National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
R01 GM45377
米国
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
R01 GM112108
米国
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
P41 GM109824
米国
引用
ジャーナル: Cell / 年: 2022 タイトル: Comprehensive structure and functional adaptations of the yeast nuclear pore complex. 著者: Christopher W Akey / Digvijay Singh / Christna Ouch / Ignacia Echeverria / Ilona Nudelman / Joseph M Varberg / Zulin Yu / Fei Fang / Yi Shi / Junjie Wang / Daniel Salzberg / Kangkang Song / ...著者: Christopher W Akey / Digvijay Singh / Christna Ouch / Ignacia Echeverria / Ilona Nudelman / Joseph M Varberg / Zulin Yu / Fei Fang / Yi Shi / Junjie Wang / Daniel Salzberg / Kangkang Song / Chen Xu / James C Gumbart / Sergey Suslov / Jay Unruh / Sue L Jaspersen / Brian T Chait / Andrej Sali / Javier Fernandez-Martinez / Steven J Ludtke / Elizabeth Villa / Michael P Rout / 要旨: Nuclear pore complexes (NPCs) mediate the nucleocytoplasmic transport of macromolecules. Here we provide a structure of the isolated yeast NPC in which the inner ring is resolved by cryo-EM at sub- ...Nuclear pore complexes (NPCs) mediate the nucleocytoplasmic transport of macromolecules. Here we provide a structure of the isolated yeast NPC in which the inner ring is resolved by cryo-EM at sub-nanometer resolution to show how flexible connectors tie together different structural and functional layers. These connectors may be targets for phosphorylation and regulated disassembly in cells with an open mitosis. Moreover, some nucleoporin pairs and transport factors have similar interaction motifs, which suggests an evolutionary and mechanistic link between assembly and transport. We provide evidence for three major NPC variants that may foreshadow functional specializations at the nuclear periphery. Cryo-electron tomography extended these studies, providing a model of the in situ NPC with a radially expanded inner ring. Our comprehensive model reveals features of the nuclear basket and central transporter, suggests a role for the lumenal Pom152 ring in restricting dilation, and highlights structural plasticity that may be required for transport.
超分子 #4: double nuclear outer ring of Nup84 complex pairs with Nup188-192
超分子
名称: double nuclear outer ring of Nup84 complex pairs with Nup188-192 タイプ: complex / ID: 4 / 親要素: 1 詳細: 3D map visualized by multibody and focused refinements on NPC
凍結剤: ETHANE / チャンバー内湿度: 100 % / チャンバー内温度: 283 K / 装置: FEI VITROBOT MARK III 詳細: Grids were floated with carbon side down on 5 uL sample drops in a humid chamber. This was followed by 3 stepwise transfers onto 20 uL drops of blotting buffer while keeping the backside of ...詳細: Grids were floated with carbon side down on 5 uL sample drops in a humid chamber. This was followed by 3 stepwise transfers onto 20 uL drops of blotting buffer while keeping the backside of the grids dry. blot buffer was removed with a manual blot from the bottom edge of the grid through a side port in the humid chamber; an appropriate volume of blot buffer was pipetted immediately onto the grid, which was double blotted and plunge frozen..
詳細
NPCs were released gently from the NE with detergent-extraction of a cell cryo-lysate and purified with a single affinity step; to minimize local domain movements NPCs were mildly cross-linked by the addition of DiSuccinimidylSuberate
フィルム・検出器のモデル: GATAN K2 SUMMIT (4k x 4k) 検出モード: SUPER-RESOLUTION / デジタル化 - サイズ - 横: 3838 pixel / デジタル化 - サイズ - 縦: 3710 pixel / デジタル化 - サンプリング間隔: 5.0 µm / デジタル化 - 画像ごとのフレーム数: 2-40 / 撮影したグリッド数: 1 / 実像数: 4015 / 平均露光時間: 0.5 sec. / 平均電子線量: 40.0 e/Å2 詳細: Navigator parameters were set to have 6-7 groups of holes per mesh, with one focus spot per group using an in-house script from Dr. Chen Xu.
電子線
加速電圧: 300 kV / 電子線源: FIELD EMISSION GUN
電子光学系
C2レンズ絞り径: 70.0 µm 最大 デフォーカス(補正後): 3.8000000000000003 µm 最小 デフォーカス(補正後): 1.5 µm / 倍率(補正後): 37651 / 照射モード: OTHER / 撮影モード: BRIGHT FIELD / Cs: 2.7 mm / 倍率(公称値): 50000
試料ステージ
ホルダー冷却材: NITROGEN
実験機器
モデル: Titan Krios / 画像提供: FEI Company
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画像解析
詳細
After data collection, movies were decompressed, gain corrected and aligned with Motioncor2 (v1.2.3). Manual triage was done with Motioncor2/GCTF power-pairs using the "eye of Gnome" viewer (eog) and awk scripts to eliminate poor micrographs (~80% remained)
粒子像選択
詳細: NPCs were picked with Gautomatch {K. Zhang} using an image stack of equi-spaced projection views that were calculated from a tomographic model with C8 symmetry using EMAN2 (e2project3d.py)