Sun Willy W, Michalak Dennis J, Sochacki Kem A, Kunamaneni Prasanthi, Alfonzo-Méndez Marco A, Arnold Andreas M, Strub Marie-Paule, Hinshaw Jenny E, Taraska Justin W
National Heart, Lung, and Blood Institute, US National Institutes of Health, Bethesda, Maryland, USA.
These authors contributed equally.
bioRxiv. 2024 Jun 28:2024.06.27.600657. doi: 10.1101/2024.06.27.600657.
Cryo-electron tomography (cryoET) provides sub-nanometer protein structure within the dense cellular environment. Existing sample preparation methods are insufficient at accessing the plasma membrane and its associated proteins. Here, we present a correlative cryo-electron tomography pipeline optimally suited to image large ultra-thin areas of isolated basal and apical plasma membranes. The pipeline allows for angstrom-scale structure determination with sub-tomogram averaging and employs a genetically-encodable rapid chemically-induced electron microscopy visible tag for marking specific proteins within the complex cell environment. The pipeline provides fast, efficient, distributable, low-cost sample preparation and enables targeted structural studies of identified proteins at the plasma membrane of cells.
冷冻电子断层扫描（cryoET）可在致密的细胞环境中提供亚纳米级别的蛋白质结构。现有的样品制备方法在获取质膜及其相关蛋白质方面存在不足。在此，我们展示了一种相关冷冻电子断层扫描流程，它最适合对分离的基底和顶端质膜的大型超薄区域进行成像。该流程允许通过亚断层平均进行埃级规模的结构测定，并采用一种可遗传编码的快速化学诱导电子显微镜可见标签，用于在复杂的细胞环境中标记特定蛋白质。该流程提供了快速、高效、可分发、低成本的样品制备方法，并能够对细胞的质膜上已鉴定的蛋白质进行靶向结构研究。
