Advanced Bioimaging Research Technology Platform, University of Warwick, Gibbet Hill Road, Coventry, CV4 7AL, UK.
Faraday Discuss. 2022 Nov 8;240(0):44-54. doi: 10.1039/d2fd00060a.
Grid freezing is a critical step for successful cryo-transmission electron microscopy, and optimising freezing conditions is a considerable bottleneck in many projects. To improve reproducibility in grid preparation, temperature- and humidity-controlled chambers were built into the second generation of plunge-freezers, including the ThermoFisherScientific Vitrobot and Leica GP. Since then, for most published structures, the proteins were plunge-frozen from a cold, humid environment. This provides two benefits: many proteins are more stable at 4 °C than room temperature, and both the low temperature and the humidity help control evaporation of the tiny drop of liquid. However, for optimal stability, certain samples may have different requirements. Here, we describe various (reversible) adaptations made to a Leica GP2 system to accommodate several samples with special handling requirements: a protein that is sensitive to both light and oxygen, a sample that needs to be kept at 37 °C throughout the plunge-freezing process, and a method to freeze a polymer that gels at 37 °C in its gelled state. While some of these methods are specific to these specimens, we hope sharing the ideas behind them will help people who are dealing with tricky protein samples.
网格冻结是成功进行低温透射电子显微镜的关键步骤，而优化冻结条件是许多项目中的一个重要瓶颈。为了提高网格制备的重现性，包括 ThermoFisherScientific Vitrobot 和 Leica GP 在内的第二代 plunge-freezers 中内置了温度和湿度控制腔。从那时起，对于大多数已发表的结构，蛋白质都是从寒冷、潮湿的环境中进行 plunge 冷冻的。这提供了两个好处：许多蛋白质在 4°C 下比在室温下更稳定，低温和湿度都有助于控制微小液滴的蒸发。然而，为了获得最佳稳定性，某些样品可能有不同的要求。在这里，我们描述了对 Leica GP2 系统进行的各种（可逆）适应性调整，以适应具有特殊处理要求的几个样品：一种对光和氧都敏感的蛋白质、一种在整个 plunge-freezing 过程中需要保持在 37°C 的样品，以及一种在其凝胶状态下在 37°C 下凝胶的聚合物的冷冻方法。虽然这些方法中的一些特定于这些标本，但我们希望分享这些方法背后的思路将有助于处理棘手蛋白质样本的人们。
