Molecular Biophysics Unit, Indian Institute of Science.
Gatan Inc.
J Vis Exp. 2021 Jul 29(173). doi: 10.3791/62832.
In the past several years, technological and methodological advancements in single-particle cryo-electron microscopy (cryo-EM) have paved a new avenue for the high-resolution structure determination of biological macromolecules. Despite the remarkable advances in cryo-EM, there is still scope for improvement in various aspects of the single-particle analysis workflow. Single-particle analysis demands a suitable software package for high-throughput automatic data acquisition. Several automatic data acquisition software packages were developed for automatic imaging for single-particle cryo-EM in the last eight years. This paper presents an application of a fully automated image acquisition pipeline for vitrified biomolecules under low-dose conditions. It demonstrates a software package, which can collect cryo-EM data fully, automatically, and precisely. Additionally, various microscopic parameters are easily controlled by this software package. This protocol demonstrates the potential of this software package in automated imaging of the severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) spike protein with a 200 keV cryo-electron microscope equipped with a direct electron detector (DED). Around 3,000 cryo-EM movie images were acquired in a single session (48 h) of data collection, yielding an atomic-resolution structure of the spike protein of SARS-CoV-2. Furthermore, this structural study indicates that the spike protein adopts two major conformations, 1-RBD (receptor-binding domain) up open and all RBD down closed conformations.
在过去的几年中，单颗粒冷冻电子显微镜（cryo-EM）技术和方法的进步为生物大分子的高分辨率结构测定开辟了新途径。尽管 cryo-EM 取得了显著进展，但在单颗粒分析工作流程的各个方面仍有改进的空间。单颗粒分析需要一个合适的软件包来进行高通量自动数据采集。在过去的八年中，开发了几个用于单颗粒 cryo-EM 自动成像的自动数据采集软件包。本文介绍了一种用于低剂量条件下玻璃化生物分子的全自动图像采集管道的应用。它演示了一个可以完全、自动和精确地收集 cryo-EM 数据的软件包。此外，该软件包还可以轻松控制各种显微镜参数。该方案展示了该软件包在配备直接电子探测器（DED）的 200keV 冷冻电子显微镜中自动成像严重急性呼吸综合征冠状病毒 2（SARS-CoV-2）刺突蛋白的潜力。在单个数据采集会话（48 小时）中，共采集了约 3000 张 cryo-EM 电影图像，获得了 SARS-CoV-2 刺突蛋白的原子分辨率结构。此外，这项结构研究表明，刺突蛋白采用两种主要构象，即 1-RBD（受体结合域）向上开放和所有 RBD 向下关闭构象。
