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

## TL;DR

This paper provides a detailed protocol for preparing a complex of SUV420H1 with a modified nucleosome for structural studies using cryo-EM.

## Contribution

A novel protocol for installing S-ethyl-cysteine into histone H4 to stabilize SUV420H1-nucleosome interactions for cryo-EM.

## Key findings

- Substituting lysine 20 in histone H4 with S-ethyl-cysteine enhances SUV420H1-nucleosome interaction stability.
- The protocol includes detailed steps for Ecx installation and cryo-EM sample preparation.
- The method is applicable for structure determination of SUV420H1 with H2A.Z-containing nucleosomes.

## 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

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.