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- PDB-8tj5: Inner spoke ring of the yeast NPC -

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Basic information

Entry
Database: PDB / ID: 8tj5
TitleInner spoke ring of the yeast NPC
Components
  • Nucleoporin 59
  • Nucleoporin NIC96
  • Nucleoporin NSP1
  • Nucleoporin NUP157
  • Nucleoporin NUP170
  • Nucleoporin NUP188
  • Nucleoporin NUP192
  • Nucleoporin NUP49/NSP49
  • Nucleoporin NUP53
  • Nucleoporin NUP57
  • Spoke connector
KeywordsTRANSPORT PROTEIN / nuclear pore complex / nucleocytoplasmic transport / nucleoporin / membrane protein / translocase
Function / homology
Function and homology information


response to spindle checkpoint signaling / nuclear pore linkers / : / regulation of protein desumoylation / mRNA export from nucleus in response to heat stress / nuclear pore inner ring / protein localization to nuclear inner membrane / transcription-dependent tethering of RNA polymerase II gene DNA at nuclear periphery / nuclear pore central transport channel / telomere tethering at nuclear periphery ...response to spindle checkpoint signaling / nuclear pore linkers / : / regulation of protein desumoylation / mRNA export from nucleus in response to heat stress / nuclear pore inner ring / protein localization to nuclear inner membrane / transcription-dependent tethering of RNA polymerase II gene DNA at nuclear periphery / nuclear pore central transport channel / telomere tethering at nuclear periphery / regulation of nucleocytoplasmic transport / nuclear pore organization / nuclear pore complex assembly / tRNA export from nucleus / post-transcriptional tethering of RNA polymerase II gene DNA at nuclear periphery / nuclear pore cytoplasmic filaments / nuclear pore nuclear basket / protein localization to kinetochore / structural constituent of nuclear pore / RNA export from nucleus / poly(A)+ mRNA export from nucleus / nucleocytoplasmic transport / nuclear localization sequence binding / regulation of mitotic nuclear division / NLS-bearing protein import into nucleus / ribosomal small subunit export from nucleus / ribosomal large subunit export from nucleus / mRNA transport / heterochromatin formation / nuclear pore / nuclear periphery / chromosome segregation / promoter-specific chromatin binding / phospholipid binding / protein import into nucleus / protein transport / single-stranded DNA binding / nuclear envelope / nuclear membrane / molecular adaptor activity / cell cycle / cell division / chromatin binding / protein-containing complex binding / positive regulation of DNA-templated transcription / DNA binding / RNA binding / identical protein binding / nucleus
Similarity search - Function
RNA-recognition motif (RRM) Nup35-type domain / Nucleoporin, NUP53 / 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 Nup188, N-terminal / : / Nucleoporin Nup188, N-terminal subdomain III ...RNA-recognition motif (RRM) Nup35-type domain / Nucleoporin, NUP53 / 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 Nup188, N-terminal / : / Nucleoporin Nup188, N-terminal subdomain III / Nucleoporin Nup54/Nup57/Nup44 / Nucleoporin p58/p45 / Nucleoporin Nup54, alpha-helical domain / Nucleoporin Nup188 / Nucleoporin complex subunit 54 / Nucleoporin, NSP1-like, C-terminal / Nucleoporin NSP1/NUP62 / Nsp1-like C-terminal region / 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, Nup133/Nup155-like, C-terminal / Nucleoporin FG repeat / Nucleoporin FG repeat region / Non-repetitive/WGA-negative nucleoporin C-terminal / Nucleoporin, Nup133/Nup155-like, N-terminal / Nup133 N terminal like / RNA-binding domain superfamily / Nucleotide-binding alpha-beta plait domain superfamily
Similarity search - Domain/homology
Nucleoporin NSP1 / Nucleoporin NIC96 / Nucleoporin NUP170 / Nucleoporin NUP157 / Nucleoporin NUP192 / Nucleoporin NUP57 / Nucleoporin NUP188 / Nucleoporin NUP49/NSP49 / Nucleoporin NUP53 / Nucleoporin ASM4
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 6.6 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 20, 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
7: Spoke connector
3: Spoke connector
8: Spoke connector
4: Spoke connector
a: Spoke connector
b: Spoke connector
k: Spoke connector
c: Spoke connector
m: Spoke connector
d: Spoke connector
e: Spoke connector
f: Spoke connector
g: Spoke connector
l: Spoke connector
h: Spoke connector
n: Spoke connector
j: Spoke connector
i: Spoke connector
Y: Nucleoporin NUP170
Z: Nucleoporin NUP157
A: Nucleoporin NSP1
B: Nucleoporin NUP57
C: Nucleoporin NUP49/NSP49
D: Nucleoporin NSP1
E: Nucleoporin NUP57
F: Nucleoporin NUP49/NSP49
M: Nucleoporin NUP192
N: Nucleoporin NUP188
Q: Nucleoporin NIC96
R: Nucleoporin NIC96
U: Nucleoporin NUP53
V: Nucleoporin 59
0: Nucleoporin NUP170
1: Nucleoporin NUP157
G: Nucleoporin NSP1
H: Nucleoporin NUP57
I: Nucleoporin NUP49/NSP49
J: Nucleoporin NSP1
K: Nucleoporin NUP57
L: Nucleoporin NUP49/NSP49
O: Nucleoporin NUP192
P: Nucleoporin NUP188
S: Nucleoporin NIC96
T: Nucleoporin NIC96
W: Nucleoporin NUP53
X: Nucleoporin 59


Theoretical massNumber of molelcules
Total (without water)2,852,49246
Polymers2,852,49246
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/peptide , 1 types, 18 molecules 7384abkcmdefglhnji

#1: Protein/peptide
Spoke connector


Mass: 3166.895 Da / Num. of mol.: 18 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / Cell line: W303

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Protein , 10 types, 28 molecules Y0Z1ADGJBEHKCFILMONPQRSTUWVX

#2: Protein Nucleoporin NUP170 / Nuclear pore protein NUP170


Mass: 169651.969 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / Cell line: W303 / Strain: MATa ade2-1 ura3-1 his3-11 / References: UniProt: P38181
#3: Protein Nucleoporin NUP157 / Nuclear pore protein NUP157


Mass: 156827.484 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / Cell line: W303 / Strain: 15 trp1-1 leu2-3 / References: UniProt: P40064
#4: Protein
Nucleoporin NSP1 / Nuclear pore protein NSP1 / Nucleoskeletal-like protein / p110


Mass: 86611.672 Da / Num. of mol.: 4 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / Cell line: W303 / Strain: 112 can1-100 MLP1-PPX-ProteinA::HIS5 / References: UniProt: P14907
#5: Protein
Nucleoporin NUP57 / Nuclear pore protein NUP57


Mass: 57547.145 Da / Num. of mol.: 4 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / Cell line: W303 / References: UniProt: P48837
#6: Protein
Nucleoporin NUP49/NSP49 / Nuclear pore protein NUP49/NSP49


Mass: 49174.762 Da / Num. of mol.: 4 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / Cell line: W303 / References: UniProt: Q02199
#7: Protein Nucleoporin NUP192 / Nuclear pore protein NUP192


Mass: 191718.125 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / Cell line: W303 / References: UniProt: P47054
#8: Protein Nucleoporin NUP188 / Nuclear pore protein NUP188


Mass: 188753.281 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / Cell line: W303 / References: UniProt: P52593
#9: Protein
Nucleoporin NIC96 / 96 kDa nucleoporin-interacting component / Nuclear pore protein NIC96


Mass: 96291.586 Da / Num. of mol.: 4 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / Cell line: W303 / References: UniProt: P34077
#10: Protein Nucleoporin NUP53 / Nuclear pore protein NUP53


Mass: 52688.668 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / Cell line: W303 / References: UniProt: Q03790
#11: Protein Nucleoporin 59 / / Nuclear pore protein NUP59


Mass: 58853.902 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / Cell line: W303 / References: UniProt: Q05166

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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

ComponentName: Nuclear Pore ComplexNuclear pore / Type: COMPLEX / Details: Protein A tagged Mlp1 pullout of NPC / Entity ID: all / Source: NATURAL
Molecular weightValue: 18 MDa / Experimental value: YES
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
Cellular location: nuclear envelope / Organelle: nucleus
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 reconstruction
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: 6.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 208392 / Algorithm: FOURIER SPACE
Details: Multibody extracted spoke image stack processed in CryoSPARC
Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Details: Rigid body docking of 3D map of Nucleoporins in the spoke complex with Chimera followed by flexible fitting and rebuilding in Coot.
Atomic model buildingDetails: none / Source name: Other / Type: experimental model

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