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- PDB-6z1v: Structure of the EC2 domain of CD9 in complex with nanobody 4E8 -

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Basic information

Entry
Database: PDB / ID: 6z1v
TitleStructure of the EC2 domain of CD9 in complex with nanobody 4E8
Components
  • CD9 antigen
  • Nanobody 4E8
KeywordsCELL ADHESION / Antibody-antigen complex / tetraspanin / EC2 domain / nanobody
Function / homology
Function and homology information


Acrosome Reaction and Sperm:Oocyte Membrane Binding / myoblast fusion involved in skeletal muscle regeneration / sperm-egg recognition / fusion of sperm to egg plasma membrane involved in single fertilization / regulation of macrophage migration / paranodal junction assembly / glial cell migration / platelet alpha granule membrane / negative regulation of platelet aggregation / Uptake and function of diphtheria toxin ...Acrosome Reaction and Sperm:Oocyte Membrane Binding / myoblast fusion involved in skeletal muscle regeneration / sperm-egg recognition / fusion of sperm to egg plasma membrane involved in single fertilization / regulation of macrophage migration / paranodal junction assembly / glial cell migration / platelet alpha granule membrane / negative regulation of platelet aggregation / Uptake and function of diphtheria toxin / cellular response to low-density lipoprotein particle stimulus / clathrin-coated endocytic vesicle membrane / receptor internalization / platelet activation / endocytic vesicle membrane / extracellular vesicle / integrin binding / Platelet degranulation / cell population proliferation / cell adhesion / negative regulation of cell population proliferation / external side of plasma membrane / focal adhesion / protein-containing complex / extracellular space / extracellular exosome / membrane / plasma membrane
Similarity search - Function
CD9, extracellular domain / Tetraspanin, conserved site / Transmembrane 4 family signature. / Tetraspanin, animals / Tetraspanin, EC2 domain superfamily / Tetraspanin/Peripherin / Tetraspanin family
Similarity search - Domain/homology
ACETIC ACID / TRIETHYLENE GLYCOL / CD9 antigen
Similarity search - Component
Biological speciesHomo sapiens (human)
Lama glama (llama)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.33 Å
AuthorsOosterheert, W. / Pearce, N.M. / Gros, P.
Funding support Netherlands, 2items
OrganizationGrant numberCountry
Netherlands Organisation for Scientific Research (NWO)731.015.201 Netherlands
Netherlands Organisation for Scientific Research (NWO)024.002.009 Netherlands
CitationJournal: Life Sci Alliance / Year: 2020
Title: Implications for tetraspanin-enriched microdomain assembly based on structures of CD9 with EWI-F.
Authors: Wout Oosterheert / Katerina T Xenaki / Viviana Neviani / Wouter Pos / Sofia Doulkeridou / Jip Manshande / Nicholas M Pearce / Loes Mj Kroon-Batenburg / Martin Lutz / Paul Mp van Bergen En ...Authors: Wout Oosterheert / Katerina T Xenaki / Viviana Neviani / Wouter Pos / Sofia Doulkeridou / Jip Manshande / Nicholas M Pearce / Loes Mj Kroon-Batenburg / Martin Lutz / Paul Mp van Bergen En Henegouwen / Piet Gros /
Abstract: Tetraspanins are eukaryotic membrane proteins that contribute to a variety of signaling processes by organizing partner-receptor molecules in the plasma membrane. How tetraspanins bind and cluster ...Tetraspanins are eukaryotic membrane proteins that contribute to a variety of signaling processes by organizing partner-receptor molecules in the plasma membrane. How tetraspanins bind and cluster partner receptors into tetraspanin-enriched microdomains is unknown. Here, we present crystal structures of the large extracellular loop of CD9 bound to nanobodies 4C8 and 4E8 and, the cryo-EM structure of 4C8-bound CD9 in complex with its partner EWI-F. CD9-EWI-F displays a tetrameric arrangement with two central EWI-F molecules, dimerized through their ectodomains, and two CD9 molecules, one bound to each EWI-F transmembrane helix through CD9-helices h3 and h4. In the crystal structures, nanobodies 4C8 and 4E8 bind CD9 at loops C and D, which is in agreement with the 4C8 conformation in the CD9-EWI-F complex. The complex varies from nearly twofold symmetric (with the two CD9 copies nearly anti-parallel) to ca. 50° bent arrangements. This flexible arrangement of CD9-EWI-F with potential CD9 homo-dimerization at either end provides a "concatenation model" for forming short linear or circular assemblies, which may explain the occurrence of tetraspanin-enriched microdomains.
History
DepositionMay 14, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Sep 23, 2020Provider: repository / Type: Initial release
Revision 1.1Sep 30, 2020Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2May 1, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: CD9 antigen
B: Nanobody 4E8
hetero molecules


Theoretical massNumber of molelcules
Total (without water)25,9485
Polymers25,6762
Non-polymers2723
Water2,414134
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration, 1 : 1 complex was purified using gel filtration prior to crystallization.
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area1880 Å2
ΔGint2 kcal/mol
Surface area9930 Å2
MethodPISA
Unit cell
Length a, b, c (Å)61.521, 89.446, 35.684
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number18
Space group name H-MP21212
Space group name HallP22ab
Symmetry operation#1: x,y,z
#2: x+1/2,-y+1/2,-z
#3: -x+1/2,y+1/2,-z
#4: -x,-y,z

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Components

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Protein / Antibody , 2 types, 2 molecules AB

#1: Protein CD9 antigen / 5H9 antigen / Cell growth-inhibiting gene 2 protein / Leukocyte antigen MIC3 / Motility-related ...5H9 antigen / Cell growth-inhibiting gene 2 protein / Leukocyte antigen MIC3 / Motility-related protein / MRP-1 / Tetraspanin-29 / Tspan-29 / p24


Mass: 10129.433 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CD9, MIC3, TSPAN29, GIG2 / Plasmid: pUPE 107.03 / Cell (production host): EMBRYONIC / Cell line (production host): HEK293EBNA / Organ (production host): KIDNEY / Production host: Homo sapiens (human) / References: UniProt: P21926
#2: Antibody Nanobody 4E8


Mass: 15546.274 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lama glama (llama) / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): Codon Plus

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Non-polymers , 4 types, 137 molecules

#3: Chemical ChemComp-PGE / TRIETHYLENE GLYCOL


Mass: 150.173 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C6H14O4
#4: Chemical ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL


Mass: 62.068 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C2H6O2
#5: Chemical ChemComp-ACY / ACETIC ACID


Mass: 60.052 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C2H4O2
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 134 / Source method: isolated from a natural source / Formula: H2O

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Details

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: 1.91 Å3/Da / Density % sol: 35.67 % / Description: 3D-diamond like shape
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 8
Details: 0.2 M sodium acetate, 0.1 M Tris pH 8.0, 30% (w/v) PEG 4,000 cryoprotected with reservoir solution supplemented with 20% (v/v) ethylene glycol.
Temp details: room temperature

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Data collection

DiffractionMean temperature: 100 K / Ambient temp details: liquid nitrogen temperature / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: Diamond / Beamline: I04-1 / Wavelength: 0.9159 Å
DetectorType: DECTRIS PILATUS 6M-F / Detector: PIXEL / Date: Aug 6, 2019
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9159 Å / Relative weight: 1
ReflectionResolution: 1.33→44.72 Å / Num. obs: 32044 / % possible obs: 92 % / Redundancy: 6.4 % / Biso Wilson estimate: 19.48 Å2 / CC1/2: 1 / Rmerge(I) obs: 0.04 / Rpim(I) all: 0.02 / Net I/σ(I): 17.6
Reflection shellResolution: 1.33→1.44 Å / Redundancy: 6.1 % / Rmerge(I) obs: 0.98 / Mean I/σ(I) obs: 1.5 / Num. unique obs: 1603 / CC1/2: 0.653 / Rpim(I) all: 0.6 / % possible all: 68.9

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Processing

Software
NameVersionClassification
REFMAC5refinement
PHENIX1.14_3260refinement
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: Structure of CD9EC2 bound to nanobody 4C8

Resolution: 1.33→44.72 Å / SU ML: 0.1369 / Cross valid method: FREE R-VALUE / σ(F): 1.35 / Phase error: 25.1549 / Stereochemistry target values: CDL v1.2
Details: For the CD9EC2 - 4E8 dataset, the autoprocessed and anisotropical-truncated (autoproc-staraniso) reflection data file provided by DLS was employed. The structure was solved by molecular ...Details: For the CD9EC2 - 4E8 dataset, the autoprocessed and anisotropical-truncated (autoproc-staraniso) reflection data file provided by DLS was employed. The structure was solved by molecular replacement using PHASER with the CD9EC2 - 4C8 structure as search model. The 4C8 residues were replaced with the corresponding 4E8 residues and the CDR regions of the nanobody were manually built in Coot. The structure was then iteratively refined using Refmac5 or Phenix, alternated with model improvement in COOT. The final refinement in Phenix yielded Rwork/Rfree = 15.1/19.0%
RfactorNum. reflection% reflection
Rfree0.1898 1570 4.9 %
Rwork0.1512 30472 -
obs0.1531 32042 69.74 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 27.7 Å2
Refinement stepCycle: LAST / Resolution: 1.33→44.72 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1570 0 18 134 1722
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00791775
X-RAY DIFFRACTIONf_angle_d0.94062429
X-RAY DIFFRACTIONf_chiral_restr0.0772261
X-RAY DIFFRACTIONf_plane_restr0.0057319
X-RAY DIFFRACTIONf_dihedral_angle_d23.8579679
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.33-1.370.5263200.2693371X-RAY DIFFRACTION9.49
1.37-1.420.3523450.2443784X-RAY DIFFRACTION20.2
1.42-1.480.2764780.2111505X-RAY DIFFRACTION38.55
1.48-1.550.24781250.1812138X-RAY DIFFRACTION54.74
1.55-1.630.24641500.16312820X-RAY DIFFRACTION72.09
1.63-1.730.21961710.15943584X-RAY DIFFRACTION91.03
1.73-1.870.18572030.14413957X-RAY DIFFRACTION99.93
1.87-2.050.17151880.13223704X-RAY DIFFRACTION93.33
2.05-2.350.18931890.13223436X-RAY DIFFRACTION86.56
2.35-2.960.19461980.1483956X-RAY DIFFRACTION97.67
2.96-44.720.17792030.15784217X-RAY DIFFRACTION99.77

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