Work

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

Year: 2024

Type: article

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. 1 • Universal approach to integrate Ecx in histones with low yield • Detailed steps for installing S-ethyl-cysteine (Ecx) into histone H4K20C • Extensive protocol detail for cryo-EM nucleosome complex preparation Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. 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.

Source: STAR Protocols

DOI: 10.1016/j.xpro.2024.103295

Authors: Li Huang, Zheng Zhou

Institutions: Chinese Academy of Sciences, Institute of Biophysics, University of Chinese Academy of Sciences

Language: English

Topic: Genomics and Chromatin Dynamics

Subfield: Molecular Biology

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

Open Access status: gold

Estimated APC paid: $1,500

Funders: National Natural Science Foundation of China, Chinese Academy of Sciences, Ministry of Science and Technology of the People's Republic of China, Institute of Biophysics, Chinese Academy of Sciences