# A method for cryo-EM analysis of eukaryotic nucleosomes reconstituted in bacterial cells

## TL;DR

This paper introduces a new method to produce nucleosomes in bacteria for cryo-EM analysis, enabling faster and more efficient structural studies.

## Contribution

A novel method for reconstituting nucleosomes in bacterial cells for cryo-EM analysis is developed.

## Key findings

- Bacterially reconstituted nucleosomes yield a 2.56 Å cryo-EM structure similar to in vitro nucleosomes.
- A non-canonical close-packed di-hexasome (CPDH) nucleosome structure was unexpectedly discovered.
- The method provides a robust platform for studying nucleosome variants and chromatin architectures.

## Abstract

Conventional methods for preparing nucleosomes are time-consuming and technically demanding. In the present study, we extended the approach of generating nucleosomes in Escherichia coli by the co-expression of all four histones, allowing nucleosomes to be assembled in cells. The bacterially reconstituted nucleosomes can be readily prepared from the E. coli cells and directly subjected to cryo-EM single particle analysis. Using this method, we obtained a 2.56 Å nucleosome structure that is highly similar to a previously reported nucleosome crystal structure, validating the use of nucleosomes formed in E. coli for cryo-EM analysis. Unexpectedly, we also discovered a non-canonical nucleosome structure, in which two hexasomes are closely packed. This system provides a robust and efficient platform for structural studies of nucleosomes and nucleosome variants, and may facilitate the discovery of chromatin architectures.

•Nucleosomes assembled within E. coli cells can be used for cryo-EM analysis•Nucleosomes formed in E. coli cells closely resemble those reconstituted in vitro•The close-packed di-hexasome (CPDH) structure is discovered

Nucleosomes assembled within E. coli cells can be used for cryo-EM analysis

Nucleosomes formed in E. coli cells closely resemble those reconstituted in vitro

The close-packed di-hexasome (CPDH) structure is discovered

Biochemistry; molecular biology; structural biology