通过低温电子显微镜确定组蛋白甲基转移酶与核小体复合物的结构。
Determination of Histone Methyltransferase Structures in Complex with the Nucleosome by Cryogenic Electron Microscopy.
机构信息
Department of Biochemistry and Biophysics, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.
Division of Chemical Biology and Medicinal Chemistry, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.
出版信息
Methods Mol Biol. 2022;2529:149-168. doi: 10.1007/978-1-0716-2481-4_8.
Cryogenic electron microscopy (cryo-EM) has recently emerged as an optimal technique for the determination of histone methyltransferase-nucleosome complex structures. Histone methyltransferases are a group of enzymes that posttranslationally methylate histone lysine and arginine residues on the nucleosome, providing important epigenetic signals that regulate gene expression. Here we describe a protocol to solve the structure of histone lysine methyltransferase Dot1L bound to a chemically ubiquitylated nucleosome, including complex reconstitution, crosslinking, grid preparation, and data collection and analysis. Throughout, we discuss key steps requiring optimization to allow this protocol to serve as a starting point for the determination of new histone methyltransferase-nucleosome complex structures.
低温电子显微镜（cryo-EM）最近成为确定组蛋白甲基转移酶-核小体复合物结构的最佳技术。组蛋白甲基转移酶是一组酶，它们在翻译后修饰核小体上的组蛋白赖氨酸和精氨酸残基，提供重要的表观遗传信号，调节基因表达。在这里，我们描述了一种解决组蛋白赖氨酸甲基转移酶 Dot1L 与化学泛素化核小体结合的结构的方案，包括复合物重建、交联、网格制备以及数据收集和分析。在整个过程中，我们讨论了需要优化的关键步骤，以使该方案成为确定新的组蛋白甲基转移酶-核小体复合物结构的起点。
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