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Development of structural analysis and electron microscopy to visualize single motor protein

Research Project

Project/Area Number 06558098
Research Category

Grant-in-Aid for Developmental Scientific Research (B)

Allocation TypeSingle-year Grants
Research Field Biophysics
Research InstitutionUniversity of Tokyo

Principal Investigator

WAKABAYASHI Takeyuki  Univ.of Tokyo, School of Science, Projessor, 大学院・理学系研究科, 教授 (90011717)

Co-Investigator(Kenkyū-buntansha) SATOH Yuji  Hitachi Co., technitian, 技師
Project Period (FY) 1994 – 1995
Project Status Completed (Fiscal Year 1995)
Budget Amount *help
¥8,400,000 (Direct Cost: ¥8,400,000)
Fiscal Year 1995: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1994: ¥7,500,000 (Direct Cost: ¥7,500,000)
KeywordsMuscle / Myosin / Actin / Tubulin / Kinesin / Electron microscopy / X-ray scattering / Site-directed mutagenesis / クライオ電子顕微鏡法 / 対物レンズ / 傾斜像シリーズ / 画像解析 / ミオシン / 筋収縮 / アンデカゴールド
Research Abstract

Our goal is to understand the molecular mechanism of motor proteins on the basis of atomic structure of proteins.
First, we inproved an electron microscope with a cold field emission gun. Especially, we developed a new objective lenz, by which we can observe high-tilted images, an anti-contaminator, a new minimum dose system.
Second, we developed new image analysis techniques. The first one is electron tomography to reconstruct three-dimensional images of cross-bridge in contracting muscle.The second one is visualization of single motor protein using a holographic image reconstruction technique. The third one is three-dimensional reconstruction without using helical symmetry in order to reconstruct three-dimensional structure of thin filaments. The forth one is a new three-dimensional structure using helical symmetry.
Third, by electron cryo-microscopy, we analyzed three-dimensional structure of kinesin-tubulin complexes, thin filaments, native thin filaments, actomyosin complexes, and actin filaments.
Last, we identified the position of the myosin N-terminus in the actin-myosin rigor complexes using protein engineering and cryo-electron microscopy.

Report

(3 results)
  • 1995 Annual Research Report   Final Research Report Summary
  • 1994 Annual Research Report
  • Research Products

    (27 results)

All Other

All Publications (27 results)

  • [Publications] 石川 他: "Calcium induced change in three-divensional structure of thinfilaments of rabbit skeletal muscle as revealed by cryo-electoron mieroscopy" Biochem.Biophys.Res.Commun.203. 951-958 (1994)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] 吉川 他: "Three-dimensional structure of tubulin-lcinesin complex" Nature. 376. 274-277 (1995)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] 孟 他: "Determination of the protein components of natiue thin filowents isdeted from natural actomyosin" J.Biochem.118. 442-427 (1995)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] 松浦 他: "Methanol traps the troponin-tropomyosin-actin complex in an eff-state" J.Biochem. 118. 1293-1296 (1995)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] 佐伯 他: "Troporaysin-bindina site(s) on the Dictyosteriun activl surface asidentified by site-dirocted mutageusis" Biochemistry. 35. 14465-14472 (1996)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] 安永 他: "Extensible and object-orieuted system Eos supplies a new environment for image avalysis" J.Struct.Biol.116. 155-160 (1996)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Ishikawa, T.& Wakabayashi T.: "Calcium induced change in three-dimensional structure of thin filaments of rabbit skeletal muscle as revealed by cryo-electron microscopy" Biochem.Biophys.Res.Commun.203. 951-958 (1994)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Kikkawa, M., Ishikawa, T., Miyazawa, A., Fujiyoshi, Y., Wakabayashi, T.& Hirokawa, N.: "Direct visualization of the microtubule lattice seam both in vitro and in vivo." J Cell Biol. 127. 1965-1971 (1994)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Ishikawa, T.Wakabayashi, T.: "Proposal of Alignment-Independent Classification of Electron Microscopic Images with Helical Symmetry and its Application to Reconstrituted Thin Filaments of Skeletal Muscle" Ultramicroscopy. 57. 91-101 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Kikkawa, M., Ishikawa, T., Miyazawa, A., Fujiyoshi, Y., Wakabayashi, T.& Hirokawa, N.: "Three-dimensional structure of tubulin-kinesin complex" Nature. 376. 274-277 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Meng, Y., Yasunaga, T., Wakabayashi, T.: "Determination of the protein components of native thin filaments isolated from natural actomyosin : nebulin and alpha-actinin are associated with actin filaments" J.Biochem.118. 422-427 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Matsuura, Y., Yoshimoto, Y., Saeki, K., and Wakabayashi, T.: "Methanol traps the troponin-troppomyosin-actin complex in an off-state" J.Biochem.118. 1293-1296 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Saeki, K., Sutoh, K.& Wakabayashi, T.: "T., Tropomyosin-binding site(s) on the Dictyosterium Actin surface as identify by site-directed mutagenesis" Biochemistry. 35. 14465-14472 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Yasunaga, T., Saeki, K., & Wakabayashi, T.: "Facilitationg effect of ADP and phosphate on the sliding movement of actin filaments on the myosin-coated surface" J.Mus.Res.Cell Motil.Abstract.17. 282 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Yasunaga, T., & Wakabayashi, T.: "Extensible and object-oriented system Eos supplies a new environment for image analysis of electron micrographs of macromolecules" J.Struct.Biol.116. 155-160 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Yamakawa, H., Seog, D.-H., Yoda, K., Yamasaki, M., & Wakabayashi, T.: "Uso1 protein is a dimer with two globular heads and a long coiled-coil tail" J.Struct.Biol.116. 356-365 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Yasunaga, T.Hashiba, S., Ueno, S., & Wakabayashi, T.: "Visualization of single myosin heads by cryo-electron microscopy and a holographic image reconstruction technique(HIRT)" Structure and function of macromolecular assmbly. 244-252 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Wakabayashi, T., Yasunaga, T., Miki, M., Saeki, K., Sutoh, & Sasaki, H.: "Structure and function of actin filaments revealed by sited-directed mutagenesis of actin and image analysis of cryo-electron micrographs" Structure and function of macromolecular assmbly. 244-252 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Matsuura, Y. et al.: "Methanol Traps the Troponin-Tropomyosin-Actin Complex in an “Off-State" J. Biochem.118. 1293-1296 (1995)

    • Related Report
      1995 Annual Research Report
  • [Publications] Kikkawa, M. et al.: "Three-dimensional structure of the kinesin head-microtubule complex" Nature. 376. 274-277 (1995)

    • Related Report
      1995 Annual Research Report
  • [Publications] Yasunaga, T. & Wakabayashi, T.: "Extensible and Object-oriented System(Eos)Supplies a New Environment for Image Analysis of Electron Micrographs of Macromolecules" J. Struct. Biol.(in press). (1996)

    • Related Report
      1995 Annual Research Report
  • [Publications] Meng, Y. et al.: "Detemination of the Protein Components of Native Thin Filaments Isolated from Natural actomyosin: Nebulin and a-Actinin Are Associated with Actin Filaments" J. Biochem.118. 422-427 (1995)

    • Related Report
      1995 Annual Research Report
  • [Publications] Ishikawa, T. & Wakabyashi, T.: "Proposal of Alignment-Independent Classification of Electron Microscopic Images with Helical Symmetry and its Application to Reconstituted Thin Filaments of Skeletal Muscle" Ultramicroscopy. 57. 91-101 (1995)

    • Related Report
      1995 Annual Research Report
  • [Publications] Ishikawa, T. & Wakabayashi, T.: "Calcium Induced Change in Three-dimensional Structure of Thin Filaments of Rabbit Skeletal Muscle as Revealed by Cryo-electron Microscopy." Biochem. Biophys. Res. Commun.203. 951-958 (1994)

    • Related Report
      1995 Annual Research Report
  • [Publications] Ishikawa,T.& Wakabayashi,T.: "Proposal of alignment-independent classification of electron microscopic images with helical symmetry and its application to reconstituted thin filaments of skeletal muscle" Ultramicroscopy. 57. 91-101 (1995)

    • Related Report
      1994 Annual Research Report
  • [Publications] Ishikawa,T.& Wakabayashi,T.: "Calcium Induced Change in Three-dimensional Structure of Thin Filaments of Rabbit Skeletal Muscle as Revealed by Cryo-electron Microscopy" Biochem.Biophys.Res.Commun.203. 951-958 (1994)

    • Related Report
      1994 Annual Research Report
  • [Publications] Kikkawa,M.et al.: "Direct Visualization of the Microtubule Lattice Seam both in vitro and vivo" J.Cell Biol.127. 1965-1971 (1994)

    • Related Report
      1994 Annual Research Report

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Published: 1994-04-01   Modified: 2016-04-21  

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