| Project/Area Number |
09480171
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Biophysics
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| Research Institution | The University of Tokyo |
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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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| Project Period (FY) |
1997 – 1998
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| Project Status |
Completed (Fiscal Year 1998)
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| Budget Amount *help |
¥13,800,000 (Direct Cost: ¥13,800,000)
Fiscal Year 1998: ¥2,300,000 (Direct Cost: ¥2,300,000)
Fiscal Year 1997: ¥11,500,000 (Direct Cost: ¥11,500,000)
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| Keywords | ion pump / sarcoplasmic reticulum / active transport / three-dimensional structure / electron crystallography / structural analysis / membrane proteins / X-ray crystallography |
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| Research Abstract |
Crystallographic study of the calcium ion pump (Ca^<2+>-ATPase) of sarcoplasmic reticulum(SR)has been performed to elucidate the structural basis of active transport. Two types of crystal of CA^<2+>-ATPase were known when we started this project : one was tubular crystal of native SR membrane formed in the presence of EGTA and vanadate ; the other was small three-dimensional crystals consisting of reconstituted membrane sheets formed in the presence of high concentration of calcium. Both were small and suitable for electron crystallography. Using tubular crystals, three-dimensional structures were obtained for three states at 8 A resolution : no ATP, with chromium-ATP and with thapsigargin (the last one was in collaboration with Stokes Lab at NY University). The one with bound thapsigargin was best resolved and clearly showed 10 transmembrane helices expected from secondary structure predictions (published in Nature). The one with chromium ATP indicated the position of ATP-binding site(published in Biophys. J.). We have developed an extensive set of programs for this purpose. Comparison of the projection along the b-axis of the small 3D crystals and 3D structure from tubular crystals revealed large scale conformational changes attributable to the binding of calcium (Biophys. J.).
By improving the crystallisation conditions, we succeeded in preparing well ordered plate-like crystals of this ion pump. The thinnest ones could be analysed by electron and the thickest ones by X-ray crystallography. We have developed a rotation camera for collecting electron diffraction intensities and learned that even the thinnest crystals have virtually the same lattice constants with the thickest ones. We therefore proceeded to collect phase information directly from the electron micrographs and obtained a very useful set of phases for certain reflections. We are now looking for good isomorphous heavy metal derivatives for solving the structure at an atomic resolution.
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