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- PDB-8ozb: Crystal structure of Nup35-Nb complex -

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Basic information

Entry
Database: PDB / ID: 8ozb
TitleCrystal structure of Nup35-Nb complex
Components
  • (Nup35 nanobody) x 2
  • Nucleoporin NUP35
KeywordsTRANSPORT PROTEIN / Nuclear pore complex Nanobody
Function / homology
Function and homology information


nuclear pore central transport channel / nuclear pore organization / Nuclear Pore Complex (NPC) Disassembly / nuclear pore nuclear basket / Transport of Ribonucleoproteins into the Host Nucleus / Regulation of Glucokinase by Glucokinase Regulatory Protein / Defective TPR may confer susceptibility towards thyroid papillary carcinoma (TPC) / Transport of the SLBP independent Mature mRNA / Transport of the SLBP Dependant Mature mRNA / NS1 Mediated Effects on Host Pathways ...nuclear pore central transport channel / nuclear pore organization / Nuclear Pore Complex (NPC) Disassembly / nuclear pore nuclear basket / Transport of Ribonucleoproteins into the Host Nucleus / Regulation of Glucokinase by Glucokinase Regulatory Protein / Defective TPR may confer susceptibility towards thyroid papillary carcinoma (TPC) / Transport of the SLBP independent Mature mRNA / Transport of the SLBP Dependant Mature mRNA / NS1 Mediated Effects on Host Pathways / SUMOylation of SUMOylation proteins / Transport of Mature mRNA Derived from an Intronless Transcript / Rev-mediated nuclear export of HIV RNA / structural constituent of nuclear pore / SUMOylation of RNA binding proteins / Nuclear import of Rev protein / Transport of Mature mRNA derived from an Intron-Containing Transcript / NEP/NS2 Interacts with the Cellular Export Machinery / tRNA processing in the nucleus / Postmitotic nuclear pore complex (NPC) reformation / nucleocytoplasmic transport / Viral Messenger RNA Synthesis / NLS-bearing protein import into nucleus / SUMOylation of ubiquitinylation proteins / Vpr-mediated nuclear import of PICs / SUMOylation of DNA replication proteins / Regulation of HSF1-mediated heat shock response / mRNA transport / SUMOylation of DNA damage response and repair proteins / nuclear pore / SUMOylation of chromatin organization proteins / HCMV Late Events / cellular response to leukemia inhibitory factor / Transcriptional regulation by small RNAs / phospholipid binding / ISG15 antiviral mechanism / HCMV Early Events / nuclear envelope / snRNP Assembly / nuclear membrane / nucleic acid binding / SARS-CoV-2 activates/modulates innate and adaptive immune responses / nucleoplasm / identical protein binding / plasma membrane / cytosol
Similarity search - Function
RNA-recognition motif (RRM) Nup35-type domain / Nucleoporin, NUP53 / Nup53/35/40-type RNA recognition motif / RNA-recognition motif (RRM) Nup35-type domain profile. / RNA-binding domain superfamily / Nucleotide-binding alpha-beta plait domain superfamily
Similarity search - Domain/homology
Biological speciesLama glama (llama)
Homo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.09 Å
AuthorsSrinivasan, V.
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
DepositionMay 8, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 28, 2024Provider: repository / Type: Initial release
Revision 1.1May 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 1.2Jun 12, 2024Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Nup35 nanobody
C: Nup35 nanobody
E: Nucleoporin NUP35
F: Nucleoporin NUP35


Theoretical massNumber of molelcules
Total (without water)42,4314
Polymers42,4314
Non-polymers00
Water1,24369
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area4660 Å2
ΔGint-27 kcal/mol
Surface area17610 Å2
Unit cell
Length a, b, c (Å)49.120, 77.220, 127.470
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number17
Space group name H-MP2221

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Components

#1: Antibody Nup35 nanobody


Mass: 12582.026 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lama glama (llama) / Production host: Escherichia coli (E. coli)
#2: Antibody Nup35 nanobody


Mass: 12669.104 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lama glama (llama) / Production host: Escherichia coli (E. coli)
#3: Protein Nucleoporin NUP35 / 35 kDa nucleoporin / Mitotic phosphoprotein 44 / MP-44 / Nuclear pore complex protein Nup53 / ...35 kDa nucleoporin / Mitotic phosphoprotein 44 / MP-44 / Nuclear pore complex protein Nup53 / Nucleoporin NUP53


Mass: 8589.972 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: NUP35, MP44, NUP53 / Production host: Escherichia coli (E. coli) / References: UniProt: Q8NFH5
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 69 / Source method: isolated from a natural source / Formula: H2O

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.85 Å3/Da / Density % sol: 56.82 %
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop
Details: 0.05 M HEPES (pH 6.5); 25% v/v PEG400; 0.05 M NaCl, 0.01 M MgCl2.

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: PETRA III, EMBL c/o DESY / Beamline: P14 (MX2) / Wavelength: 0.9763 Å
DetectorType: DECTRIS EIGER R 4M / Detector: PIXEL / Date: Dec 5, 2019
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9763 Å / Relative weight: 1
ReflectionResolution: 2.09→77.02 Å / Num. obs: 29113 / % possible obs: 98.6 % / Redundancy: 12.5 % / CC1/2: 1 / Net I/σ(I): 8.1
Reflection shellResolution: 2.09→2.15 Å / Num. unique obs: 1993 / CC1/2: 1

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Processing

Software
NameVersionClassification
REFMAC5.8.0257refinement
XDSdata reduction
XSCALEdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2.09→20 Å / Cor.coef. Fo:Fc: 0.954 / Cor.coef. Fo:Fc free: 0.938 / SU B: 7.021 / SU ML: 0.171 / Cross valid method: THROUGHOUT / ESU R: 0.23 / ESU R Free: 0.19 / Stereochemistry target values: MAXIMUM LIKELIHOOD / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.2674 1493 5.1 %RANDOM
Rwork0.24168 ---
obs0.24306 27532 98.3 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso mean: 37.928 Å2
Baniso -1Baniso -2Baniso -3
1--0.27 Å20 Å20 Å2
2---1.04 Å20 Å2
3---1.31 Å2
Refinement stepCycle: 1 / Resolution: 2.09→20 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2976 0 0 69 3045
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0090.0133036
X-RAY DIFFRACTIONr_bond_other_d0.0010.0182764
X-RAY DIFFRACTIONr_angle_refined_deg1.6751.634100
X-RAY DIFFRACTIONr_angle_other_deg1.3231.5776386
X-RAY DIFFRACTIONr_dihedral_angle_1_deg7.6195381
X-RAY DIFFRACTIONr_dihedral_angle_2_deg32.19321.558154
X-RAY DIFFRACTIONr_dihedral_angle_3_deg16.77915505
X-RAY DIFFRACTIONr_dihedral_angle_4_deg19.8641520
X-RAY DIFFRACTIONr_chiral_restr0.0680.2387
X-RAY DIFFRACTIONr_gen_planes_refined0.0080.023433
X-RAY DIFFRACTIONr_gen_planes_other0.0010.02683
X-RAY DIFFRACTIONr_nbd_refined
X-RAY DIFFRACTIONr_nbd_other
X-RAY DIFFRACTIONr_nbtor_refined
X-RAY DIFFRACTIONr_nbtor_other
X-RAY DIFFRACTIONr_xyhbond_nbd_refined
X-RAY DIFFRACTIONr_xyhbond_nbd_other
X-RAY DIFFRACTIONr_metal_ion_refined
X-RAY DIFFRACTIONr_metal_ion_other
X-RAY DIFFRACTIONr_symmetry_vdw_refined
X-RAY DIFFRACTIONr_symmetry_vdw_other
X-RAY DIFFRACTIONr_symmetry_hbond_refined
X-RAY DIFFRACTIONr_symmetry_hbond_other
X-RAY DIFFRACTIONr_symmetry_metal_ion_refined
X-RAY DIFFRACTIONr_symmetry_metal_ion_other
X-RAY DIFFRACTIONr_mcbond_it3.2383.8271536
X-RAY DIFFRACTIONr_mcbond_other3.2343.8261535
X-RAY DIFFRACTIONr_mcangle_it4.7395.731913
X-RAY DIFFRACTIONr_mcangle_other4.745.7321914
X-RAY DIFFRACTIONr_scbond_it4.3894.2691500
X-RAY DIFFRACTIONr_scbond_other4.3884.2711501
X-RAY DIFFRACTIONr_scangle_it
X-RAY DIFFRACTIONr_scangle_other6.6646.1922188
X-RAY DIFFRACTIONr_long_range_B_refined12.68644.6323329
X-RAY DIFFRACTIONr_long_range_B_other12.98144.6533326
X-RAY DIFFRACTIONr_rigid_bond_restr
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
LS refinement shellResolution: 2.09→2.144 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.398 90 -
Rwork0.42 1766 -
obs--87.51 %

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