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- PDB-8cdt: Crystal structure of the xNup93-Nb2t VHH antibody -

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Basic information

Entry
Database: PDB / ID: 8cdt
TitleCrystal structure of the xNup93-Nb2t VHH antibody
Components15-Nup93 tracking VHH antibody
KeywordsIMMUNE SYSTEM / VHH antibody / nanobody
Biological speciesVicugna pacos (alpaca)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.41 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: 15-Nup93 tracking VHH antibody
hetero molecules


Theoretical massNumber of molelcules
Total (without water)13,1063
Polymers12,9221
Non-polymers1842
Water2,252125
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)99.968, 99.968, 30.707
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number173
Space group name H-MP63
Space group name HallP6c
Symmetry operation#1: x,y,z
#2: x-y,x,z+1/2
#3: y,-x+y,z+1/2
#4: -y,x-y,z
#5: -x+y,-x,z
#6: -x,-y,z+1/2

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Components

#1: Antibody 15-Nup93 tracking VHH antibody


Mass: 12921.641 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-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL / Glycerol


Mass: 92.094 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C3H8O3
#3: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 125 / 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: 3.43 Å3/Da / Density % sol: 64.17 %
Crystal growTemperature: 293.15 K / Method: vapor diffusion, sitting drop / pH: 10.5
Details: 0.1 M CAPS (pH 10.5), 1.2 M sodium phosphate, 0.8 M potassium phosphate, 0.2 M lithium sulfate

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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 EIGER2 X 16M / Detector: PIXEL / Date: Mar 21, 2021
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 1.33→43.29 Å / Num. obs: 79377 / % possible obs: 99.5 % / Redundancy: 19.8 % / Biso Wilson estimate: 20.56 Å2 / CC1/2: 1 / Rmerge(I) obs: 0.045 / Rrim(I) all: 0.047 / Net I/σ(I): 22.9
Reflection shell
Resolution (Å)Rmerge(I) obsNum. unique obsCC1/2Rrim(I) allDiffraction-ID
1.33-1.411.818124880.7151.9191
1.41-1.50.587120960.9560.6191
1.5-1.620.246113180.990.2591
1.62-1.780.121103880.9970.1271
1.78-1.990.06493700.9990.0681
1.99-2.290.04782900.9990.0491
2.29-2.810.04270190.9990.0441
2.81-3.960.03654060.9990.0381
3.96-43.290.03430010.9990.0361

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Processing

Software
NameVersionClassification
PHENIX1.14-3260refinement
PHASER2.8.2phasing
XDSJan 31, 2020 BUILT=20200417data scaling
XDSJan 31, 2020 BUILT=20200417data reduction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 1.41→32.72 Å / SU ML: 0.1684 / Cross valid method: FREE R-VALUE / σ(F): 1.38 / Phase error: 17.7705 / Stereochemistry target values: CDL v1.2
RfactorNum. reflection% reflection
Rfree0.1882 1674 4.89 %
Rwork0.1579 32542 -
obs0.1593 34216 99.95 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 25.24 Å2
Refinement stepCycle: LAST / Resolution: 1.41→32.72 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms898 0 6 125 1029
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0191996
X-RAY DIFFRACTIONf_angle_d1.53471353
X-RAY DIFFRACTIONf_chiral_restr0.113147
X-RAY DIFFRACTIONf_plane_restr0.0096177
X-RAY DIFFRACTIONf_dihedral_angle_d12.4346384
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.41-1.450.3871330.35662693X-RAY DIFFRACTION99.79
1.45-1.50.22191360.2212655X-RAY DIFFRACTION99.93
1.5-1.550.1831350.15542692X-RAY DIFFRACTION99.89
1.55-1.610.18011390.152711X-RAY DIFFRACTION100
1.61-1.690.17831390.14872689X-RAY DIFFRACTION99.93
1.69-1.780.17961460.1472689X-RAY DIFFRACTION100
1.78-1.890.17051400.14222696X-RAY DIFFRACTION100
1.89-2.030.1541370.14312704X-RAY DIFFRACTION99.96
2.03-2.240.16191400.14632713X-RAY DIFFRACTION100
2.24-2.560.2311430.16142740X-RAY DIFFRACTION100
2.56-3.230.21591430.16822730X-RAY DIFFRACTION100
3.23-32.720.17291430.15282830X-RAY DIFFRACTION99.87

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