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Yorodumi- PDB-7tdz: Cryo-EM model of protomer of the cytoplasmic ring of the nuclear ... -
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-Basic information
Entry | Database: PDB / ID: 7tdz | ||||||
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Title | Cryo-EM model of protomer of the cytoplasmic ring of the nuclear pore complex from Xenopus laevis | ||||||
Components |
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Keywords | NUCLEAR PROTEIN / Nuclear pore complex | ||||||
Function / homology | Function and homology information macromolecule localization / nitrogen compound transport / GATOR2 complex / nephron development / macromolecule metabolic process / protein exit from endoplasmic reticulum / protein localization to nuclear inner membrane / COPII-coated vesicle budding / nuclear pore inner ring / nuclear pore central transport channel ...macromolecule localization / nitrogen compound transport / GATOR2 complex / nephron development / macromolecule metabolic process / protein exit from endoplasmic reticulum / protein localization to nuclear inner membrane / COPII-coated vesicle budding / nuclear pore inner ring / nuclear pore central transport channel / transcription-dependent tethering of RNA polymerase II gene DNA at nuclear periphery / nuclear pore outer ring / nuclear pore organization / COPII vesicle coat / : / post-transcriptional tethering of RNA polymerase II gene DNA at nuclear periphery / attachment of mitotic spindle microtubules to kinetochore / structural constituent of nuclear pore / RNA export from nucleus / nucleocytoplasmic transport / poly(A)+ mRNA export from nucleus / nuclear localization sequence binding / mitotic metaphase chromosome alignment / NLS-bearing protein import into nucleus / ribosomal large subunit export from nucleus / positive regulation of TOR signaling / mRNA transport / cellular response to nutrient levels / mRNA export from nucleus / nuclear pore / ribosomal small subunit export from nucleus / positive regulation of TORC1 signaling / cellular response to amino acid starvation / GTPase activator activity / phospholipid binding / kinetochore / protein import into nucleus / protein transport / nuclear membrane / cell division / lysosomal membrane / positive regulation of DNA-templated transcription / structural molecule activity / metal ion binding / cytoplasm / cytosol Similarity search - Function | ||||||
Biological species | Xenopus laevis (African clawed frog) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 6.9 Å | ||||||
Authors | Fontana, P. / Wu, H. | ||||||
Funding support | 1items
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Citation | Journal: Science / Year: 2022 Title: Structure of cytoplasmic ring of nuclear pore complex by integrative cryo-EM and AlphaFold. Authors: Pietro Fontana / Ying Dong / Xiong Pi / Alexander B Tong / Corey W Hecksel / Longfei Wang / Tian-Min Fu / Carlos Bustamante / Hao Wu / Abstract: INTRODUCTION The nuclear pore complex (NPC) is the molecular conduit in the nuclear membrane of eukaryotic cells that regulates import and export of biomolecules between the nucleus and the cytosol, ...INTRODUCTION The nuclear pore complex (NPC) is the molecular conduit in the nuclear membrane of eukaryotic cells that regulates import and export of biomolecules between the nucleus and the cytosol, with vertebrate NPCs ~110 to 125 MDa in molecular mass and ~120 nm in diameter. NPCs are organized into four main rings: the cytoplasmic ring (CR) at the cytosolic side, the inner ring and the luminal ring on the plane of the nuclear membrane, and the nuclear ring facing the nucleus. Each ring possesses an approximate eightfold symmetry and is composed of multiple copies of different nucleoporins. NPCs have been implicated in numerous biological processes, and their dysfunctions are associated with a growing number of serious human diseases. However, despite pioneering studies from many groups over the past two decades, we still lack a full understanding of NPCs' organization, dynamics, and complexity. RATIONALE We used the oocyte as a model system for the structural characterization because each oocyte possesses a large number of NPC particles that can be visualized on native nuclear membranes without the aid of detergent extraction. We used single-particle cryo-electron microscopy (cryo-EM) analysis on data collected at different stage tilt angles for three-dimensional reconstruction and structure prediction with AlphaFold for model building. RESULTS We reconstructed the CR map of NPC at 6.9 and 6.7 Å resolutions for the full CR protomer and a core region, respectively, and predicted the structures of the individual nucleoporins using AlphaFold because no high-resolution models of Nups were available. For any ambiguous subunit interactions, we also predicted complex structures, which further guided model fitting of the CR protomer. We placed the nucleoporin or complex structures into the CR density to obtain an almost full CR atomic model, composed of the inner and outer Y-complexes, two copies of Nup205, two copies of the Nup214-Nup88-Nup62 complex, one Nup155, and five copies of Nup358. In particular, we predicted the largest protein in the NPC, Nup358, as having an S-shaped globular domain, a coiled-coil domain, and a largely disordered C-terminal region containing phenylalanine-glycine (FG) repeats previously shown to form a gel-like condensate phase for selective cargo passage. Four of the Nup358 copies clamp around the inner and outer Y-complexes to stabilize the CR, and the fifth Nup358 situates in the center of the cluster of clamps. AlphaFold also predicted a homo-oligomeric, likely specifically pentameric, coiled-coil structure of Nup358 that may provide the avidity for Nup358 recruitment to the NPC and for lowering the threshold for Nup358 condensation in NPC biogenesis. CONCLUSION Our studies offer an example of integrative cryo-EM and structure prediction as a general approach for attaining more precise models of megadalton protein complexes from medium-resolution density maps. The more accurate and almost complete model of the CR presented here expands our understanding of the molecular interactions in the NPC and represents a substantial step forward toward the molecular architecture of a full NPC, with implications for NPC function, biogenesis, and regulation. [Figure: see text]. | ||||||
History |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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PDBx/mmCIF format | 7tdz.cif.gz | 6.9 MB | Display | PDBx/mmCIF format |
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PDB format | pdb7tdz.ent.gz | Display | PDB format | |
PDBx/mmJSON format | 7tdz.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 7tdz_validation.pdf.gz | 1.2 MB | Display | wwPDB validaton report |
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Full document | 7tdz_full_validation.pdf.gz | 1.3 MB | Display | |
Data in XML | 7tdz_validation.xml.gz | 437.9 KB | Display | |
Data in CIF | 7tdz_validation.cif.gz | 712.7 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/td/7tdz ftp://data.pdbj.org/pub/pdb/validation_reports/td/7tdz | HTTPS FTP |
-Related structure data
Related structure data | 25817MC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
-Assembly
Deposited unit |
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-Components
-Protein , 13 types, 28 molecules HhtTsSrRgGLlEeDdUPOQNMBbAaIi
#1: Protein | Mass: 105398.547 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Xenopus laevis (African clawed frog) / References: UniProt: A2RV69 #2: Protein | Mass: 55969.496 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Xenopus laevis (African clawed frog) / References: UniProt: Q91349 #3: Protein | Mass: 209080.406 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Xenopus laevis (African clawed frog) / References: UniProt: Q9PVZ2 #4: Protein | Mass: 82573.148 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Xenopus laevis (African clawed frog) / References: UniProt: Q4KLQ6 #5: Protein | Mass: 33914.844 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Xenopus laevis (African clawed frog) / References: UniProt: Q7ZYJ8 #6: Protein | Mass: 227854.141 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Xenopus laevis (African clawed frog) / References: UniProt: Q642R6 #7: Protein | Mass: 36037.664 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Xenopus laevis (African clawed frog) / References: UniProt: Q4FZW5 #8: Protein | Mass: 41744.512 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Xenopus laevis (African clawed frog) / References: UniProt: Q05AW3 #10: Protein | | Mass: 154922.422 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Xenopus laevis (African clawed frog) / References: UniProt: Q7ZWL0 #11: Protein | Mass: 322784.344 Da / Num. of mol.: 5 / Source method: isolated from a natural source / Source: (natural) Xenopus laevis (African clawed frog) / References: UniProt: A0A1L8HGL2 #12: Protein | Mass: 36588.625 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Xenopus laevis (African clawed frog) / References: UniProt: Q66IZ6 #13: Protein | Mass: 162658.234 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Xenopus laevis (African clawed frog) / References: UniProt: A0A1L8GIX3 #14: Protein | Mass: 127551.250 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Xenopus laevis (African clawed frog) / References: UniProt: A0A1L8H1I9 |
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-Nuclear pore complex protein ... , 2 types, 4 molecules CcFf
#9: Protein | Mass: 75160.047 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Xenopus laevis (African clawed frog) / References: UniProt: Q68FJ0 #15: Protein | Mass: 77887.562 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Xenopus laevis (African clawed frog) / References: UniProt: A0A1L8HBE3 |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: Cytoplasmic ring of nuclear pore complex / Type: COMPLEX / Entity ID: all / Source: NATURAL |
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Source (natural) | Organism: Xenopus laevis (African clawed frog) |
Buffer solution | pH: 7.5 |
Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Vitrification | Cryogen name: ETHANE |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: TFS KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Nominal defocus max: 3000 nm / Nominal defocus min: 1000 nm |
Image recording | Electron dose: 1.25 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) |
-Processing
CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION |
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3D reconstruction | Resolution: 6.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 333214 / Symmetry type: POINT |