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- EMDB-41119: Cytoplasmic outer ring of the yeast NPC -

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Basic information

Entry
Database: EMDB / ID: EMD-41119
TitleCytoplasmic outer ring of the yeast NPC
Map datac8 map of cytoplasmic outer ring reconstructed from multibody volume
Sample
  • Complex: 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: 22.0 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_41119.png

Downloads & links

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Map

FileDownload / File: emd_41119.map.gz / Format: CCP4 / Size: 421.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationc8 map of cytoplasmic outer ring reconstructed from multibody volume
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.66 Å/pix.
x 480 pix.
= 1276.8 Å		2.66 Å/pix.
x 480 pix.
= 1276.8 Å		2.66 Å/pix.
x 480 pix.
= 1276.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.66 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0164
Minimum - Maximum-0.11530132 - 0.3653888
Average (Standard dev.)0.0005079929 (±0.008120659)
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: half map of cytoplasmic outer ring multibody volume

Fileemd_41119_additional_1.map
Annotationhalf map of cytoplasmic outer ring multibody volume
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Additional map: half map of cytoplasmic outer ring multibody volume

Fileemd_41119_additional_2.map
Annotationhalf map of cytoplasmic outer ring multibody volume
Projections & Slices
AxesZYX

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Additional map: 2.5x multibody map of cytoplasmic outer ring

Fileemd_41119_additional_3.map
Annotation2.5x multibody map of cytoplasmic outer ring
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map of c8 cytoplasmic outer ring

Fileemd_41119_half_map_1.map
Annotationhalf map of c8 cytoplasmic outer ring
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map of c8 cytoplasmic outer ring

Fileemd_41119_half_map_2.map
Annotationhalf map of c8 cytoplasmic outer 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 : Nuclear Pore Complex

EntireName: Nuclear Pore Complex
Components
  • Complex: Nuclear Pore Complex

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Supramolecule #1: Nuclear Pore Complex

SupramoleculeName: 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 - Time: 30 sec. / Pretreatment - Atmosphere: OTHER
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 283 K / Instrument: FEI VITROBOT MARK III
DetailsOne step affinity purified

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
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
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 FIELD / Cs: 2.7 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.5 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 26049
Startup modelType of model: OTHER
Details: C8 symmetrized NPC map segmented in CHIMERA with Segger to provide initial 3D map of the cytoplasmic outer ring, which was low pass filtered for the first reference.
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 22.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0.7) / Details: Multibody in RELION / Number images used: 34576
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0.7) / Details: C8 symmetry used
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0.7) / Details: C1 symmetry used to refine with 2.5x Multibody
Final 3D classificationSoftware - Name: RELION (ver. 3.0.7)
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
DetailsRigid body docking of 3D map of cytoplasmic outer ring into NPC 3D map with Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: cross correlation

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