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- EMDB-41116: Lumenal ring of the isolated yeast NPC -

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Basic information

Entry
Database: EMDB / ID: EMD-41116
TitleLumenal ring of the isolated yeast NPC
Map dataMain map reconstructed from Multibody volume
Sample
  • Complex: Lumenal rig of Nuclear Pore Complex
Keywordsnuclear pore complex / nucleocytoplasmic transport / nucleoporin / membrane protein / TRANSPORT PROTEIN
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 14.7 Å
AuthorsAkey CW / Echeverria I / Ouch C / Fernandez-Martinez J / Rout MP
Funding support United States, 4 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)NIH R01 GM45377 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)NIH P41 GM109824 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)NIH R01 GM112108 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)NIH GM117212 United States
CitationJournal: Mol Cell / Year: 2023
Title: Implications of a multiscale structure of the yeast nuclear pore complex.
Authors: Christopher W Akey / Ignacia Echeverria / Christna Ouch / Ilona Nudelman / Yi Shi / Junjie Wang / Brian T Chait / Andrej Sali / Javier Fernandez-Martinez / Michael P Rout /
Abstract: Nuclear pore complexes (NPCs) direct the nucleocytoplasmic transport of macromolecules. Here, we provide a composite multiscale structure of the yeast NPC, based on improved 3D density maps from ...Nuclear pore complexes (NPCs) direct the nucleocytoplasmic transport of macromolecules. Here, we provide a composite multiscale structure of the yeast NPC, based on improved 3D density maps from cryogenic electron microscopy and AlphaFold2 models. Key features of the inner and outer rings were integrated into a comprehensive model. We resolved flexible connectors that tie together the core scaffold, along with equatorial transmembrane complexes and a lumenal ring that anchor this channel within the pore membrane. The organization of the nuclear double outer ring reveals an architecture that may be shared with ancestral NPCs. Additional connections between the core scaffold and the central transporter suggest that under certain conditions, a degree of local organization is present at the periphery of the transport machinery. These connectors may couple conformational changes in the scaffold to the central transporter to modulate transport. Collectively, this analysis provides insights into assembly, transport, and NPC evolution.
History
DepositionJun 24, 2023-
Header (metadata) releaseOct 11, 2023-
Map releaseOct 11, 2023-
UpdateOct 11, 2023-
Current statusOct 11, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_41116.png

Downloads & links

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Map

FileDownload / File: emd_41116.map.gz / Format: CCP4 / Size: 421.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMain map reconstructed from Multibody volume
Voxel sizeX=Y=Z: 2.66 Å
Density
Contour LevelBy AUTHOR: 0.73
Minimum - Maximum-0.66977376 - 1.8079556
Average (Standard dev.)0.005565968 (±0.06463531)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions480480480
Spacing480480480
CellA=B=C: 1276.8 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: multibody half map

Fileemd_41116_additional_1.map
Annotationmultibody half map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: post processed multibody volume bf -300 local resolution box 50

Fileemd_41116_additional_2.map
Annotationpost processed multibody volume bf -300 local resolution box 50
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: multibody half map

Fileemd_41116_additional_3.map
Annotationmultibody half map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map full D8 lumenal ring

Fileemd_41116_half_map_1.map
Annotationhalf map full D8 lumenal ring
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map full D8 lumenal ring

Fileemd_41116_half_map_2.map
Annotationhalf map full D8 lumenal ring
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Lumenal rig of Nuclear Pore Complex

EntireName: Lumenal rig of Nuclear Pore Complex
Components
  • Complex: Lumenal rig of Nuclear Pore Complex

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Supramolecule #1: Lumenal rig of Nuclear Pore Complex

SupramoleculeName: Lumenal rig of Nuclear Pore Complex / type: complex / ID: 1 / Parent: 0 / Details: Protein A tagged Mlp1 pullout of NPC
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Strain: MATa ade2-1 ura3-1 his3-11,15 trp1-1 leu2-3,112 can1-100 MLP1-PPX-ProteinA::HIS5
Organelle: nucleus / Location in cell: nuclear envelope

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.1 mg/mL
BufferpH: 7.5
Details: 20mM HEPES,50mM Potassium acetate,20mM NaCl,2mM MgCl2,1mM DTT
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Support film - Film thickness: 5 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: OTHER
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 283 K / Instrument: FEI VITROBOT MARK III
DetailsOne step affinity purified with NPC

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Calibrated magnification: 37651 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.5 µm
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
DetailsPreliminary grid screening done manually with individual images of low magnification montages of candidate meshes.
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Digitization - Frames/image: 2-40 / Number grids imaged: 1 / Number real images: 4015 / Average electron dose: 40.0 e/Å2 / Details: 3218 images retained after triage
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 85322 / Details: from Multibody with 2x protomer
Startup modelType of model: OTHER
Details: low resolution composite 3D map of spoke and lumenal ring
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0.7)
Details: C1 symmetry in refinemet, C2 enforced 3D reference.
Final 3D classificationSoftware - Name: RELION (ver. 3.0.7)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0 7)
Details: C1 symmetry with appropriate mask in stepwise refinements gradually excluding proximal regions of the spoke used to refine with 2.5x Multibody
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 14.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0.7)
Details: half maps were masked to remove contaminating density from the Multibody step.
Number images used: 85322
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Source name: Other / Chain - Initial model type: experimental model / Details: none
DetailsThe 2x protomer was fitted together to form a complete c8 lumenal ring with imposed 2-fold symmetry.
RefinementSpace: REAL / Protocol: OTHER / Target criteria: cross correlation

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