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

Abstract

Conventional methods for preparing nucleosomes are time-consuming and technically demanding. In the present study, we extended the approach of generating nucleosomes in <i>Escherichia coli</i> 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 <i>E. coli</i> 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 <i>E. coli</i> 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.