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

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  • Un Seng Chio
  • Eugene Palovcak
  • Anton A.A. Smith
  • Autzen, Henriette Elisabeth
  • Elise N. Muñoz
  • Zanlin Yu
  • Feng Wang
  • David A. Agard
  • Jean Paul Armache
  • Geeta J. Narlikar
  • Yifan Cheng
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). Here, to address this issue, we develop 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 facilitate 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.
OriginalsprogEngelsk
Artikelnummer2225
TidsskriftNature Communications
Vol/bind15
Udgave nummer1
Antal sider12
ISSN2041-1723
DOI
StatusUdgivet - 2024

Bibliografisk note

Funding Information:
We thank Nathan Gamarra and Laura Hsieh for nucleosome samples for initial experiments, Maxine Bi and Shawn Zheng for assistance with cryo-ET, Julia Tretyakova and Yongqiang (John) Wang for managing the Narlikar and Cheng labs respectively, Junrui Li, Chengmin Li, and Matt Harrington for maintaining computational resources, David Bulkley and Glenn Gilbert for maintaining the UCSF EM facility, and Gregory D. Bowman, Ilana Nodelman, and members of both the Cheng and Narlikar labs for critical discussions. This work was supported by grants from the National Institute of Health (R35GM140847 to Y.C. and R35GM127020 to G.J.N.) and a NIH NIGMS fellowship F32GM137463 to U.S.C. A.A.A.A. was supported by grants NNF18OC0030896 from the Novo Nordisk Foundation and the Stanford Bio-X program and 0171-00081B from Independent Research Fund Denmark. H.E.A was funded by grant R265-2017-4015 from the Lundbeck Foundation. The UCSF cryo-EM facility was partially supported by NIH grants (S10OD020054, S10OD021741, and S10OD025881). Y.C. is an investigator of the Howard Hughes Medical Institute. The figures for this manuscript were generated using UCSF ChimeraX v1.4. UCSF ChimeraX is developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco, with support from National Institutes of Health R01-GM129325 and the Office of Cyber Infrastructure and Computational Biology, National Institute of Allergy and Infectious Diseases.

Funding Information:
We thank Nathan Gamarra and Laura Hsieh for nucleosome samples for initial experiments, Maxine Bi and Shawn Zheng for assistance with cryo-ET, Julia Tretyakova and Yongqiang (John) Wang for managing the Narlikar and Cheng labs respectively, Junrui Li, Chengmin Li, and Matt Harrington for maintaining computational resources, David Bulkley and Glenn Gilbert for maintaining the UCSF EM facility, and Gregory D. Bowman, Ilana Nodelman, and members of both the Cheng and Narlikar labs for critical discussions. This work was supported by grants from the National Institute of Health (R35GM140847 to Y.C. and R35GM127020 to G.J.N.) and a NIH NIGMS fellowship F32GM137463 to U.S.C. A.A.A.A. was supported by grants NNF18OC0030896 from the Novo Nordisk Foundation and the Stanford Bio-X program and 0171-00081B from Independent Research Fund Denmark. H.E.A was funded by grant R265-2017-4015 from the Lundbeck Foundation. The UCSF cryo-EM facility was partially supported by NIH grants (S10OD020054, S10OD021741, and S10OD025881). Y.C. is an investigator of the Howard Hughes Medical Institute. The figures for this manuscript were generated using UCSF ChimeraX v1.4. UCSF ChimeraX is developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco, with support from National Institutes of Health R01-GM129325 and the Office of Cyber Infrastructure and Computational Biology, National Institute of Allergy and Infectious Diseases.

Publisher Copyright:
© The Author(s) 2024.

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