Institute of Biological Chemistry, Academia Sinica; Academia Sinica Cryo-EM Facility, Academia Sinica.
Department of Life Sciences, National Central University; Hubei University.
J Vis Exp. 2021 Jul 26(173). doi: 10.3791/62772.
The sample grids for cryo-electron microscopy (cryo-EM) experiments are usually prepared at a temperature optimal for the storage of biological samples, mostly at 4 °C and occasionally at room temperature. Recently, we discovered that the protein structure solved at low temperature may not be functionally relevant, particularly for proteins from thermophilic archaea. A procedure was developed to prepare protein samples at higher temperatures (up to 70 °C) for cryo-EM analysis. We showed that the structures from samples prepared at higher temperatures are functionally relevant and temperature dependent. Here we describe a detailed protocol for preparing sample grids at high temperature, using 55 °C as an example. The experiment made use of a vitrification apparatus modified using an additional centrifuge tube, and samples were incubated at 55 °C. The detailed procedures were fine-tuned to minimize vapor condensation and obtain a thin layer of ice on the grid. Examples of successful and unsuccessful experiments are provided.
用于冷冻电子显微镜（cryo-EM）实验的样品网格通常在最适合储存生物样品的温度下制备，大多数情况下为 4°C，偶尔也在室温下。最近，我们发现低温下解决的蛋白质结构可能与功能无关，特别是对于来自嗜热古菌的蛋白质。我们开发了一种在较高温度（高达 70°C）下制备 cryo-EM 分析用蛋白质样品的方法。我们表明，在较高温度下制备的样品的结构在功能上是相关的，并且与温度有关。这里我们描述了一个在高温下制备样品网格的详细方案，以 55°C 为例。该实验使用了一个经过改进的玻璃化设备，其中使用了一个额外的离心管，并且将样品在 55°C 下孵育。详细的程序经过了微调，以最大程度地减少蒸汽冷凝并在网格上获得薄冰层。提供了成功和不成功实验的示例。
