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- EMDB-24224: Combined 3D structure of the isolated yeast NPC -

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Entry
Database: EMDB / ID: EMD-24224
TitleCombined 3D structure of the isolated yeast NPC
Map datainner spoke ring
Sample
  • Complex: isolated yeast NPC
    • Complex: inner ring of spokes from NPC
    • Complex: Nsp1 complex connections to FG domains
    • Complex: double nuclear outer ring of Nup84 complex pairs with Nup188-192
    • Complex: central transporter
    • Complex: Pom152 ring
Function / homology
Function and homology information


response to spindle checkpoint signaling / nuclear pore linkers / : / peroxisomal importomer complex / regulation of protein desumoylation / mRNA export from nucleus in response to heat stress / nuclear pore inner ring / Seh1-associated complex / protein localization to nuclear inner membrane / positive regulation of ER to Golgi vesicle-mediated transport ...response to spindle checkpoint signaling / nuclear pore linkers / : / peroxisomal importomer complex / regulation of protein desumoylation / mRNA export from nucleus in response to heat stress / nuclear pore inner ring / Seh1-associated complex / protein localization to nuclear inner membrane / positive regulation of ER to Golgi vesicle-mediated transport / protein exit from endoplasmic reticulum / COPII-coated vesicle budding / COPII-mediated vesicle transport / transcription-dependent tethering of RNA polymerase II gene DNA at nuclear periphery / nuclear pore central transport channel / regulation of TORC1 signaling / adenyl-nucleotide exchange factor activity / nuclear pore localization / telomere tethering at nuclear periphery / regulation of nucleocytoplasmic transport / establishment of mitotic spindle localization / nuclear migration along microtubule / nuclear pore organization / nuclear pore complex assembly / nuclear pore outer ring / tRNA export from nucleus / post-transcriptional tethering of RNA polymerase II gene DNA at nuclear periphery / nuclear pore cytoplasmic filaments / COPII vesicle coat / positive regulation of protein exit from endoplasmic reticulum / nuclear pore nuclear basket / cytoplasmic dynein complex / RNA export from nucleus / protein localization to kinetochore / structural constituent of nuclear pore / silent mating-type cassette heterochromatin formation / nucleocytoplasmic transport / vacuolar membrane / poly(A)+ mRNA export from nucleus / nuclear localization sequence binding / regulation of mitotic nuclear division / NLS-bearing protein import into nucleus / dynein intermediate chain binding / establishment of mitotic spindle orientation / subtelomeric heterochromatin formation / ribosomal small subunit export from nucleus / ribosomal large subunit export from nucleus / Hydrolases; Acting on peptide bonds (peptidases); Serine endopeptidases / positive regulation of TOR signaling / mRNA transport / cytoplasmic microtubule / mRNA export from nucleus / heterochromatin formation / nuclear pore / : / positive regulation of TORC1 signaling / cellular response to amino acid starvation / Neutrophil degranulation / protein export from nucleus / nuclear periphery / chromosome segregation / cell periphery / promoter-specific chromatin binding / phospholipid binding / protein import into nucleus / transcription corepressor activity / double-strand break repair / protein transport / single-stranded DNA binding / nuclear envelope / nuclear membrane / chromosome, telomeric region / molecular adaptor activity / hydrolase activity / cell cycle / cell division / chromatin binding / protein-containing complex binding / structural molecule activity / endoplasmic reticulum membrane / positive regulation of DNA-templated transcription / negative regulation of transcription by RNA polymerase II / endoplasmic reticulum / positive regulation of transcription by RNA polymerase II / DNA binding / RNA binding / identical protein binding / nucleus / cytosol / cytoplasm
Similarity search - Function
Nucleoporin Nup159/Nup146, N-terminal / Nucleoporin or Nuclear pore complex subunit NUP214=Nup159 / RNA-recognition motif (RRM) Nup35-type domain / Nucleoporin, NUP53 / Nucleoporin NUP88/NUP82 / Nup53/35/40-type RNA recognition motif / RNA-recognition motif (RRM) Nup35-type domain profile. / Nuclear pore protein Nup188, C-terminal / Nuclear pore protein NUP188 C-terminal domain / Nucleoporin Nup188, N-terminal ...Nucleoporin Nup159/Nup146, N-terminal / Nucleoporin or Nuclear pore complex subunit NUP214=Nup159 / RNA-recognition motif (RRM) Nup35-type domain / Nucleoporin, NUP53 / Nucleoporin NUP88/NUP82 / Nup53/35/40-type RNA recognition motif / RNA-recognition motif (RRM) Nup35-type domain profile. / Nuclear pore protein Nup188, C-terminal / Nuclear pore protein NUP188 C-terminal domain / Nucleoporin Nup188, N-terminal / Nucleoporin p58/p45 / Nucleoporin Nup188, N-terminal / : / Nucleoporin Nup188, N-terminal subdomain III / Nucleoporin, NSP1-like, C-terminal / Nucleoporin Nup54/Nup57/Nup44 / Nucleoporin Nup54, alpha-helical domain / Nucleoporin NSP1/NUP62 / Nucleoporin Nup188 / Nsp1-like C-terminal region / Nucleoporin complex subunit 54 / Nucleoporin Nup85-like / Nucleoporin Nup120/160 / Nup85 Nucleoporin / Nuclear pore protein 84/107 / Nuclear pore protein 84 / 107 / Nuclear pore complex protein Nup133-like / Nucleoporin, Nup155-like / Nucleoporin, Nup155-like, C-terminal, subdomain 1 / Nucleoporin, Nup155-like, C-terminal, subdomain 2 / Nucleoporin Nup186/Nup192/Nup205 / Nuclear pore complex scaffold, nucleoporins 186/192/205 / Nucleoporin interacting component Nup93/Nic96 / Nup93/Nic96 / Nucleoporin FG repeat / Nucleoporin FG repeat region / Nucleoporin, Nup133/Nup155-like, C-terminal / Non-repetitive/WGA-negative nucleoporin C-terminal / Dynein light chain, type 1/2, conserved site / Dynein light chain type 1 signature. / Nucleoporin, Nup133/Nup155-like, N-terminal / Nup133 N terminal like / Sec13/Seh1 family / Nuclear pore complex protein NUP96, C-terminal domain / Nuclear protein 96 / Nuclear pore complex protein Nup98-Nup96-like, autopeptidase S59 domain / Nuclear pore complex protein Nup98-Nup96-like, autopeptidase S59 domain superfamily / Nucleoporin autopeptidase / NUP C-terminal domain profile. / Dynein light chain type 1 / Nucleoporin peptidase S59-like / Dynein light chain, type 1/2 / Dynein light chain superfamily / Dynein light chain type 1 / RNA-binding domain superfamily / Nucleotide-binding alpha-beta plait domain superfamily / WD domain, G-beta repeat / WD40 repeats / WD40 repeat / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily
Similarity search - Domain/homology
Nucleoporin NSP1 / Nucleoporin NIC96 / Nucleoporin NUP120 / Nucleoporin NUP133 / Nucleoporin NUP170 / Nucleoporin NUP157 / Nucleoporin NUP82 / Nucleoporin NUP159 / Nucleoporin NUP85 / Nucleoporin NUP192 ...Nucleoporin NSP1 / Nucleoporin NIC96 / Nucleoporin NUP120 / Nucleoporin NUP133 / Nucleoporin NUP170 / Nucleoporin NUP157 / Nucleoporin NUP82 / Nucleoporin NUP159 / Nucleoporin NUP85 / Nucleoporin NUP192 / Nucleoporin NUP57 / Nucleoporin NUP145 / Nucleoporin NUP188 / Nucleoporin NUP84 / Nucleoporin SEH1 / Nucleoporin NUP49/NSP49 / Dynein light chain 1, cytoplasmic / Nucleoporin NUP53 / Protein transport protein SEC13 / Nucleoporin ASM4
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 7.6 Å
AuthorsAkey CW / Rout MP / Ouch C / Echeverria I / Fernandez-Martinez J / Nudelman I
Funding support United States, 3 items
OrganizationGrant numberCountry
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
CitationJournal: 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
DepositionJun 10, 2021-
Header (metadata) releaseJan 26, 2022-
Map releaseJan 26, 2022-
UpdateFeb 2, 2022-
Current statusFeb 2, 2022Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.595
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.595
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_24224.png

Downloads & links

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Map

FileDownload / File: emd_24224.map.gz / Format: CCP4 / Size: 421.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationinner spoke ring
Voxel sizeX=Y=Z: 2.66 Å
Density
Contour LevelBy AUTHOR: 0.595 / Movie #1: 0.595
Minimum - Maximum0.0 - 4.327462
Average (Standard dev.)0.018599069 (±0.121410735)
SymmetrySpace group: 1
Details

EMDB XML:

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

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.662.662.66
M x/y/z480480480
origin x/y/z0.0000.0000.000
length x/y/z1276.8001276.8001276.800
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS480480480
D min/max/mean0.0004.3270.019

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Supplemental data

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Additional map: central transporter class average

Fileemd_24224_additional_1.map
Annotationcentral transporter class average
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: recombined double nuclear outer ring

Fileemd_24224_additional_2.map
Annotationrecombined double nuclear outer ring
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Cutout Pom152 ring (zoned from low resolution 3D NPC map)

Fileemd_24224_additional_3.map
AnnotationCutout Pom152 ring (zoned from low resolution 3D NPC map)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Cutout detergent-lipid ring

Fileemd_24224_additional_4.map
AnnotationCutout detergent-lipid ring
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: cutout center map with transporter connections from refined...

Fileemd_24224_additional_5.map
Annotationcutout center map with transporter connections from refined class average
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: hook ring of nup82 complexes

Fileemd_24224_additional_6.map
Annotationhook ring of nup82 complexes
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : isolated yeast NPC

EntireName: isolated yeast NPC
Components
  • Complex: isolated yeast NPC
    • Complex: inner ring of spokes from NPC
    • Complex: Nsp1 complex connections to FG domains
    • Complex: double nuclear outer ring of Nup84 complex pairs with Nup188-192
    • Complex: central transporter
    • Complex: Pom152 ring

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Supramolecule #1: isolated yeast NPC

SupramoleculeName: isolated yeast NPC / type: complex / ID: 1 / Parent: 0

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Supramolecule #2: inner ring of spokes from NPC

SupramoleculeName: inner ring of spokes from NPC / type: complex / ID: 2 / Parent: 1
Details: 3D map from 1-step affinity isolation of NPCs, multibody and focused refinements
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Organelle: nucleus / Location in cell: nuclear envelope
Molecular weightTheoretical: 9.0 MDa

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Supramolecule #3: Nsp1 complex connections to FG domains

SupramoleculeName: Nsp1 complex connections to FG domains / type: complex / ID: 3 / Parent: 1
Details: focused and zoned map for central channel connections
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Organelle: nucleus / Location in cell: nuclear envelope

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Supramolecule #4: double nuclear outer ring of Nup84 complex pairs with Nup188-192

SupramoleculeName: double nuclear outer ring of Nup84 complex pairs with Nup188-192
type: complex / ID: 4 / Parent: 1
Details: 3D map visualized by multibody and focused refinements on NPC
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Organelle: nucleus / Location in cell: nuclear envelope

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Supramolecule #5: central transporter

SupramoleculeName: central transporter / type: complex / ID: 5 / Parent: 1 / Details: 3D map of refined central channel density in NPC
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Organelle: nucleus / Location in cell: nuclear envelope

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Supramolecule #6: Pom152 ring

SupramoleculeName: Pom152 ring / type: complex / ID: 6 / Parent: 1
Details: density map cutout from low resolution full NPC that traces the lumenal Pom152 ring
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / 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

Concentration0.3 mg/mL
BufferpH: 7.5
Component:
ConcentrationNameFormula
20.0 mMHEPES
50.0 mMpotassium acetate
20.0 mMsodium chlorideNaClSodium chloride
2.0 mMmagnesium chlorideMgCl2
1.0 mMDTT
0.1 percent wt/wtDeoxyBigChaps
GridModel: 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: AIR
VitrificationCryogen 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..
DetailsNPCs 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

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Calibrated defocus max: 3.8000000000000003 µm / Calibrated defocus min: 1.5 µm / Calibrated magnification: 37651 / Illumination mode: OTHER / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal magnification: 50000
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Sample stageCooling holder cryogen: NITROGEN
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 - 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.
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionDetails: 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)
CTF correctionSoftware - Name: Gctf (ver. 1.18)
Startup modelType of model: EMDB MAP
EMDB ID:

7321


Details: tomographic NPC 3D map
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.0)
Final 3D classificationNumber classes: 50 / Software - Name: RELION (ver. 3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0) / Software - details: multibody and focused 3D refinements
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 7.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0)
Details: C8 protomer subunits: initial and final (208393/145000)for spoke ring protomers from double outer ring: initial and final (208393/45000)
Number images used: 26049
DetailsAfter 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)

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