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- PDB-8t9l: Pom34-Pom152 membrane attachment site yeast NPC -

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Basic information

Entry
Database: PDB / ID: 8t9l
TitlePom34-Pom152 membrane attachment site yeast NPC
Components
  • Nucleoporin POM152
  • Nucleoporin POM34
KeywordsTRANSPORT PROTEIN / nuclear pore complex / nucleocytoplasmic transport / nucleoporin / membrane protein / translocase
Function / homology
Function and homology information


nuclear pore transmembrane ring / spindle pole body duplication / nuclear pore organization / structural constituent of nuclear pore / nuclear outer membrane / nucleocytoplasmic transport / nuclear envelope lumen / mRNA transport / nuclear pore / protein-membrane adaptor activity ...nuclear pore transmembrane ring / spindle pole body duplication / nuclear pore organization / structural constituent of nuclear pore / nuclear outer membrane / nucleocytoplasmic transport / nuclear envelope lumen / mRNA transport / nuclear pore / protein-membrane adaptor activity / nuclear periphery / cell periphery / protein import into nucleus / nuclear envelope / nuclear membrane / mitochondrion
Similarity search - Function
Nuclear pore complex component / Nuclear pore complex component / Nucleoporin Pom152
Similarity search - Domain/homology
Nucleoporin POM152 / Nucleoporin POM34
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 7 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
DepositionJun 24, 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
B: Nucleoporin POM152
A: Nucleoporin POM34
D: Nucleoporin POM152
C: Nucleoporin POM34


Theoretical massNumber of molelcules
Total (without water)372,2264
Polymers372,2264
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

#1: Protein Nucleoporin POM152 / Nuclear pore protein POM152 / P150 / Pore membrane protein POM152


Mass: 151833.891 Da / Num. of mol.: 2 / Source method: isolated from a natural source
Details: Pore outer membrane protein and anchor for the lumenal ring
Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / Cell line: W303 / Plasmid details: nucleus / Strain: MATa ade2-1 ura3-1 his3-11 / References: UniProt: P39685
#2: Protein Nucleoporin POM34 / Nuclear pore protein POM34 / Pore membrane protein POM34


Mass: 34279.301 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Details: Pore membrane protein and inner ring anchor / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / Cell line: W303 / Plasmid details: nucleus / Strain: 15 trp1-1 leu2-3 / References: UniProt: Q12445

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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
2Pom34-Pom152 Inner ring anchor complexCOMPLEXnative complex resolved by multibody image processing1NATURAL
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
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R2/2
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
10RELION3.0.7initial Euler assignment
11RELION3.0.7final Euler assignment
12RELION3.0.7classification
13RELION3.0.73D 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: C2 (2 fold cyclic)
3D reconstructionResolution: 7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 208392 / Algorithm: FOURIER SPACE / Details: local resolution for the TMDs 10.5-12 Angstroms / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL / Target criteria: cross correlation
Details: Rigid body docking and manual building with Chimera and coot
Atomic model buildingDetails: none / Source name: AlphaFold / Type: in silico model

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