High-Resolution Cryo-EM Analyses of Nucleosomes.

The fundamental chromatin unit is the nucleosome, in which approximately 150 base pairs of DNA are bound to the surface of a symmetric histone octamer containing 2 copies each of histones H2A, H2B, H3, and H4. Over the years, numerous structures of nucleosomes have been determined by X-ray crystallography. However, their structural and functional versatility may not have been fully revealed, due to crystal packing effects. Various structures of nucleosomes and their complexes with nucleosome-binding proteins are now being determined by cryo-electron microscopy (cryo-EM) single-particle analysis, allowing the visualization of their structural diversity. In this report, we present a method for high-resolution structural analyses of nucleosomes by cryo-EM and describe the detailed procedures for nucleosome purification, cryo-EM grid preparation, data collection, and data processing. This method can serve as a good starting point for cryo-EM investigations of nucleosomes and their wide range of complexes.