用于结构测定的含有 H2A.Z 和 H4K20Ecx 的核小体与 SUV420H1 复合物的制备方案。

Protocol for preparing SUV420H1 in complex with the nucleosome containing H2A.Z and H4K20Ecx for structure determination.

机构信息

National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China; Key Laboratory of Epigenetic Regulation and Intervention, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China.

出版信息

STAR Protoc. 2024 Sep 20;5(3):103295. doi: 10.1016/j.xpro.2024.103295. Epub 2024 Sep 6.

DOI:10.1016/j.xpro.2024.103295

PMID:39243379

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC11408314/

Abstract

The histone lysine methyltransferase SUV420H1 preferentially targets the H2A.Z-containing nucleosome core particle (H2A.Z-NCP) and catalyzes the H4K20me2 modification at replication origins. Here, we present a protocol for preparing SUV420H1 in complex with the nucleosome containing H2A.Z and H4K20Ecx for structure determination. We describe steps for the installation of S-ethyl-cysteine (Ecx), nucleosome and complex preparation, and performing the cryoelectron microscopy (cryo-EM) sample check. This protocol substitutes lysine 20 in histone H4 with S-ethyl-cysteine (H4K20Ecx), which enhances the stability of the interaction between SUV420H1 and nucleosomes. For complete details on the use and execution of this protocol, please refer to Huang et al..

摘要

组蛋白赖氨酸甲基转移酶 SUV420H1 优先靶向含有 H2A.Z 的核小体核心颗粒（H2A.Z-NCP），并催化复制起始处的 H4K20me2 修饰。在这里，我们提供了一种制备与含有 H2A.Z 和 H4K20Ecx 的核小体复合的 SUV420H1 的方案，用于结构测定。我们描述了安装 S-乙基半胱氨酸（Ecx）、核小体和复合物制备以及进行冷冻电镜（cryo-EM）样品检查的步骤。该方案用 S-乙基半胱氨酸（H4K20Ecx）取代组蛋白 H4 中的赖氨酸 20，增强了 SUV420H1 与核小体之间相互作用的稳定性。有关此方案使用和执行的完整详细信息，请参阅 Huang 等人的研究。