1994 Fiscal Year Final Research Report Summary
Development of technologise for three-dimensional structural analysis of protein crystals using electron microscope
| Project/Area Number |
05508004
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| Research Category |
Grant-in-Aid for Developmental Scientific Research (A)
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| Allocation Type | Single-year Grants |
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| Research Field |
Biophysics
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| Research Institution | The University of Tokyo (1994)
Tokyo Institute of Technology (1993) |
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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)
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| 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
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| Project Period (FY) |
1993 – 1994
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| Keywords | Electron microscope / Protein crystals / Electron diffraction / Ice embedding / Structural analysis / Radiation damage / Cryo-transfer |
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| 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.
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[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
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[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
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