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- PDB-8tie: Double nuclear outer ring of Nup84-complexes from the yeast NPC -

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Basic information

Entry
Database: PDB / ID: 8tie
TitleDouble nuclear outer ring of Nup84-complexes from the yeast NPC
Components
  • NUP133 isoform 1
  • NUP145 isoform 1
  • Nucleoporin NUP120
  • Nucleoporin NUP84
  • Nucleoporin NUP85
  • Nucleoporin Seh1
  • Protein transport protein SEC13Protein targeting
KeywordsTRANSPORT PROTEIN / nuclear pore complex / nucleocytoplasmic transport / nucleoporin / membrane protein / translocase
Function / homology
Function and homology information


mRNA export from nucleus in response to heat stress / Seh1-associated complex / positive regulation of ER to Golgi vesicle-mediated transport / protein exit from endoplasmic reticulum / COPII-coated vesicle budding / COPII-mediated vesicle transport / nuclear pore central transport channel / nuclear pore localization / telomere tethering at nuclear periphery / regulation of TORC1 signaling ...mRNA export from nucleus in response to heat stress / Seh1-associated complex / positive regulation of ER to Golgi vesicle-mediated transport / protein exit from endoplasmic reticulum / COPII-coated vesicle budding / COPII-mediated vesicle transport / nuclear pore central transport channel / nuclear pore localization / telomere tethering at nuclear periphery / regulation of TORC1 signaling / nuclear pore outer ring / post-transcriptional tethering of RNA polymerase II gene DNA at nuclear periphery / COPII vesicle coat / positive regulation of protein exit from endoplasmic reticulum / structural constituent of nuclear pore / silent mating-type cassette heterochromatin formation / nucleocytoplasmic transport / vacuolar membrane / subtelomeric heterochromatin formation / ribosomal large subunit export from nucleus / positive regulation of TOR signaling / mRNA transport / mRNA export from nucleus / nuclear pore / : / positive regulation of TORC1 signaling / protein export from nucleus / cell periphery / protein import into nucleus / double-strand break repair / nuclear envelope / nuclear membrane / chromosome, telomeric region / endoplasmic reticulum membrane / structural molecule activity / positive regulation of DNA-templated transcription / negative regulation of transcription by RNA polymerase II / endoplasmic reticulum / positive regulation of transcription by RNA polymerase II / identical protein binding
Similarity search - Function
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 FG repeat / Nucleoporin FG repeat region / Nucleoporin, Nup133/Nup155-like, N-terminal / Nup133 N terminal like ...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 FG repeat / Nucleoporin FG repeat region / 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. / Nucleoporin peptidase S59-like / 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
NUP133 isoform 1 / NUP145 isoform 1 / Nucleoporin NUP120 / Nucleoporin NUP85 / Nucleoporin NUP84 / Protein transport protein SEC13
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 8.1 Å
AuthorsAkey, C.W. / Echeverria, I. / Ouch, C. / Fernandez-Martinez, J. / Rout, M.P.
Funding support United States, 4items
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
DepositionJul 19, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 11, 2023Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
a: Nucleoporin NUP120
b: Nucleoporin NUP85
c: NUP145 isoform 1
d: Protein transport protein SEC13
e: Nucleoporin Seh1
f: Nucleoporin NUP84
g: NUP133 isoform 1
l: Nucleoporin NUP120
m: Nucleoporin NUP85
n: NUP145 isoform 1
o: Protein transport protein SEC13
p: Nucleoporin Seh1
q: Nucleoporin NUP84
r: NUP133 isoform 1


Theoretical massNumber of molelcules
Total (without water)1,272,50014
Polymers1,272,50014
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 7 types, 14 molecules albmcndoepfqgr

#1: Protein Nucleoporin NUP120 / Nuclear pore protein NUP120


Mass: 120560.328 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Details: long arm of Nup84 Y-complex / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / Cell line: W303 / Plasmid details: nucleus / Strain: MATa ade2-1 ura3-1 his3-11 / References: UniProt: P35729
#2: Protein Nucleoporin NUP85 / Nuclear pore protein NUP85


Mass: 84972.438 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Details: short arm of Nup84 Y-complex / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / Cell line: W303 / References: UniProt: P46673
#3: Protein NUP145 isoform 1


Mass: 145810.578 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Details: stem junction of Nup84 Y-complex / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / Cell line: W303 / References: UniProt: A0A8H4C085
#4: Protein Protein transport protein SEC13 / Protein targeting


Mass: 33082.965 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Details: cofactor of Nup145C on stem of Nup84 Y-complex / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / Cell line: W303 / References: UniProt: Q04491
#5: Protein Nucleoporin Seh1


Mass: 34652.078 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Details: cofactor Nup85 / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / Cell line: W303
#6: Protein Nucleoporin NUP84 / Nuclear pore protein NUP84


Mass: 83718.867 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Details: tail of Nup84 Y-complex / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / Cell line: W303 / References: UniProt: P52891
#7: Protein NUP133 isoform 1


Mass: 133452.672 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Details: tail Nup84 Y-complex / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / Cell line: W303 / References: UniProt: A0A6V8RYD2

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeDetailsEntity IDParent-IDSource
1Nuclear Pore ComplexNuclear poreCOMPLEXProtein A tagged Mlp1 pullout of NPCall0NATURAL
2double Nup84 Y-complexCOMPLEXNup84 Y-complexes in nuclear double outer ringall1NATURAL
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
1160 MDaYES
21NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-IDStrainCellular locationOrganelle
21Saccharomyces cerevisiae (brewer's yeast)4932MATa ade2-1 ura3-1 his3-11,15 trp1-1 leu2-3,112 can1-100 MLP1-PPX-ProteinA::HIS5nuclear envelopenucleus
32Saccharomyces cerevisiae (brewer's yeast)4932MATa ade2-1 ura3-1 his3-11,15 trp1-1 leu2-3,112 can1-100 MLP1-PPX-ProteinA::HIS5nuclear envelopenucleus
Buffer solutionpH: 7.5
Details: 20mM HEPES,50mM Potassium acetate,20mM NaCl,2mM MgCl2,1mM DTT
SpecimenConc.: 0.1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: One step affinity purified
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 283 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Details: Preliminary grid screening done manually with individual images of low magnification montages of candidate meshes.
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Calibrated magnification: 37651 X / Nominal defocus max: 3500 nm / Nominal defocus min: 1500 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 40 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 4015 / Details: 3218 images retained after triage
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scansWidth: 3838 / Height: 3710 / Movie frames/image: 40 / Used frames/image: 2-40

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Processing

EM software
IDNameVersionCategoryDetails
1GautomatchGautomatch_v0.56_sm61_cu7.5particle selectionlow resolution model used for picking
2SerialEMimage acquisition
4GctfGctf-v0.1.06_sm_20_cu7.5_x86_64CTF correctioninitial CTF estimate per micrograph
5RELION3.0.7CTF correctionper particle CTF estimate
8UCSF Chimera1.14model fitting
10MDFFmodel refinement
11Coot0.8.9.2model refinement
12RELION3.0.7initial Euler assignment
13cryoSPARC3.3.2final Euler assignmentnon-uniform refinement
14RELION3.0.7classification
15cryoSPARC3.3.23D reconstructionnon-uniform refinement
CTF correctionDetails: CTF correction applied in RELION during the alignment and reconstruction
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 26049
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 8.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 29655 / Algorithm: FOURIER SPACE
Details: multibody in RELION followed by refinements in cryosparc
Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Target criteria: cross correlation
Details: Molecular dynamics flexible fitting after docking Alphafold2 CSMs for Nups into the 3D map of double Y complex ring with Chimera
Atomic model buildingDetails: none / Source name: Other / Type: experimental model

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