A method for cryo-EM analysis of eukaryotic nucleosomes reconstituted in bacterial cells
Cheng-Han Ho, Yuki Kobayashi, Mitsuo Ogasawara, Yoshimasa Takizawa, Hitoshi Kurumizaka
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.
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…
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Taxonomy
TopicsAdvanced Electron Microscopy Techniques and Applications · Bacterial Genetics and Biotechnology · Genomics and Chromatin Dynamics