New cryo-electron microscopy method for elucidation of G-protein coupled receptor supercomplexes
| Project/Area Number |
21F20764
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| Research Category |
Grant-in-Aid for JSPS Fellows
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| Allocation Type | Single-year Grants |
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| Section | 外国 |
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| Review Section |
Basic Section 43020:Structural biochemistry-related
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| Research Institution | The University of Tokyo |
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Principal Investigator |
Danev Radostin 東京大学, 大学院医学系研究科(医学部), 教授 (50415931)
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| Co-Investigator(Kenkyū-buntansha) |
EISENSTEIN FABIAN 東京大学, 医学(系)研究科(研究院), 外国人特別研究員
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| Project Period (FY) |
2021-04-28 – 2023-03-31
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| Project Status |
Completed (Fiscal Year 2022)
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| Budget Amount *help |
¥2,300,000 (Direct Cost: ¥2,300,000)
Fiscal Year 2022: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 2021: ¥1,200,000 (Direct Cost: ¥1,200,000)
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| Keywords | cryo-EM / cryo-ET / cryo-FIB / in situ / visual proteomics / cryo-electron microscopy / single particle analysis / GPCR / cryo-electron tomography |
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| Outline of Research at the Start |
Important membrane protein complexes like G-protein coupled receptors are involved in signaling pathways of many eukaryotic cells. Malfunctions in these pathways can cause chronic diseases like obesity, diabetes, nervous system disorders and more, making them attractive targets for drug development. This project aims to develop methods suited to gain structural and organizational insight into proteins in their native environment inside cell membranes. These methods will include cryo-electron microscopy, cryo-electron tomography, cryo-focused ion beam milling and correlative light microscopy.
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| Outline of Annual Research Achievements |
For this research project, it was our goal to improve and develop methods and workflows making routine in situ structure determination of membrane associated macromolecular complexes feasible. We combined cryo-electron tomography (cryoET) with correlative light microscopy and cryo-focused ion beam (cryoFIB) milling to develop a robust workflow.
To obtain data from a sufficiently large number of target molecules, cryoET data acquisition was one of the major bottlenecks. To overcome this, we developed new software to drastically speed up data collection by considering the 3D geometry of the sample to parallelize the data acquisition (Eisenstein et al., Nature Methods, 2023). The use of optical image shift allowed for a 3-5x speed up of cryoET data collection optimising the use of state-of-the-art electron microscopes on top of maximising the data output per sample area. This new technique benefitted not only my own research but also the research of other researchers using the facilities. For example, we could image the architecture of epithelial cell-cell boundaries in collaboration with Prof. Sachiko Tsukita (manuscript in preparation), which will lay the groundwork for future research of a variety of epithelial cell barrier related diseases.
While I worked on collection and processing of in vitro structural data of GPCRs (Danev et al., Nature Communications, 2021), it was not possible yet to solve the structure of small membrane proteins like GPCRs in situ. Nonetheless, important foundations towards this goal have been laid and future research will benefit from our work.
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| Research Progress Status |
令和4年度が最終年度であるため、記入しない。
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| Strategy for Future Research Activity |
令和4年度が最終年度であるため、記入しない。
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Report
(2 results)
Research Products
(10 results)
