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- EMDB-14849: Nup93 in complex with 5-Nup93 inhibitory NB and 15-Nup93 tracking NB -

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Open data


ID or keywords:

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Basic information

Entry
Database: EMDB / ID: EMD-14849
TitleNup93 in complex with 5-Nup93 inhibitory NB and 15-Nup93 tracking NBNucleoporin 93
Map dataNup93 in complex with inhibitory NanobodyNucleoporin 93
Sample
  • Complex: Nup93 in complex with 5-Nup93 inhibitory NB and 15-Nup93 tracking NBNucleoporin 93
    • Protein or peptide: Nuclear pore complex protein Nup93Nuclear pore
    • Protein or peptide: xhNup93-Nb4i
    • Protein or peptide: xNup93-Nb2t
KeywordsNUP93 / Nuclearporin / inhibitory NB / tracking NB / NUCLEAR PROTEIN
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, Vicugna pacos / Xenopus laevis (African clawed frog) / Vicugna pacos (alpaca)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.4 Å
AuthorsFu Z / Guttler T / Colom MS
Funding support Germany, 1 items
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, 2022-
Header (metadata) releaseNov 8, 2023-
Map releaseNov 8, 2023-
UpdateMay 22, 2024-
Current statusMay 22, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_14849.png

Downloads & links

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Map

FileDownload / File: emd_14849.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNup93 in complex with inhibitory Nanobody
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 256 pix.
= 213.504 Å		0.83 Å/pix.
x 256 pix.
= 213.504 Å		0.83 Å/pix.
x 256 pix.
= 213.504 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.834 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.01
Minimum - Maximum-0.034531042 - 0.05808816
Average (Standard dev.)0.00013910343 (±0.0015824556)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 213.504 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: half map of Nup93 in complex with inhibitory Nanobody

Fileemd_14849_half_map_1.map
Annotationhalf map of Nup93 in complex with inhibitory Nanobody
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map of Nup93 in complex with inhibitory Nanobody

Fileemd_14849_half_map_2.map
Annotationhalf map of Nup93 in complex with inhibitory Nanobody
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Nup93 in complex with 5-Nup93 inhibitory NB and 15-Nup93 tracking NB

EntireName: Nup93 in complex with 5-Nup93 inhibitory NB and 15-Nup93 tracking NBNucleoporin 93
Components
  • Complex: Nup93 in complex with 5-Nup93 inhibitory NB and 15-Nup93 tracking NBNucleoporin 93
    • Protein or peptide: Nuclear pore complex protein Nup93Nuclear pore
    • Protein or peptide: xhNup93-Nb4i
    • Protein or peptide: xNup93-Nb2t

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Supramolecule #1: Nup93 in complex with 5-Nup93 inhibitory NB and 15-Nup93 tracking NB

SupramoleculeName: Nup93 in complex with 5-Nup93 inhibitory NB and 15-Nup93 tracking NB
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Xenopus laevis, Vicugna pacos
Molecular weightTheoretical: 100 KDa

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Macromolecule #1: Nuclear pore complex protein Nup93

MacromoleculeName: Nuclear pore complex protein Nup93 / type: protein_or_peptide / ID: 1 / Details: Xenopus laevis Nup93(residues 168-820) / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Xenopus laevis (African clawed frog)
Molecular weightTheoretical: 74.782289 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: SESGAPGRSS LDNVEMAYAR QMYMYNEKVV SGHLQPSLVD LCTEAAERLD DKNVSDLWVM VKQMTDVPLI PASDTLKSRC SGQMQMAFV RQALNYLEQS YKNYTLISVF ANLQQAQLGG VPGTYNLVRS FLNIRLPTPI PGLQDGEIEG YPVWALIYYC M RCGDLMAA ...String:
SESGAPGRSS LDNVEMAYAR QMYMYNEKVV SGHLQPSLVD LCTEAAERLD DKNVSDLWVM VKQMTDVPLI PASDTLKSRC SGQMQMAFV RQALNYLEQS YKNYTLISVF ANLQQAQLGG VPGTYNLVRS FLNIRLPTPI PGLQDGEIEG YPVWALIYYC M RCGDLMAA QQVVNRAQHQ LGDFKNCFQE YIHNKDRRLS PTTENKLRLH YRRAVRASTD PYKRAVYCII GRCDVSDNHS EV ADKTEDY LWLKLSQVCF EDEANSSPQD RLTLPQFQKQ LFEDYGESHF AVNQQPYLYF QVLFLTAQFE AAIAFLFRLE RTR CHAVHV ALALFELKLL LKSTGQSAQL LSQEPGEPQG VRRLNFIRLL MLYTRKFEPT DPREALQYFY FLRNEKDNQG ESMF LRCVS ELVIESREFD MLLGKLEKDG SRKPGAIDKF TRDTKTIINK VASVAENKGL FEEAAKLYDL AKNPDKVLEL TNKLL SPVV SQISAPQSNR ERLKNMALAI AERYKSQGVS AEKSINSTFY LLLDLITFFD EYHAGHIDLS FDVIERLKLV PLSQDS VEE RVAAFRNFSD EIRHNLSEIL LATMNILFTQ YKRLKGSGPT TLGRPQRVQE DKDSVLRSQA RALITFAGMI PYRMSGD TN ARLVQMEVLM N

UniProtKB: Nuclear pore complex protein Nup93

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Macromolecule #2: xhNup93-Nb4i

MacromoleculeName: xhNup93-Nb4i / type: protein_or_peptide / ID: 2 / Details: 5-Nup93 inhibitory NB / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Vicugna pacos (alpaca)
Molecular weightTheoretical: 13.811384 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
GQVQLVESGG GVVQTGDSLM LSCTVSGHTI DNWAMGWFRQ TPRKQREFVA AIDKRGTGAI YGNSVKGRFT VSRDNAKNMV YLRMNSLKP EDTAVYFCAV DQLNAGLGDV SYDYDYWGQG TQVTVSS

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Macromolecule #3: xNup93-Nb2t

MacromoleculeName: xNup93-Nb2t / type: protein_or_peptide / ID: 3 / Details: 15-Nup93 tracking NB / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Vicugna pacos (alpaca)
Molecular weightTheoretical: 12.921641 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
GDVQLVESGG GLVQPGGSLR LSCVASGFTF SRVGMGWVRQ APGKGLEWVS DINASGGTGY ADSVKGRFAI SRDNAKNTLY LQMNRLKPE DTAVYYCAKM MDTAMIEAGI IKPAGQGTQV TVSS

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration1.0 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
50.0 mMNaClSodium chloridesodium chloride
20.0 mMTris/HCl
2.0 mMDTTDithiothreitol
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.04 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV
Details: Sample volume: 2.0 microliters blotting time: 5 s blot force setting: 6.
DetailsThe 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.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 81000
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number grids imaged: 1 / Number real images: 12320 / Average exposure time: 2.0 sec. / Average electron dose: 54.0 e/Å2 / Details: Counting mode 5 images per hole( beam-image shift)
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 808840
Startup modelType of model: OTHER
Details: the ab-initio 3D model was generated from particles from good 2D classes using RELION(version 3.1)
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final 3D classificationNumber classes: 4 / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.4 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 202725
FSC plot (resolution estimation)

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