Setting up and operating a cryo-EM laboratory.

IF 7.2 2区 生物学 Q1 BIOPHYSICS
Deryck J Mills
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引用次数: 8

Abstract

Cryo-electron microscopy (cryo-EM) has become the technique of choice for structural biology of macromolecular assemblies, after the 'resolution revolution' that has occurred in this field since 2012. With a suitable instrument, an appropriate electron detector and, last but not least, a cooperative sample it is now possible to collect images from which macromolecular structures can be determined to better than 2 Å resolution, where reliable atomic models can be built. By electron tomography and sub-tomogram averaging of cryo-samples, it is also possible to reconstruct subcellular structures to sub-nanometre resolution. This review describes the infrastructure that is needed to achieve this goal. Ideally, a cryo-EM lab will have a dedicated 300 kV electron microscope for data recording and a 200 kV instrument for screening cryo-samples, both with direct electron detectors, and at least one 120 kV EM for negative-stain screening at room temperature. Added to this should be ancillary equipment for specimen preparation, including a light microscope, carbon coater, plasma cleaner, glow discharge unit, a device for fast, robotic sample freezing, liquid nitrogen storage Dewars and a ready supply of clean liquid nitrogen. In practice, of course, the available budget will determine the number and types of microscopes and how elaborate the lab can be. The cryo-EM lab should be designed with adequate space for the electron microscopes and ancillary equipment, and should allow for sufficient storage space. Each electron microscope room should be connected to the image-processing computers by fibre-optic cables for the rapid transfer of large datasets. The cryo-EM lab should be overseen by a facility manager whose responsibilities include the day-to-day tasks to ensure that all microscopes are operating perfectly, organising service and repairs to minimise downtime, and controlling the budget. Large facilities will require additional support staff who help to oversee the operation of the facility and instruct new users.

建立和操作低温电子显微镜实验室。
自2012年该领域发生“分辨率革命”后,冷冻电子显微镜(cryo-EM)已成为大分子组装结构生物学的首选技术。有了合适的仪器,合适的电子探测器,最后但并非最不重要的是,一个合作的样品,现在可以收集图像,从中可以确定大分子结构,分辨率高于2 Å,在那里可以建立可靠的原子模型。通过电子断层扫描和亚层析成像平均冷冻样品,也可以重建亚细胞结构到亚纳米分辨率。此回顾描述了实现此目标所需的基础结构。理想情况下,冷冻电镜实验室将有一台专用的300千伏电子显微镜用于数据记录,一台200千伏仪器用于筛选冷冻样品,两者都有直接电子探测器,至少一台120千伏电子显微镜用于室温下的阴性染色筛选。除此之外,还应该有用于标本制备的辅助设备,包括光学显微镜、碳涂层机、等离子体清洁器、辉光放电装置、快速机器人样品冷冻装置、液氮储存杜瓦瓶和现成的清洁液氮供应。当然,在实践中,可用的预算将决定显微镜的数量和类型,以及实验室的复杂程度。低温电镜实验室应设计为电子显微镜和辅助设备提供足够的空间,并应允许足够的存储空间。每个电子显微镜室应通过光纤电缆连接到图像处理计算机,以便快速传输大型数据集。低温电镜实验室应由一名设施经理监督,其职责包括日常任务,以确保所有显微镜完美运行,组织服务和维修以尽量减少停机时间,并控制预算。大型设施将需要额外的支助人员来帮助监督设施的运作并指导新用户。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
Quarterly Reviews of Biophysics
Quarterly Reviews of Biophysics 生物-生物物理
CiteScore
12.90
自引率
1.60%
发文量
16
期刊介绍: Quarterly Reviews of Biophysics covers the field of experimental and computational biophysics. Experimental biophysics span across different physics-based measurements such as optical microscopy, super-resolution imaging, electron microscopy, X-ray and neutron diffraction, spectroscopy, calorimetry, thermodynamics and their integrated uses. Computational biophysics includes theory, simulations, bioinformatics and system analysis. These biophysical methodologies are used to discover the structure, function and physiology of biological systems in varying complexities from cells, organelles, membranes, protein-nucleic acid complexes, molecular machines to molecules. The majority of reviews published are invited from authors who have made significant contributions to the field, who give critical, readable and sometimes controversial accounts of recent progress and problems in their specialty. The journal has long-standing, worldwide reputation, demonstrated by its high ranking in the ISI Science Citation Index, as a forum for general and specialized communication between biophysicists working in different areas. Thematic issues are occasionally published.
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