研究実績の概要 |
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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