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Development of technologise for three-dimensional structural analysis of protein crystals using electron microscope

Research Project

Project/Area Number 05508004
Research Category

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

Allocation TypeSingle-year Grants
Research Field Biophysics
Research InstitutionThe University of Tokyo (1994)
Tokyo Institute of Technology (1993)

Principal Investigator

TOYOSHIMA Chikashi  The University of Tokyo, Institute of Molecular and Cellular Biosciences, Professor, 分子細胞生物学研究所, 教授 (70172210)

Co-Investigator(Kenkyū-buntansha) OOBAYASHI Toshihide  Hitachi, Ltd.Instrument Division, lst Design Department, Chief manager, 計測器事業部・第一設計部, 部長
Project Period (FY) 1993 – 1994
Project Status Completed (Fiscal Year 1994)
Budget Amount *help
¥43,000,000 (Direct Cost: ¥43,000,000)
Fiscal Year 1994: ¥35,000,000 (Direct Cost: ¥35,000,000)
Fiscal Year 1993: ¥8,000,000 (Direct Cost: ¥8,000,000)
KeywordsElectron microscope / Protein crystals / Electron diffraction / Ice embedding / Structural analysis / Radiation damage / Cryo-transfer / 結晶解析 / 電子線回析 / 低電子線量法 / CCDカメラ / 電子線 / 三次元構造解析
Research Abstract

The aim of this project is to develop technologies necessary for analyzing three-dimensional structure of proteins using electron crystallography. As to the hardwave, it is to construct an electron microscope optimized for electron crystallography. Conventional electron microscopes have been designed for mostly material scientists and histologists. Protein crystals are special in that they are very sensitive to radiation damage. Hence specimens must be kept in frozen-hydrated conditions and data taking should avoid unnecessary electron irradiation as much as possible. High resolution data collection can be done using the microscope as a diffractometer. To obtain phase data efficiently, image must be obtained from the same field. Thus integration of diffraction and imaging is needed. Many modifications to the existing microscope were made, in particular to allow easy and accurate low dose microscopy. We have optimizad microscope firmware to do so and developed a new method for blanking and focusing at multiple points. Electron diffraction is now integrated in the low-dose procedure. Several aspects of the cryo-transfer and low temperature examination were examined intensively. As a result, new pumping system gas been employed, which will greatly reduce ice contamination to the specimen at low temperature. As to the software side, a set of computer program has been developed for determining the orientation of a three-dimensional crystal to the electron beam. The program has been applied to micro crystals of calcium ATPase of sarcoplasmic reticulum.

Report

(3 results)
  • 1994 Annual Research Report   Final Research Report Summary
  • 1993 Annual Research Report
  • Research Products

    (26 results)

All Other

All Publications (26 results)

  • [Publications] M.Nakasako: "A crystal mounting device made from capillary for cryogenic macromolecular crystallography." J. appl. crystallography. 28. In press (1995)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] Y.Mimori: "The Structure of the R-type Straight Flagellar Filament of Salmonella at 9 Å Resolution by Electron Cryomicroscopy." J. Mol. Biol.249. 69-87 (1995)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] 豊島 近: "電子顕微鏡で膜蛋白質を見る." 化学と生物. 32. 791-796 (1994)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] 豊島 近: "アセチルコリン受容体の構造と機能" ブレインサイエンス. 5. 149-155 (1994)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] C.Toyoshima: "Three-dimensional cryo-electron microscopy of the calcium ion pump in the sarcoplamisc reticulum memebrane." Nature. 362. 469-471 (1993)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] C.Toyoshima: "Contrast transfer for the frozen hydrated specimen.II : Amplitude contrast at very low frequencies." Ultramicroscopy. 48. 165-176 (1993)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] A.Sato: "Two-dimensional crystallization of catalase on a monolayer film of poly(l-benzyl-L-histidine) spread at the air/water interface." Biochim. Biophys. Acta. 1162. 54-60 (1993)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] 豊島 近: "膜蛋白質の電子線三次元構造解析" 蛋白質・核酸・酵素. 38. 1276-1286 (1993)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] Bamberg: "The Sodium Pump : Structure,Mechanisms,Hormonal control and its role in disease." W.Springer, 120-130 (1994)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] 井村裕夫: "レセプター-基礎と臨床-" 朝倉書店, 288-296 (1993)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] Nakasako, M., Ueki, T., Toyoshima, C.and Umeda, Y.: "A crystal mounting device mada from capillary for cryogenic macromolecular crystallography" J.appl.cryst. 28 (In press).

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] Mimori, Y., Yamashita, I., Murata, K., Fujiyoshi, Y., Yonekura, K., Yoyoshima, C.and K.Namba: "The structure of the R-type straight flagellar filament of Salmonella at 9 * resolution by electron cryomicroscopy" J.Mol.Biol.249. 69-87 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] Toyoshima, C.: "Visualizing proteins by electron microscopy" Chemistry and Biology. 32. 791-796 (1994)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] Toyoshima, C.: "Structure and function of the acetylcholine receptor" Brain Science. 5. 149-155 (1994)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] Toyoshima, C., Sasabe, H., and Stokes, D.L.: "Three-dimensional cryo-electron microscopy of the calcium ion pump in the sarcoplamisc reticulum membrane" Nature. 362. 469-471 (1993)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] Toyoshima, C., Yonekura, K., and Sasabe, H.: "Contrast transfer for the frozen hydratd specimen. II : Amplitude contrast at very low frequencies" Ultramicroscopy. 48. 165-176 (1993)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] Sato, A., Furuno, T., Toyoshima, C., and Sasabe, H.: "Two-dimensional crystallization of catalase on a monlayr film of poly (1-benzyl-L-histidine) spread at the air/water interface" Bichim.Biophys.Acta. 1162. 54-60 (1993)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] Toyoshima, C.: "Three-dimensional electron microscopy of membrane proteins" Proteins, Nucleic Acids and Enzymes. 38. 1276-1286 (1993)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] Stokes, D.L., Green, N.M., Toyoshima, C.: The Sodium Pump : Structure, Mechanisms, Hormonal Control and Its Role in Disease (Bamberg and Schoner eds), "Structure studies of Ca^<2+>-ATPase by Cryoelectron Microscopy". W.Springer, New York., 120-130 (1994)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] Toyoshima, C.: Receptors--Basic and Clinical Aspects (Imura et al eda.), "Three-dimensional structural analysis". Asakura Shoten, 288-296 (1993)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] 豊島近: "アセチルコリン受容体の構造と機能" ブレインサイエンス. 5. 149-155 (1994)

    • Related Report
      1994 Annual Research Report
  • [Publications] 豊島近: "電子顕微鏡で膜蛋白質を見る" 化学と生物. 32. 791-796 (1994)

    • Related Report
      1994 Annual Research Report
  • [Publications] C.Toyoshima: "Three-dimensional cryo-electron microscopy of the calcium ion pump in the sarcoplamisc reticulum membrane." Nature. 362. 469-471 (1993)

    • Related Report
      1993 Annual Research Report
  • [Publications] C.Toyoshima: "Contrast transfer for the frozen hydrated specimen.II:Amplitude contrast at very low frequencies." Ultramicroscopy. 48. 165-176 (1993)

    • Related Report
      1993 Annual Research Report
  • [Publications] A.Sato: "Two-dimensional crystallization of catalase on a monolayer film of poly (1-benzyl-L-histidine)spread at the air/water interface." Biochim.Biophys.Acta. 1162. 54-60 (1993)

    • Related Report
      1993 Annual Research Report
  • [Publications] 豊島 近: "膜蛋白質の電子線三次元構造解析" 蛋白質・核酸・酵素. 38. 1276-1286 (1993)

    • Related Report
      1993 Annual Research Report

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Published: 1993-04-01   Modified: 2016-04-21  

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