Single-particle cryo-electron microscopy (cryoEM) provides an attractive avenue for advancing our atomic resolution understanding of materials, molecules and living systems. However, the vast majority of published cryoEM methodologies focus on the characterization of aerobically purified samples. Air-sensitive enzymes and microorganisms represent important yet understudied systems in structural biology. We have recently demonstrated the success of an anaerobic single-particle cryoEM workflow applied to the air-sensitive nitrogenase enzymes. In this protocol, we detail the use of Schlenk lines and anaerobic chambers to prepare samples, including a protein tag for monitoring sample exposure to oxygen in air. We describe how to use a plunge freezing apparatus inside of a soft-sided vinyl chamber of the type we routinely use for anaerobic biochemistry and crystallography of oxygen-sensitive proteins. Manual control of the airlock allows for introduction of liquid cryogens into the tent. A custom vacuum port provides slow, continuous evacuation of the tent atmosphere to avoid accumulation of flammable vapors within the enclosed chamber. These methods allowed us to obtain high-resolution structures of both nitrogenase proteins using single-particle cryoEM. The procedures involved can be generally subdivided into a 4 d anaerobic sample generation procedure, and a 1 d anaerobic cryoEM sample preparation step, followed by conventional cryoEM imaging and processing steps. As nitrogen is a substrate for nitrogenase, the Schlenk lines and anaerobic chambers described in this procedure are operated under an argon atmosphere; however, the system and these procedures are compatible with other controlled gas environments.

单粒子冷冻电子显微镜（cryoEM）为推进我们对材料、分子和生命系统的原子分辨率理解提供了一个有吸引力的途径。然而，绝大多数已发表的冷冻电镜方法都侧重于有氧纯化样品的表征。空气敏感酶和微生物代表了结构生物学中重要但尚未得到充分研究的系统。我们最近展示了应用于空气敏感固氮酶的厌氧单颗粒冷冻电镜工作流程的成功。在本协议中，我们详细介绍了使用 Schlenk 线和厌氧室来制备样品，包括用于监测样品暴露于空气中氧气的蛋白质标签。我们描述了如何在软面乙烯基室内使用浸入式冷冻装置，这种装置通常用于氧敏感蛋白的厌氧生物化学和晶体学。气闸室的手动控制允许将液体冷冻剂引入帐篷中。定制的真空端口可缓慢、连续地抽空帐篷内的空气，以避免封闭室内积聚易燃蒸气。这些方法使我们能够使用单颗粒冷冻电镜获得两种固氮酶蛋白的高分辨率结构。所涉及的程序通常可分为 4 d 厌氧样品生成程序和 1 d 厌氧冷冻电镜样品制备步骤，然后是传统的冷冻电镜成像和处理步骤。由于氮气是固氮酶的底物，因此本程序中描述的 Schlenk 生产线和厌氧室在氩气气氛下运行；然而，该系统和这些程序与其他受控气体环境兼容。