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

Un Seng Chio; Eugene Palovcak; Anton A A Autzen; Henriette E Autzen; Elise N Muñoz; Zanlin Yu; Feng Wang; David A Agard; Jean-Paul Armache; Geeta J Narlikar; Yifan Cheng

doi:10.1101/2023.06.20.545796

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

bioRxiv [Preprint]. 2023 Jun 20:2023.06.20.545796. doi: 10.1101/2023.06.20.545796.

Authors

Un Seng Chio¹, Eugene Palovcak², Anton A A Autzen^{3

4}, Henriette E Autzen^{1

5}, Elise N Muñoz⁶, Zanlin Yu¹, Feng Wang¹, David A Agard¹, Jean-Paul Armache⁷, Geeta J Narlikar¹, Yifan Cheng^{1

8}

Affiliations

¹ Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, CA, USA.
² Biophysics Graduate Program, University of California San Francisco, San Francisco, CA, USA.
³ Department of Materials Science & Engineering, Stanford University, Stanford, CA, USA.
⁴ Current: Department of Health Technology, Technical University of Denmark.
⁵ Linderstrom-Lang Centre for Protein Science, Department of Biology, University of Copenhagen, Denmark.
⁶ Tetrad Graduate Program, University of California, San Francisco, San Francisco, CA, USA.
⁷ Department of Biochemistry and Molecular Biology and the Huck Institutes of the Life Sciences, Pennsylvania State University, University Park, PA, USA.
⁸ Howard Hughes Medical Institute, University of California San Francisco, San Francisco, CA, USA.

Abstract

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.

Keywords: ISWI; SNF2h; chromatin remodeler; graphene oxide; nucleosome; single-particle cryo-EM.

Publication types

Preprint

Abstract

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