, Shigeru Matsuoka, Nobuaki Matsumori, Eriko Nango, Jun Kobayashi, Tatsuro Shimamura, Kanako Kimura, Chihiro Mori, Naoki Kunishima, Michihiro Sugahara, Yoko Takakyu, Shigeyuki Inoue, Tetsuya MasudaToshiaki Hosaka, Kensuke Tono, Yasumasa Joti, Takashi Kameshima, Takaki Hatsui, Makina Yabashi, Tsuyoshi Inoue, Osamu Nureki, So Iwata, Michio Murata, Eiichi Mizohata

Research output: Contribution to journalArticle

22 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)13039-13044
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume113
Issue number46
DOIs
Publication statusPublished - Nov 15 2016

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Crystallography
Iodine
Bacteriorhodopsins
Membrane Proteins
Lasers
X-Rays
Electrons
Detergents
Magic
Purinergic P1 Receptors
X Ray Crystallography
GTP-Binding Proteins

All Science Journal Classification (ASJC) codes

  • General

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Membrane protein structure determination by SAD, SIR, or SIRAS phasing in serial femtosecond crystallography using an iododetergent. / Nakane, Takanori; Hanashima, Shinya; Suzuki, Mamoru; Saiki, Haruka; Hayashi, Taichi; Kakinouchi, Keisuke; Sugiyama, Shigeru; Kawatake, Satoshi; Matsuoka, Shigeru; Matsumori, Nobuaki; Nango, Eriko; Kobayashi, Jun; Shimamura, Tatsuro; Kimura, Kanako; Mori, Chihiro; Kunishima, Naoki; Sugahara, Michihiro; Takakyu, Yoko; Inoue, Shigeyuki; Masuda, Tetsuya; Hosaka, Toshiaki; Tono, Kensuke; Joti, Yasumasa; Kameshima, Takashi; Hatsui, Takaki; Yabashi, Makina; Inoue, Tsuyoshi; Nureki, Osamu; Iwata, So; Murata, Michio; Mizohata, Eiichi.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 113, No. 46, 15.11.2016, p. 13039-13044.

Research output: Contribution to journalArticle

Nakane, T, Hanashima, S, Suzuki, M, Saiki, H, Hayashi, T, Kakinouchi, K, Sugiyama, S, Kawatake, S, Matsuoka, S, Matsumori, N, Nango, E, Kobayashi, J, Shimamura, T, Kimura, K, Mori, C, Kunishima, N, Sugahara, M, Takakyu, Y, Inoue, S, Masuda, T, Hosaka, T, Tono, K, Joti, Y, Kameshima, T, Hatsui, T, Yabashi, M, Inoue, T, Nureki, O, Iwata, S, Murata, M & Mizohata, E 2016, 'Membrane protein structure determination by SAD, SIR, or SIRAS phasing in serial femtosecond crystallography using an iododetergent', Proceedings of the National Academy of Sciences of the United States of America, vol. 113, no. 46, pp. 13039-13044. https://doi.org/10.1073/pnas.1602531113
Nakane, Takanori ; Hanashima, Shinya ; Suzuki, Mamoru ; Saiki, Haruka ; Hayashi, Taichi ; Kakinouchi, Keisuke ; Sugiyama, Shigeru ; Kawatake, Satoshi ; Matsuoka, Shigeru ; Matsumori, Nobuaki ; Nango, Eriko ; Kobayashi, Jun ; Shimamura, Tatsuro ; Kimura, Kanako ; Mori, Chihiro ; Kunishima, Naoki ; Sugahara, Michihiro ; Takakyu, Yoko ; Inoue, Shigeyuki ; Masuda, Tetsuya ; Hosaka, Toshiaki ; Tono, Kensuke ; Joti, Yasumasa ; Kameshima, Takashi ; Hatsui, Takaki ; Yabashi, Makina ; Inoue, Tsuyoshi ; Nureki, Osamu ; Iwata, So ; Murata, Michio ; Mizohata, Eiichi. / Membrane protein structure determination by SAD, SIR, or SIRAS phasing in serial femtosecond crystallography using an iododetergent. In: Proceedings of the National Academy of Sciences of the United States of America. 2016 ; Vol. 113, No. 46. pp. 13039-13044.
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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 {\AA}) 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-{\AA} 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-{\AA} 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.",
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T1 - Membrane protein structure determination by SAD, SIR, or SIRAS phasing in serial femtosecond crystallography using an iododetergent

AU - Nakane, Takanori

AU - Hanashima, Shinya

AU - Suzuki, Mamoru

AU - Saiki, Haruka

AU - Hayashi, Taichi

AU - Kakinouchi, Keisuke

AU - Sugiyama, Shigeru

AU - Kawatake, Satoshi

AU - Matsuoka, Shigeru

AU - Matsumori, Nobuaki

AU - Nango, Eriko

AU - Kobayashi, Jun

AU - Shimamura, Tatsuro

AU - Kimura, Kanako

AU - Mori, Chihiro

AU - Kunishima, Naoki

AU - Sugahara, Michihiro

AU - Takakyu, Yoko

AU - Inoue, Shigeyuki

AU - Masuda, Tetsuya

AU - Hosaka, Toshiaki

AU - Tono, Kensuke

AU - Joti, Yasumasa

AU - Kameshima, Takashi

AU - Hatsui, Takaki

AU - Yabashi, Makina

AU - Inoue, Tsuyoshi

AU - Nureki, Osamu

AU - Iwata, So

AU - Murata, Michio

AU - Mizohata, Eiichi

PY - 2016/11/15

Y1 - 2016/11/15

N2 - 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.

AB - 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.

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