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- PDB-8cds: Crystal structure of the xhNup93-Nb4i VHH antibody -

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Basic information

Entry
Database: PDB / ID: 8cds
TitleCrystal structure of the xhNup93-Nb4i VHH antibody
Components5-Nup93 inhibitory VHH antibody
KeywordsIMMUNE SYSTEM / inhibitory VHH antibody / nanobody
Function / homologyDI(HYDROXYETHYL)ETHER
Function and homology information
Biological speciesVicugna pacos (alpaca)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.53 Å
AuthorsGuttler, T. / Colom, M.S. / Gorlich, D.
Funding support Germany, 2items
OrganizationGrant numberCountry
Max Planck Society Germany
Boehringer Ingelheim Fonds (BIF) 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
DepositionFeb 1, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 21, 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: 5-Nup93 inhibitory VHH antibody
hetero molecules


Theoretical massNumber of molelcules
Total (without water)14,0673
Polymers13,8981
Non-polymers1682
Water2,594144
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)26.877, 42.105, 44.946
Angle α, β, γ (deg.)90.000, 93.055, 90.000
Int Tables number4
Space group name H-MP1211
Space group name HallP2yb
Symmetry operation#1: x,y,z
#2: -x,y+1/2,-z

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Components

#1: Antibody 5-Nup93 inhibitory VHH antibody


Mass: 13898.463 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vicugna pacos (alpaca) / Production host: Escherichia coli (E. coli)
#2: Chemical ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL


Mass: 62.068 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C2H6O2
#3: Chemical ChemComp-PEG / DI(HYDROXYETHYL)ETHER


Mass: 106.120 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C4H10O3
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 144 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestN

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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: 1.83 Å3/Da / Density % sol: 32.68 %
Crystal growTemperature: 293.15 K / Method: vapor diffusion, sitting drop / pH: 8
Details: 0.1 M imidazole pH 8, 30% (w/v) PEG 8,000, 0.2 M NaCl

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SLS / Beamline: X10SA / Wavelength: 1 Å
DetectorType: DECTRIS EIGER X 16M / Detector: PIXEL / Date: Mar 29, 2021
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 1.36→30.71 Å / Num. obs: 40137 / % possible obs: 94.5 % / Redundancy: 6.2187 % / Biso Wilson estimate: 19.32 Å2 / CC1/2: 0.997 / Rmerge(I) obs: 0.053 / Rrim(I) all: 0.064 / Net I/σ(I): 9.94
Reflection shell
Resolution (Å)Rmerge(I) obsNum. unique obsCC1/2Rrim(I) allDiffraction-ID
1.36-1.442.48962890.3123.0711
1.44-1.540.6460010.7150.7791
1.54-1.660.27356290.9230.3321
1.66-1.820.13651740.9760.1651
1.82-2.040.07447660.9910.091
2.04-2.350.05742650.9930.0671
2.35-2.880.05436030.9940.0631
2.88-4.060.04528160.9960.0531
4.06-30.710.0415940.9970.0471

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Processing

Software
NameVersionClassification
PHENIX1.14-3260refinement
PHASER2.8.2phasing
XDSJan 31, 2020 BUILT=20200417data reduction
XSCALEJan 31, 2020 BUILT=20200417data scaling
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 1.53→30.71 Å / SU ML: 0.1756 / Cross valid method: FREE R-VALUE / σ(F): 1.39 / Phase error: 18.8197 / Stereochemistry target values: CDL v1.2
RfactorNum. reflection% reflection
Rfree0.1755 1436 9.76 %
Rwork0.1333 13284 -
obs0.1376 14720 96.37 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 22.05 Å2
Refinement stepCycle: LAST / Resolution: 1.53→30.71 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms971 0 11 144 1126
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00591058
X-RAY DIFFRACTIONf_angle_d0.80371442
X-RAY DIFFRACTIONf_chiral_restr0.0573157
X-RAY DIFFRACTIONf_plane_restr0.0043187
X-RAY DIFFRACTIONf_dihedral_angle_d9.1987640
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.53-1.580.25411400.17491313X-RAY DIFFRACTION94.05
1.58-1.650.22791430.14821274X-RAY DIFFRACTION95.1
1.65-1.720.21851350.15331312X-RAY DIFFRACTION94.95
1.72-1.810.21141440.13231303X-RAY DIFFRACTION95.01
1.81-1.930.19231420.1231322X-RAY DIFFRACTION96.32
1.93-2.080.18611320.12541338X-RAY DIFFRACTION96.65
2.08-2.290.19881470.13321326X-RAY DIFFRACTION97.36
2.29-2.620.20021500.14691342X-RAY DIFFRACTION97.77
2.62-3.30.18631580.14431348X-RAY DIFFRACTION97.79
3.3-30.710.12411450.1191406X-RAY DIFFRACTION98.6

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