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Structural study of ion-driven energizers by cryo-EM and X-ray crystallography

Research Project

Project/Area Number 16H04757
Research Category

Grant-in-Aid for Scientific Research (B)

Allocation TypeSingle-year Grants
Section一般
Research Field Structural biochemistry
Research InstitutionInstitute of Physical and Chemical Research

Principal Investigator

Koji Yonekura  国立研究開発法人理化学研究所, 放射光科学研究センター, グループディレクター (50346144)

Research Collaborator Maki Saori (Yonekura Saori)  
Project Period (FY) 2016-04-01 – 2019-03-31
Project Status Completed (Fiscal Year 2018)
Budget Amount *help
¥17,550,000 (Direct Cost: ¥13,500,000、Indirect Cost: ¥4,050,000)
Fiscal Year 2018: ¥3,510,000 (Direct Cost: ¥2,700,000、Indirect Cost: ¥810,000)
Fiscal Year 2017: ¥7,540,000 (Direct Cost: ¥5,800,000、Indirect Cost: ¥1,740,000)
Fiscal Year 2016: ¥6,500,000 (Direct Cost: ¥5,000,000、Indirect Cost: ¥1,500,000)
Keywordsクライオ電子顕微鏡 / X線結晶構造解析 / 単粒子解析 / 電子線三次元結晶構造解析 / イオンチャネル / プロトン駆動力 / 構造多型 / ダイナミクス / 電荷可視化 / 結晶解析 / イオン駆動力
Outline of Final Research Achievements

Gram-negative bacteria import essential nutrients such as iron and vitamin B12 through outer membrane receptors. This process utilizes proton motive force harvested by the Ton system made up of three inner membrane proteins, ExbB, ExbD and TonB. ExbB and ExbD form the proton channel that energizes uptake through TonB. We present structures of hexameric complexes of ExbB and ExbD revealed by X-ray crystallography and single particle cryo-EM. Image analysis shows that hexameric and pentameric complexes coexist, with the proportion of hexamer increasing with pH. Channel current measurement and 2D crystallography support the existence and transition of the two oligomeric states in membranes. The hexameric complex consists of six ExbB subunits and three ExbD transmembrane helices enclosed within the central channel. We propose models for activation / inactivation associated with hexamer and pentamer formation and utilization of proton motive force.

Academic Significance and Societal Importance of the Research Achievements

本研究で、生命の根源的なエネルギーであるプロトン駆動力を利用するExbBDの構造をX線結晶構造解析と単粒子クライオ電子顕微鏡法により決定した。また、これまで知られていなかった膜蛋白質のダイナミックな形態変化という新しい現象の一端が明らかになった。同様の機構は、他の膜蛋白質でも機能の制御などに利用されている可能性も考えられる。このような研究を通じて、実際に薬剤が機能していく段階や過程を可視化することで、新しい創薬技術の創出へと発展することも期待できる。今後、導入した新型クライオ電子顕微鏡を用いて、より高い分解能での単粒子解析と高精度な電子線三次元結晶構造解析を進め、研究を加速したい。

Report

(4 results)
  • 2018 Annual Research Report   Final Research Report ( PDF )
  • 2017 Annual Research Report
  • 2016 Annual Research Report
  • Research Products

    (45 results)

All 2019 2018 2017 2016 Other

All Int'l Joint Research (2 results) Journal Article (9 results) (of which Peer Reviewed: 7 results,  Open Access: 5 results,  Acknowledgement Compliant: 1 results) Presentation (29 results) (of which Int'l Joint Research: 8 results,  Invited: 22 results) Book (1 results) Remarks (3 results) Patent(Industrial Property Rights) (1 results)

  • [Int'l Joint Research] UCSF(米国)

    • Related Report
      2018 Annual Research Report
  • [Int'l Joint Research] UCSF(米国)

    • Related Report
      2017 Annual Research Report
  • [Journal Article] A new cryo-EM system for single particle analysis2019

    • Author(s)
      Hamaguchi Tasuku、Maki-Yonekura Saori、Naitow Hisashi、Matsuura Yoshinori、Ishikawa Tetsuya、Yonekura Koji
    • Journal Title

      Journal of Structural Biology

      Volume: in press Issue: 1 Pages: 1-9

    • DOI

      10.1016/j.jsb.2019.04.011

    • Related Report
      2018 Annual Research Report
    • Peer Reviewed
  • [Journal Article] A new cryo-EM system for electron 3D crystallography by eEFD2019

    • Author(s)
      Yonekura Koji、Ishikawa Tetsuya、Maki-Yonekura Saori
    • Journal Title

      Journal of Structural Biology

      Volume: 206 Issue: 2 Pages: 243-253

    • DOI

      10.1016/j.jsb.2019.03.009

    • Related Report
      2018 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] Ionic scattering factors of atoms that compose biological molecules2018

    • Author(s)
      Yonekura Koji、Matsuoka Rei、Yamashita Yoshiki、Yamane Tsutomu、Ikeguchi Mitsunori、Kidera Akinori、Maki-Yonekura Saori
    • Journal Title

      IUCrJ

      Volume: 5 Issue: 3 Pages: 348-353

    • DOI

      10.1107/s2052252518005237

    • Related Report
      2018 Annual Research Report 2017 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] Hexameric and pentameric complexes of the ExbBD energizer in the Ton system2018

    • Author(s)
      Maki-Yonekura Saori、Matsuoka Rei、Yamashita Yoshiki、Shimizu Hirofumi、Tanaka Maiko、Iwabuki Fumie、Yonekura Koji
    • Journal Title

      eLife

      Volume: 7 Pages: 1-24

    • DOI

      10.7554/elife.35419

    • Related Report
      2018 Annual Research Report 2017 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] 三次元結晶,単粒子複合解析による膜タンパク質の構造ー荷電・プロトン化状態,構造変化・多型を可視 化する2018

    • Author(s)
      米倉功治,眞木さおり
    • Journal Title

      実験医学

      Volume: 36 Pages: 1333-1338

    • Related Report
      2018 Annual Research Report
  • [Journal Article] 三次元結晶,単粒子複合解析による膜タンパク質の構造ー荷電・プロトン化状態,構造変化・多型を可視化する2018

    • Author(s)
      米倉功治,眞木さおり
    • Journal Title

      実験医学

      Volume: 36 Pages: 1333-1338

    • Related Report
      2017 Annual Research Report
  • [Journal Article] タンパク質の電子線三次元結晶構造解析と電荷の精密化2017

    • Author(s)
      米倉功治,眞木さおり
    • Journal Title

      日本結晶学会誌

      Volume: 59 Pages: 88-95

    • NAID

      130006767778

    • Related Report
      2017 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] “Refinement of cryo-EM structures using scattering factors of charged atoms2016

    • Author(s)
      K. Yonekura, S. Maki-Yonekura
    • Journal Title

      J. Appl. Cryst.

      Volume: 49 Issue: 5 Pages: 1517-1523

    • DOI

      10.1107/s1600576716011274

    • Related Report
      2016 Annual Research Report
    • Peer Reviewed / Acknowledgement Compliant
  • [Journal Article] Taste substance binding elicits conformational change of taste receptor T1r heterodimer extracellular domains2016

    • Author(s)
      E. Nango, S. Akiyama, S. Maki-Yonekura, Y. Ashikawa, Y. Kusakabe, E. Krayukhina, T. Maruno, S. Uchiyama, N. Nuemket, K. Yonekura, M. Shimizu, N. Atsumi, N. Yasui, T. Hikima, M. Yamamoto, Y. Kobayashi, A. Yamashita
    • Journal Title

      Sci. Rep

      Volume: 6 Issue: 1 Pages: 25745-25745

    • DOI

      10.1038/srep25745

    • Related Report
      2016 Annual Research Report
    • Peer Reviewed / Open Access
  • [Presentation] A new cryo-EM system for higher-resolution single particle analysis and electron 3D crystallography2019

    • Author(s)
      Koji Yonekura
    • Organizer
      RSC Cryo-Electron Microscopy Symposium
    • Related Report
      2018 Annual Research Report
    • Invited
  • [Presentation] 300kV CFEG cryo-EMによる電子線三次元結晶構造解析と高分解能単粒子解析2019

    • Author(s)
      米倉功治
    • Organizer
      東京大学 第1257回生物科学セミナー
    • Related Report
      2018 Annual Research Report
    • Invited
  • [Presentation] クライオEM, EDによる構造生物学2019

    • Author(s)
      米倉功治
    • Organizer
      RSCミニシンポジウム
    • Related Report
      2018 Annual Research Report
    • Invited
  • [Presentation] クライオEMと放射光による構造生物研究2019

    • Author(s)
      米倉功治
    • Organizer
      第32回日本放射光学会年会・放射光科学合同シンポジウム
    • Related Report
      2018 Annual Research Report
    • Invited
  • [Presentation] プロトン駆動力供給チャネルのクライオEM解析 -- 構造多型、荷電状態の解析2018

    • Author(s)
      米倉功治
    • Organizer
      平成30年度膜タンパク質研究会
    • Related Report
      2018 Annual Research Report
    • Invited
  • [Presentation] クライオ電子顕微鏡と放射光による構造生物研究2018

    • Author(s)
      米倉功治
    • Organizer
      第8回JASRIワークショップ「高分解能イメージング技術」
    • Related Report
      2018 Annual Research Report
    • Invited
  • [Presentation] Electron 3D crystallography and single particle cryo-EM of membrane proteins --- Analysis of charge and Damage2018

    • Author(s)
      Koji Yonekura, Saori Maki-Yonekura
    • Organizer
      The 10th international workshop on X-ray damage to biological samples
    • Related Report
      2018 Annual Research Report
    • Int'l Joint Research / Invited
  • [Presentation] プロトン駆動力変換チャネルExbBDの作動機構の解明 --- クライオEMと放射光の相補的利用2018

    • Author(s)
      米倉功治
    • Organizer
      第9回SPRUC放射光構造生物学研究会&第18回日本蛋白質科学会
    • Related Report
      2018 Annual Research Report
    • Invited
  • [Presentation] Cryo-EM and crystallography of an energizer membrane protein ExbBD in the Ton system --- Visualization of multi-conformational states and charges2018

    • Author(s)
      Koji Yonekura
    • Organizer
      Takada Lab Seminar, Kyoto University
    • Related Report
      2018 Annual Research Report
    • Invited
  • [Presentation] クライオ電子顕微鏡と電荷の可視化2018

    • Author(s)
      米倉功治
    • Organizer
      分野横断ワークショップ
    • Related Report
      2017 Annual Research Report
    • Invited
  • [Presentation] Visualization and structures of membrane proteins by cryo-electron microscopy2018

    • Author(s)
      濵口祐、眞木さおり、米倉功治
    • Organizer
      第9回OCARINA国際シンポジウム
    • Related Report
      2017 Annual Research Report
    • Int'l Joint Research / Invited
  • [Presentation] プロトン駆動力利用機構のクライオEMと結晶構造解析2018

    • Author(s)
      米倉功治
    • Organizer
      第91回日本細菌学会総会 「WS13 機能の直接可視化によって解き明かすバクテリア膜タンパク質デバイスの動作原理」
    • Related Report
      2017 Annual Research Report
    • Invited
  • [Presentation] Structure determination of membrane proteins by cryo-electron microscopy and X-ray crystallography2017

    • Author(s)
      K. Yonekura
    • Organizer
      University of Strasbourg-RIKEN Workshop on Membrane Lipidology
    • Place of Presentation
      Strasbourg, France
    • Year and Date
      2017-03-10
    • Related Report
      2016 Annual Research Report
    • Int'l Joint Research
  • [Presentation] Single Particle Analysis of Membrane Protein by Cryo-electron Microscopy2017

    • Author(s)
      Y. Yamashita, S. Maki-Yonekura, R. Matsuoka, K. Yonekura
    • Organizer
      University of Strasbourg-RIKEN Workshop on Membrane Lipidology
    • Place of Presentation
      Strasbourg, France
    • Year and Date
      2017-03-10
    • Related Report
      2016 Annual Research Report
  • [Presentation] Protein Expression, Purification, Crystalization and Electron Microscopy Analysis of a Protein Channel2017

    • Author(s)
      R. Matsuoka, K. Yonekura
    • Organizer
      University of Strasbourg-RIKEN Workshop on Membrane Lipidology
    • Place of Presentation
      Strasbourg, France
    • Year and Date
      2017-03-10
    • Related Report
      2016 Annual Research Report
  • [Presentation] Single-particle cryo-EM and crystallography of a channel protein2017

    • Author(s)
      米倉功治
    • Organizer
      Pioneering Project "Dynamic Structural Biology" 第1回研究報告会
    • Related Report
      2017 Annual Research Report
  • [Presentation] クライオ電子顕微鏡のIntegrated lipidologyへの展開2017

    • Author(s)
      米倉功治
    • Organizer
      第3回 Integrated Liqidology 領域会議
    • Related Report
      2017 Annual Research Report
  • [Presentation] Electron 3D crystallography of protein crystals for visualization of charges2017

    • Author(s)
      Koji Yonekura
    • Organizer
      IUCr
    • Related Report
      2017 Annual Research Report
    • Int'l Joint Research / Invited
  • [Presentation] Electron 3D crystallography and single particle cryo-EM of membrane proteins for visualization of charges2017

    • Author(s)
      Koji Yonekura
    • Organizer
      IBS seminar
    • Related Report
      2017 Annual Research Report
    • Int'l Joint Research / Invited
  • [Presentation] Electron 3D crystallography and single particle cryo-EM of membrane proteins for visualization of charges2017

    • Author(s)
      Koji Yonekura
    • Organizer
      the PSI / CCP4 Workshop , "3D Electron Crystallography for Macromolecular Compounds"
    • Related Report
      2017 Annual Research Report
    • Int'l Joint Research / Invited
  • [Presentation] 膜蛋白質の単粒子クライオ電子顕微鏡解析と電子線三次元結晶構造解析2017

    • Author(s)
      米倉功治
    • Organizer
      TIA「かけはし」セミナー 第3回クライオTEMセミナー
    • Related Report
      2017 Annual Research Report
    • Invited
  • [Presentation] 膜蛋白質の結晶構造解析および単粒子解析2017

    • Author(s)
      米倉功治
    • Organizer
      東北大学工学研究科応用物理学専攻数理物理学研究室セミナー
    • Place of Presentation
      東北大学 (仙台)
    • Related Report
      2016 Annual Research Report
    • Invited
  • [Presentation] Refinement of Cryo-EM Structures Using Scattering Factors of Charged Atoms2016

    • Author(s)
      Koji Yonekura, Saori Maki-Yonekura, Rei Matsuoka, Yoshiki Yamashita, Fumie Iwabuki and Maiko Tanaka
    • Organizer
      第54回日本生物物理学会年会
    • Place of Presentation
      つくば国際会議場 (つくば市)
    • Year and Date
      2016-11-27
    • Related Report
      2016 Annual Research Report
  • [Presentation] Oligomeric structure of the ExbB-ExbD complex revealed by X-ray crystallography and cryo-EM2016

    • Author(s)
      Saori Maki-Yonekura, Yoshiki Yamashita, Rei Matsuoka, Maiko Tanaka, Fumie Iwabuki and Koji Yonekura
    • Organizer
      第54回日本生物物理学会年会
    • Place of Presentation
      つくば国際会議場 (つくば市)
    • Year and Date
      2016-11-25
    • Related Report
      2016 Annual Research Report
  • [Presentation] Cryo EM infrastructure at SR facility2016

    • Author(s)
      K. Yonekura
    • Organizer
      Three-Way Meeting 2016
    • Place of Presentation
      Hamburg, Germany
    • Related Report
      2016 Annual Research Report
    • Int'l Joint Research / Invited
  • [Presentation] 低温電子顕微鏡法による三次元結晶と単粒子構造解析2016

    • Author(s)
      米倉功治
    • Organizer
      第58回構造生物応用研究会
    • Place of Presentation
      東京大学 (東京)
    • Related Report
      2016 Annual Research Report
    • Invited
  • [Presentation] 膜蛋白質の電子線三次元結晶構造解析と単粒子解析2016

    • Author(s)
      米倉功治、眞木さおり、山下良樹
    • Organizer
      第16回日本蛋白質科学会年会
    • Place of Presentation
      福岡国際会議場 (福岡)
    • Related Report
      2016 Annual Research Report
    • Invited
  • [Presentation] 膜蛋白質の電子線単粒子及び結晶構造解析2016

    • Author(s)
      米倉功治
    • Organizer
      理研シンポジウム「クライオ電子顕微鏡が拓く未来-構造生物学研究のパラダイムシフト-」
    • Place of Presentation
      横浜理研 (横浜)
    • Related Report
      2016 Annual Research Report
    • Invited
  • [Presentation] Cryo-EM single particle analysis and crystallography for pioneering structural biology2016

    • Author(s)
      K. Yonekura
    • Organizer
      Symposium for exploring prospective research Pioneering New Fields: Forefront of RIKEN’s Science and Beyond
    • Place of Presentation
      理研 (埼玉県 和光市)
    • Related Report
      2016 Annual Research Report
    • Int'l Joint Research / Invited
  • [Book] SPring-8/SACLA Research Frontiers 20152016

    • Author(s)
      Y. Takayama, K. Yonekura
    • Total Pages
      150
    • Publisher
      JASRI
    • Related Report
      2016 Annual Research Report
  • [Remarks] 膜タンパク質のダイナミックな構造変化を解明

    • URL

      http://www.riken.jp/pr/press/2018/20180417_2/

    • Related Report
      2018 Annual Research Report
  • [Remarks] 生体分子を構成する原子のイオンの散乱因子の決定

    • URL

      http://www.riken.jp/pr/press/2018/20180427_1/

    • Related Report
      2018 Annual Research Report 2017 Annual Research Report
  • [Remarks] 膜タンパク質のダイナミックな構造変化を解明 -クライオ電子顕微鏡、放射光での新しい生命現象の可視化-

    • URL

      http://www.riken.jp/pr/press/2018/20180417_2/

    • Related Report
      2017 Annual Research Report
  • [Patent(Industrial Property Rights)] 結晶構造解析システム及び結晶構造解析方法2018

    • Inventor(s)
      Y. Nishiyama, K. Yonekura
    • Industrial Property Rights Holder
      Y. Nishiyama, K. Yonekura
    • Industrial Property Rights Type
      特許
    • Filing Date
      2018
    • Related Report
      2018 Annual Research Report

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Published: 2016-04-21   Modified: 2022-05-20  

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