Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, United Kingdom; Central Oxford Structural Microscopy and Imaging Centre, South Parks Road, Oxford OX1 3RE, United Kingdom.
Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, United Kingdom; Central Oxford Structural Microscopy and Imaging Centre, South Parks Road, Oxford OX1 3RE, United Kingdom.
Prog Biophys Mol Biol. 2021 Mar;160:97-103. doi: 10.1016/j.pbiomolbio.2020.05.010. Epub 2020 Jun 21.
Electron cryotomography is a rapidly evolving method for imaging macromolecules directly within the native environment of cells and tissues. Combined with sub-tomogram averaging, it allows structural and cell biologists to obtain sub-nanometre resolution structures in situ. However, low throughput in cryo-ET sample preparation and data acquisition, as well as difficulties in target localisation and sub-tomogram averaging image processing, limit its widespread usability. In this review, we discuss new advances in the field that address these throughput and technical problems. We focus on recent efforts made to resolve issues in sample thinning, improvement in data collection speed at the microscope, strategies for localisation of macromolecules using correlated light and electron microscopy and advancements made to improve resolution in sub-tomogram averaging. These advances will considerably decrease the amount of time and effort required for cryo-ET and sub-tomogram averaging, ushering in a new era of structural biology where in situ macromolecular structure determination will be routine.
电子晶体学断层扫描是一种快速发展的方法，可直接在细胞和组织的天然环境中对大分子进行成像。与亚断层平均法相结合，它使结构和细胞生物学家能够原位获得亚纳米分辨率的结构。然而，低温电子断层扫描样品制备和数据采集的低通量，以及目标定位和亚断层平均图像处理的困难，限制了它的广泛应用。在这篇综述中，我们讨论了该领域解决这些通量和技术问题的新进展。我们专注于最近在解决样品变薄、显微镜数据收集速度提高、利用相关光和电子显微镜对大分子进行定位的策略以及提高亚断层平均分辨率方面的努力。这些进展将大大减少低温电子断层扫描和亚断层平均所需的时间和精力，迎来结构生物学的新时代，届时原位测定大分子结构将成为常规。
