Samanta Amrita, Hughes Taylor E T, Moiseenkova-Bell Vera Y
Department of Systems Pharmacology and Translational Therapeutics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.
Methods Mol Biol. 2019;1987:39-50. doi: 10.1007/978-1-4939-9446-5_3.
Cryo electron microscopy (cryo-EM) is a powerful technique that can be used to elucidate the structural architecture of a protein molecule in a physiologically relevant environment. In this method, purified protein is frozen in its aqueous buffer in a thin layer of vitreous ice in which the biological macromolecules are embedded in various orientations. Images of this frozen sample are collected with an electron microscope, and the data is processed using different software algorithms resulting in high-resolution structures of the protein. Proteins in the presence of various ligands or other macromolecular complexes can also be studied by this method. Here, we present a protocol for the purification and vitrification of TRP channels for single particle cryo-EM.
冷冻电子显微镜（cryo-EM）是一种强大的技术，可用于在生理相关环境中阐明蛋白质分子的结构架构。在该方法中，纯化的蛋白质在其水性缓冲液中被冷冻在一层玻璃态冰中，生物大分子以各种取向嵌入其中。用电子显微镜收集该冷冻样品的图像，并使用不同的软件算法处理数据，从而得到蛋白质的高分辨率结构。该方法还可用于研究存在各种配体或其他大分子复合物时的蛋白质。在此，我们展示了一种用于TRP通道单颗粒冷冻电子显微镜纯化和玻璃化的方案。
