Functionalized graphene-oxide grids enable high-resolution cryo-EM structures of the SNF2h-nucleosome complex without crosslinking.

Autor:	Chio US; Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, CA, USA., Palovcak E; Biophysics Graduate Program, University of California San Francisco, San Francisco, CA, USA., Autzen AAA; Department of Materials Science & Engineering, Stanford University, Stanford, CA, USA.; Current: Department of Health Technology, Technical University of Denmark., Autzen HE; Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, CA, USA.; Linderstrom-Lang Centre for Protein Science, Department of Biology, University of Copenhagen, Denmark., Muñoz EN; Tetrad Graduate Program, University of California, San Francisco, San Francisco, CA, USA., Yu Z; Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, CA, USA., Wang F; Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, CA, USA., Agard DA; Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, CA, USA., Armache JP; Department of Biochemistry and Molecular Biology and the Huck Institutes of the Life Sciences, Pennsylvania State University, University Park, PA, USA., Narlikar GJ; Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, CA, USA., Cheng Y; Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, CA, USA.; Howard Hughes Medical Institute, University of California San Francisco, San Francisco, CA, USA.
Jazyk:	angličtina
Zdroj:	BioRxiv : the preprint server for biology [bioRxiv] 2023 Jun 20. Date of Electronic Publication: 2023 Jun 20.
DOI:	10.1101/2023.06.20.545796
Abstrakt:	Single-particle cryo-EM is widely used to determine enzyme-nucleosome complex structures. However, cryo-EM sample preparation remains challenging and inconsistent due to complex denaturation at the air-water interface (AWI). To address this issue, we developed graphene-oxide-coated EM grids functionalized with either single-stranded DNA (ssDNA) or thiol-poly(acrylic acid-co-styrene) (TAASTY) co-polymer. These grids protect complexes between the chromatin remodeler SNF2h and nucleosomes from the AWI and facilitated collection of high-quality micrographs of intact SNF2h-nucleosome complexes in the absence of crosslinking. The data yields maps ranging from 2.3 to 3 Å in resolution. 3D variability analysis reveals nucleotide-state linked conformational changes in SNF2h bound to a nucleosome. In addition, the analysis provides structural evidence for asymmetric coordination between two SNF2h protomers acting on the same nucleosome. We envision these grids will enable similar detailed structural analyses for other enzyme-nucleosome complexes and possibly other protein-nucleic acid complexes in general. Competing Interests: Competing Interests The authors declare no competing interests.
Databáze:	MEDLINE
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