+Open data
-Basic information
Entry | Database: PDB / ID: 7zox | ||||||
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Title | Nup93 in complex with xhNup93-Nb4i and xNup93-Nb2t | ||||||
Components |
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Keywords | NUCLEAR PROTEIN / NUP93 / Nuclearporin / inhibitory NB / tracking NB | ||||||
Function / homology | Function and homology information nuclear pore complex assembly / structural constituent of nuclear pore / poly(A)+ mRNA export from nucleus / nuclear pore / nuclear periphery / protein import into nucleus / nuclear membrane / cytosol Similarity search - Function | ||||||
Biological species | Xenopus laevis (African clawed frog) Vicugna pacos (alpaca) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.4 Å | ||||||
Authors | Fu, Z. / Guttler, T. / Colom, M.S. | ||||||
Funding support | Germany, 1items
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Citation | Journal: EMBO J / Year: 2024 Title: A checkpoint function for Nup98 in nuclear pore formation suggested by novel inhibitory nanobodies. Authors: Mireia Solà Colom / Zhenglin Fu / Philip Gunkel / Thomas Güttler / Sergei Trakhanov / Vasundara Srinivasan / Kathrin Gregor / Tino Pleiner / Dirk Görlich / Abstract: Nuclear pore complex (NPC) biogenesis is a still enigmatic example of protein self-assembly. We now introduce several cross-reacting anti-Nup nanobodies for imaging intact nuclear pore complexes from ...Nuclear pore complex (NPC) biogenesis is a still enigmatic example of protein self-assembly. We now introduce several cross-reacting anti-Nup nanobodies for imaging intact nuclear pore complexes from frog to human. We also report a simplified assay that directly tracks postmitotic NPC assembly with added fluorophore-labeled anti-Nup nanobodies. During interphase, NPCs are inserted into a pre-existing nuclear envelope. Monitoring this process is challenging because newly assembled NPCs are indistinguishable from pre-existing ones. We overcame this problem by inserting Xenopus-derived NPCs into human nuclear envelopes and using frog-specific anti-Nup nanobodies for detection. We further asked whether anti-Nup nanobodies could serve as NPC assembly inhibitors. Using a selection strategy against conserved epitopes, we obtained anti-Nup93, Nup98, and Nup155 nanobodies that block Nup-Nup interfaces and arrest NPC assembly. We solved structures of nanobody-target complexes and identified roles for the Nup93 α-solenoid domain in recruiting Nup358 and the Nup214·88·62 complex, as well as for Nup155 and the Nup98 autoproteolytic domain in NPC scaffold assembly. The latter suggests a checkpoint linking pore formation to the assembly of the Nup98-dominated permeability barrier. | ||||||
History |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 7zox.cif.gz | 162.5 KB | Display | PDBx/mmCIF format |
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PDB format | pdb7zox.ent.gz | 125 KB | Display | PDB format |
PDBx/mmJSON format | 7zox.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 7zox_validation.pdf.gz | 374.4 KB | Display | wwPDB validaton report |
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Full document | 7zox_full_validation.pdf.gz | 391.6 KB | Display | |
Data in XML | 7zox_validation.xml.gz | 19.2 KB | Display | |
Data in CIF | 7zox_validation.cif.gz | 28.8 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/zo/7zox ftp://data.pdbj.org/pub/pdb/validation_reports/zo/7zox | HTTPS FTP |
-Related structure data
Related structure data | 14849MC 7nowC 7nqaC 8cdsC 8cdtC 8ozbC 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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1 |
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-Components
#1: Protein | Mass: 74782.289 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Details: Xenopus laevis Nup93(residues 168-820) / Source: (gene. exp.) Xenopus laevis (African clawed frog) / Gene: nup93 / Production host: Escherichia coli (E. coli) / References: UniProt: Q7ZX96 |
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#2: Antibody | Mass: 13811.384 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Details: 5-Nup93 inhibitory NB / Source: (gene. exp.) Vicugna pacos (alpaca) / Production host: Escherichia coli (E. coli) |
#3: Antibody | Mass: 12921.641 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Details: 15-Nup93 tracking NB / Source: (gene. exp.) Vicugna pacos (alpaca) / Production host: Escherichia coli (E. coli) |
-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: Nup93 in complex with 5-Nup93 inhibitory NB and 15-Nup93 tracking NB Type: COMPLEX / Entity ID: all / Source: RECOMBINANT | ||||||||||||||||||||
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Molecular weight | Value: 0.1 MDa / Experimental value: NO | ||||||||||||||||||||
Source (natural) | Organism: Xenopus laevis, Vicugna pacos | ||||||||||||||||||||
Source (recombinant) | Organism: Escherichia coli (E. coli) | ||||||||||||||||||||
Buffer solution | pH: 7.5 | ||||||||||||||||||||
Buffer component |
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Specimen | Conc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES Details: The complex was further purified by size exclusion chromatography using a HiLoad 26/60 Superdex 200 column. The purified complex was applied to a glow-discharged grid after being diluted to 1 mg/ml. | ||||||||||||||||||||
Specimen support | Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/2 | ||||||||||||||||||||
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K Details: Sample volume: 2.0 microliters blotting time: 5 s blot force setting: 6 |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 81000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1500 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Average exposure time: 2 sec. / Electron dose: 54 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 12320 / Details: Counting mode 5 images per hole( beam-image shift) |
EM imaging optics | Energyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV |
Image scans | Width: 5760 / Height: 4092 |
-Processing
EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||
Particle selection | Num. of particles selected: 808840 | ||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | ||||||||||||||||||||||||
3D reconstruction | Resolution: 4.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 202725 / Symmetry type: POINT |