New cryo-electron microscopy method for elucidation of G-protein coupled receptor supercomplexes

用于阐明 G 蛋白偶联受体超复合物的新冷冻电子显微镜方法

• 批准号: 21F20764
• 负责人: Danev Radostin
• 金额: $ 1.47万
• 依托单位: The University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for JSPS Fellows
• 财政年份: 2021
• 资助国家: 日本
• 起止时间: 2021-04-28 至 2023-03-31
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-21F20764/
• 关键词: cryo-EM; cryo-ET; cryo-FIB; in situ; visual proteomics; cryo-electron microscopy; single particle analysis; GPCR; cryo-electron tomography

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.

在这个研究项目中，我们的目标是改进和开发方法和工作流程，使膜相关大分子复合物的常规原位结构测定可行。我们将冷冻电子断层扫描（cryoET）与相关的光学显微镜和冷冻聚焦离子束（cryoFIB）铣削相结合，以开发一个强大的工作流程。为了从足够多的目标分子中获取数据，低温et数据采集是主要的瓶颈之一。为了克服这个问题，我们开发了新的软件，通过考虑样品的3D几何形状来并行化数据采集，从而大大加快数据收集速度（Eisenstein等人，Nature Methods, 2023）。光学图像移位的使用允许3-5倍的cryoET数据收集速度加快，优化了最先进的电子显微镜的使用，最大限度地提高了每个样品区域的数据输出。这项新技术不仅有利于我自己的研究，也有利于使用该设备的其他研究人员的研究。例如，我们可以与Sachiko Tsukita教授合作成像上皮细胞-细胞边界的结构（手稿正在准备中），这将为未来各种上皮细胞屏障相关疾病的研究奠定基础。当我从事gpcr体外结构数据的收集和处理时（Danev et al., Nature Communications, 2021），尚无法原位解决像gpcr这样的小膜蛋白的结构。尽管如此，实现这一目标的重要基础已经奠定，未来的研究将受益于我们的工作。

项目成果

Parallel cryo electron tomography

并行冷冻电子断层扫描

• 发表时间: 2022
• 作者: de Bernardi;Cecilia;Edelheim;Johan;& Crossley;Emilie;Fabian Eisenstein
• 通讯作者: Fabian Eisenstein

High throughput tomography data acquisition

高通量断层扫描数据采集

• 发表时间: 2022
• 作者: Fabian Eisenstein

PACEtomo: Parallel cryo electron tomography

PACEtomo：并行冷冻电子断层扫描

Parallel cryo electron tomography on in situ lamellae

• DOI: 10.1038/s41592-022-01690-1
• 发表时间: 2022-12-01
• 期刊: NATURE METHODS
• 影响因子: 48
• 作者: Eisenstein, Fabian;Yanagisawa, Haruaki;Danev, Radostin
• 通讯作者: Danev, Radostin