Materials and Structural Analysis Division, Thermo Fisher Scientific.
Max Planck Institute of Biochemistry, Molecular Structural Biology.
J Vis Exp. 2022 Mar 16(181). doi: 10.3791/63519.
Cryo-electron microscopy (cryo-EM) has been established as a routine method for protein structure determination during the past decade, taking an ever-increasing share of published structural data. Recent advances in TEM technology and automation have boosted both the speed of data collection and quality of acquired images while simultaneously decreasing the required level of expertise for obtaining cryo-EM maps at sub-3 Å resolutions. While most of such high-resolution structures have been obtained using state-of-the-art 300 kV cryo-TEM systems, high-resolution structures can be also obtained with 200 kV cryo-TEM systems, especially when equipped with an energy filter. Additionally, automation of microscope alignments and data collection with real-time image quality assessment reduces system complexity and assures optimal microscope settings, resulting in increased yield of high-quality images and overall throughput of data collection. This protocol demonstrates the implementation of recent technological advances and automation features on a 200 kV cryo-transmission electron microscope and shows how to collect data for the reconstruction of 3D maps that are sufficient for de novo atomic model building. We focus on best practices, critical variables, and common issues that must be considered to enable the routine collection of such high-resolution cryo-EM datasets. Particularly the following essential topics are reviewed in detail: i) automation of microscope alignments, ii) selection of suitable areas for data acquisition, iii) optimal optical parameters for high-quality, high-throughput data collection, iv) energy filter tuning for zero-loss imaging, and v) data management and quality assessment. Application of the best practices and improvement of achievable resolution using an energy filter will be demonstrated on the example of apo-ferritin that was reconstructed to 1.6 Å, and Thermoplasma acidophilum 20S proteasome reconstructed to 2.1-Å resolution using a 200 kV TEM equipped with an energy filter and a direct electron detector.
冷冻电子显微镜（cryo-EM）在过去十年中已成为蛋白质结构测定的常规方法，发表的结构数据中越来越多地采用 cryo-EM 方法。透射电子显微镜（TEM）技术和自动化的最新进展提高了数据采集的速度和获取图像的质量，同时降低了获得亚 3Å分辨率 cryo-EM 图谱所需的专业水平。虽然大多数这样的高分辨率结构都是使用最先进的 300kV cryo-TEM 系统获得的，但也可以使用 200kV cryo-TEM 系统获得高分辨率结构，特别是配备能量过滤器时。此外，显微镜对准和数据采集的自动化以及实时图像质量评估降低了系统的复杂性，并确保了最佳的显微镜设置，从而提高了高质量图像的产量和数据采集的整体通量。本方案展示了在 200kV cryo 传输电子显微镜上实施最新技术进步和自动化功能，并展示了如何收集用于重建足以从头开始构建原子模型的 3D 图谱的数据。我们重点介绍了实现此类高分辨率 cryo-EM 数据集常规采集所必须考虑的最佳实践、关键变量和常见问题。特别是详细回顾了以下重要主题：i）显微镜对准的自动化，ii）适合数据采集的区域选择，iii）用于高质量、高通量数据采集的最佳光学参数，iv）用于零损耗成像的能量过滤器调谐，以及 v）数据管理和质量评估。将最佳实践的应用和使用能量过滤器提高可实现的分辨率的方法将以apo-铁蛋白（重建到 1.6Å）和嗜热嗜酸古菌 20S 蛋白酶体（重建到 2.1-Å 分辨率）的重建为例进行演示，这两个结构都是使用配备能量过滤器和直接电子探测器的 200kV TEM 获得的。
