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- PDB-7zox: Nup93 in complex with xhNup93-Nb4i and xNup93-Nb2t -

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Basic information

Entry
Database: PDB / ID: 7zox
TitleNup93 in complex with xhNup93-Nb4i and xNup93-Nb2t
Components
  • Nuclear pore complex protein Nup93
  • xNup93-Nb2t
  • xhNup93-Nb4i
KeywordsNUCLEAR 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
Nucleoporin interacting component Nup93/Nic96 / Nup93/Nic96
Similarity search - Domain/homology
Nuclear pore complex protein Nup93
Similarity search - Component
Biological speciesXenopus laevis (African clawed frog)
Vicugna pacos (alpaca)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.4 Å
AuthorsFu, Z. / Guttler, T. / Colom, M.S.
Funding support Germany, 1items
OrganizationGrant numberCountry
Max Planck Society Germany
CitationJournal: 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
DepositionApr 26, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 8, 2023Provider: repository / Type: Initial release
Revision 1.1Feb 28, 2024Group: Structure summary / Category: entity / struct / Item: _entity.pdbx_description / _struct.title
Revision 1.2May 22, 2024Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.3Nov 6, 2024Group: Data collection / Structure summary
Category: em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Nuclear pore complex protein Nup93
B: xhNup93-Nb4i
C: xNup93-Nb2t


Theoretical massNumber of molelcules
Total (without water)101,5153
Polymers101,5153
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area3480 Å2
ΔGint-15 kcal/mol
Surface area37700 Å2
MethodPISA

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Components

#1: Protein Nuclear pore complex protein Nup93 / 93 kDa nucleoporin / An4a / Nucleoporin Nup93


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
#2: Antibody xhNup93-Nb4i


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 xNup93-Nb2t


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)
Has protein modificationY

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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: Nup93 in complex with 5-Nup93 inhibitory NB and 15-Nup93 tracking NB
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.1 MDa / Experimental value: NO
Source (natural)Organism: Xenopus laevis, Vicugna pacos
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMsodium chlorideNaCl1
220 mMTris/HCl1
32 mMDithiothreitolDTT1
SpecimenConc.: 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 supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: 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

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: 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 holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage 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 opticsEnergyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV
Image scansWidth: 5760 / Height: 4092

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Processing

EM software
IDNameVersionCategory
1Warpparticle selection
4WarpCTF correction
10RELION3.1initial Euler assignment
11RELION3.1final Euler assignment
12RELION3.1classification
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 808840
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 202725 / Symmetry type: POINT

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