Laboratory of Membrane Protein Biology, National Centre for Cell Science, NCCS Complex, S. P. Pune University, Pune, Maharashtra 411007, India.
Emerg Top Life Sci. 2021 May 14;5(1):151-168. doi: 10.1042/ETLS20200295.
Cryo-electron microscopy (CryoEM) has superseded X-ray crystallography and NMR to emerge as a popular and effective tool for structure determination in recent times. It has become indispensable for the characterization of large macromolecular assemblies, membrane proteins, or samples that are limited, conformationally heterogeneous, and recalcitrant to crystallization. Besides, it is the only tool capable of elucidating high-resolution structures of macromolecules and biological assemblies in situ. A state-of-the-art electron microscope operable at cryo-temperature helps preserve high-resolution details of the biological sample. The structures can be determined, either in isolation via single-particle analysis (SPA) or helical reconstruction, electron diffraction (ED) or within the cellular environment via cryo-electron tomography (cryoET). All the three streams of SPA, ED, and cryoET (along with subtomogram averaging) have undergone significant advancements in recent times. This has resulted in breaking the boundaries with respect to both the size of the macromolecules/assemblies whose structures could be determined along with the visualization of atomic details at resolutions unprecedented for cryoEM. In addition, the collection of larger datasets combined with the ability to sort and process multiple conformational states from the same sample are providing the much-needed link between the protein structures and their functions. In overview, these developments are helping scientists decipher the molecular mechanism of critical cellular processes, solve structures of macromolecules that were challenging targets for structure determination until now, propelling forward the fields of biology and biomedicine. Here, we summarize recent advances and key contributions of the three cryo-electron microscopy streams of SPA, ED, and cryoET.
冷冻电子显微镜（CryoEM）已经取代了 X 射线晶体学和 NMR，成为近年来用于结构测定的一种流行且有效的工具。它已经成为研究大分子组装体、膜蛋白或数量有限、构象多样且不易结晶的样品的不可或缺的工具。此外，它是唯一能够原位阐明大分子和生物组装体高分辨率结构的工具。一台可在低温下运行的最先进的电子显微镜有助于保留生物样品的高分辨率细节。可以通过单颗粒分析（SPA）、螺旋重建、电子衍射（ED）或在细胞环境中通过冷冻电子断层扫描（cryoET）来确定结构。SPA、ED 和 cryoET 的所有三个流（以及亚结构平均）在最近都取得了重大进展。这使得在大分子/组装体的大小方面取得了突破，可以确定其结构，并且可以在 cryoEM 前所未有的分辨率下可视化原子细节。此外，更大数据集的收集以及从同一样品中对多个构象状态进行分类和处理的能力，为蛋白质结构与其功能之间提供了急需的联系。总之，这些发展正在帮助科学家破译关键细胞过程的分子机制，解决迄今为止结构测定具有挑战性的大分子的结构，推动生物学和生物医学领域的发展。在这里，我们总结了 SPA、ED 和 cryoET 这三个 cryoEM 流的最新进展和关键贡献。
