National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
R01 GM45377
United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
R01 GM112108
United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
P41 GM109824
United States
Citation
Journal: Cell / Year: 2022 Title: Comprehensive structure and functional adaptations of the yeast nuclear pore complex. Authors: 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 / ...Authors: 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 / Abstract: 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.
History
Deposition
Jun 10, 2021
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Header (metadata) release
Jan 26, 2022
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Map release
Jan 26, 2022
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Update
Jan 26, 2022
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Current status
Jan 26, 2022
Processing site: RCSB / Status: Released
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Structure visualization
Movie
Surface view with section colored by density value
Name: 3D map of yeast NPC / type: complex / ID: 1 / Parent: 0 Details: low resolution 3D map from 1-step affinity isolation of NPCs, multibody and focused refinements
Molecular weight
Experimental: 52.0 MDa
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Supramolecule #2: inner spoke ring of yeast NPC
Supramolecule
Name: inner spoke ring of yeast NPC / type: complex / ID: 2 / Parent: 1 / Details: focused 3D map of inner ring
Model: Quantifoil R2/2 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 5.0 nm / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: OTHER
Vitrification
Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 283 K / Instrument: FEI VITROBOT MARK III Details: 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 ...Details: 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..
Details
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
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Electron microscopy
Microscope
FEI TITAN KRIOS
Specialist optics
Energy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recording
Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Digitization - Sampling interval: 5.0 µm / Digitization - Frames/image: 2-40 / Number grids imaged: 1 / Number real images: 4015 / Average exposure time: 0.5 sec. / Average electron dose: 40.0 e/Å2 Details: 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.
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
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)
Particle selection
Details: 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)
Applied symmetry - Point group: C8 (8 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 24.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) Details: ring sub maps used a central transporter density subtracted particle set and masks that focused on the different rings with C8 symmetry during the refinements. Number images used: 26049
Initial angle assignment
Type: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.0)
Final angle assignment
Type: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0) / Software - details: multibody and focused 3D refinements
Final 3D classification
Number classes: 50 / Software - Name: RELION (ver. 3.0)
FSC plot (resolution estimation)
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