Takizawa Yoshimasa, Ho Cheng-Han, Sato Shoko, Danev Radostin, Kurumizaka Hitoshi
Laboratory of Chromatin Structure and Function, Institute for Quantitative Biosciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan.
Graduate School of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo, Japan.
Methods Mol Biol. 2025;2919:91-107. doi: 10.1007/978-1-0716-4486-7_6.
The fundamental chromatin unit is the nucleosome, in which approximately 150 base pairs of DNA are bound to the surface of a symmetric histone octamer containing 2 copies each of histones H2A, H2B, H3, and H4. Over the years, numerous structures of nucleosomes have been determined by X-ray crystallography. However, their structural and functional versatility may not have been fully revealed, due to crystal packing effects. Various structures of nucleosomes and their complexes with nucleosome-binding proteins are now being determined by cryo-electron microscopy (cryo-EM) single-particle analysis, allowing the visualization of their structural diversity. In this report, we present a method for high-resolution structural analyses of nucleosomes by cryo-EM and describe the detailed procedures for nucleosome purification, cryo-EM grid preparation, data collection, and data processing. This method can serve as a good starting point for cryo-EM investigations of nucleosomes and their wide range of complexes.
基本的染色质单位是核小体，其中约150个碱基对的DNA与一个对称的组蛋白八聚体表面结合，该八聚体包含组蛋白H2A、H2B、H3和H4各两个拷贝。多年来，通过X射线晶体学已经确定了核小体的众多结构。然而，由于晶体堆积效应，它们的结构和功能多样性可能尚未完全揭示。现在，通过冷冻电子显微镜（cryo-EM）单颗粒分析正在确定核小体及其与核小体结合蛋白的复合物的各种结构，从而能够可视化它们的结构多样性。在本报告中，我们介绍了一种通过cryo-EM对核小体进行高分辨率结构分析的方法，并描述了核小体纯化、cryo-EM网格制备、数据收集和数据处理的详细步骤。该方法可作为对核小体及其广泛复合物进行cryo-EM研究的良好起点。
