High-resolution cryoEM of nucleosomes in nuclear extracts of mammalian cells

Abstract

Frontier Structural Biology methods are transitioning from analysis of reconstituted macromolecular complexes in vitro to imaging of macromolecular assemblies within the physiological confines of the cell. Preparation of samples for in situ cryoEM analysis requires FIB milling or ultramicrotome sectioning, laborious and technically challenging procedures that are low-throughput and require a high degree of technical skills. We have devised a simple approach for cryoEM of nuclear macromolecular complexes that preserves to a high degree their physiological environment while removing the need for thin sectioning of the sample. The method requires only the preparation of nuclear extracts without additional purification or enrichment steps. We applied the method to obtain a 2.3 [A] cryoEM structure of nucleosomes visualised directly in the nuclear lysate of human cells. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=105 SRC="FIGDIR/small/732463v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@1062c07org.highwire.dtl.DTLVardef@34a2f8org.highwire.dtl.DTLVardef@1b75432org.highwire.dtl.DTLVardef@f8b2fc_HPS_FORMAT_FIGEXP M_FIG C_FIG