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

对于本研究项目，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）与相关光学显微镜和冷冻聚焦离子束（cryoFIB）铣削相结合，开发了一个强大的工作流程。为了克服这一点，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，从而大大加快了数据收集（Eisenstein等人，Nature Methods，2023）。光学图像偏移的使用使得cryoET数据收集的速度提高了3- 5倍，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本区域的数据输出。这项新技术不仅使我自己的研究受益，也使其他研究人员的研究受益。例如，我们可以与Sachiko Tsukita教授合作对上皮细胞-细胞边界的结构进行成像（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。当我致力于收集和处理GPCR的体外结构数据时（Danev et al.，Nature Communications，2021），但还不可能原位解决像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。

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.