Determination of Histone Methyltransferase Structures in Complex with the Nucleosome by Cryogenic Electron Microscopy.
Determination of Histone Methyltransferase Structures in Complex with the Nucleosome by Cryogenic Electron Microscopy.
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DOI：
10.1007/978-1-0716-2481-4_8
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发表时间：
2022
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低温电子显微镜（cryo-EM）最近成为测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翻译后甲基化核小体上的组蛋白赖氨酸和精氨酸残基的酶，提供调控基因表达的重要表观遗传信号。在这里，我们描述了一种方案来解决与化学泛素化核小体结合的组蛋白赖氨酸甲基转移酶Dot1L的结构，包括复杂的重构、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，以允许该方案作为确定新的组蛋白甲基转移酶-核小体复合物结构的起点。
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 post-translationally 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.