National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
GM140847
米国
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
GM127020
米国
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
GM137463
米国
引用
ジャーナル: Nat Commun / 年: 2024 タイトル: Functionalized graphene-oxide grids enable high-resolution cryo-EM structures of the SNF2h-nucleosome complex without crosslinking. 著者: Un Seng Chio / Eugene Palovcak / Anton A A Smith / Henriette Autzen / Elise N Muñoz / Zanlin Yu / Feng Wang / David A Agard / Jean-Paul Armache / Geeta J Narlikar / Yifan Cheng / 要旨: 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 ...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.
選択した数: 8509125 詳細: Particles picked using cryoSPARC's template picker with templates generated from a nucleosome.
初期モデル
モデルのタイプ: OTHER / 詳細: cryoSPARC ab initio model
最終 再構成
想定した対称性 - 点群: C1 (非対称) / 解像度のタイプ: BY AUTHOR / 解像度: 3.2 Å / 解像度の算出法: FSC 0.143 CUT-OFF / ソフトウェア - 名称: cryoSPARC / ソフトウェア - 詳細: Local refinement 詳細: cryoSPARC local refinement was used for reconstruction. Gold standard FSC determined by cryoSPARC using auto FSC mask generation and tightening. 使用した粒子像数: 14666