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

对于这个研究项目，我们的目标是改进和发展方法和工作流程，使常规的膜相关大分子络合物的结构原位测定成为可能。我们将低温电子断层扫描(CryoET)与相关光学显微镜和低温聚焦离子束(Cryo-FIB)球磨相结合，开发了一个健壮的工作流程。为了从足够多的靶分子中获得数据，低温电子断层扫描数据采集是主要瓶颈之一。为了克服这个问题，我们开发了新的软件，通过考虑样本的3D几何形状来并行数据采集，从而大大加快了数据收集的速度(Eisenstein等人，自然方法，2023)。光学图像移位的使用可以将CryoET数据收集的速度提高3-5倍，优化最先进的电子显微镜的使用，并最大限度地提高每个样品区域的数据输出。这项新技术不仅使我自己的研究受益，也使其他使用该设备的研究人员的研究受益。例如，我们可以与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)，这将为未来各种与上皮细胞屏障相关的疾病的研究奠定基础。当我致力于收集和处理GPCRs的体外结构数据(Danev等人，自然通讯，2021年)时，还不可能原位解决像GPCRs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，未来的研究将从我们的工作中受益。

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.