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- PDB-6z20: Structure of the EC2 domain of CD9 in complex with nanobody 4C8 -

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Basic information

Entry
Database: PDB / ID: 6z20
TitleStructure of the EC2 domain of CD9 in complex with nanobody 4C8
Components
  • CD9 antigen
  • Nanobody 4C8
KeywordsCELL ADHESION / Antibody-antigen complex / tetraspanin / CD9 / 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
Biological speciesHomo sapiens (human)
Lama glama (llama)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.68 Å
AuthorsOosterheert, W. / Manshande, J. / Pearce, N.M. / Lutz, 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 1.2Jan 24, 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 4C8
C: CD9 antigen
D: Nanobody 4C8
hetero molecules


Theoretical massNumber of molelcules
Total (without water)48,6607
Polymers48,4404
Non-polymers2203
Water1086
1
A: CD9 antigen
B: Nanobody 4C8
hetero molecules


Theoretical massNumber of molelcules
Total (without water)24,3123
Polymers24,2202
Non-polymers921
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
C: CD9 antigen
D: Nanobody 4C8
hetero molecules


Theoretical massNumber of molelcules
Total (without water)24,3484
Polymers24,2202
Non-polymers1282
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)75.645, 121.415, 129.249
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number24
Space group name H-MI212121
Space group name HallI2b2c
Symmetry operation#1: x,y,z
#2: x,-y,-z+1/2
#3: -x+1/2,y,-z
#4: -x,-y+1/2,z
#5: x+1/2,y+1/2,z+1/2
#6: x+1/2,-y+1/2,-z+1
#7: -x+1,y+1/2,-z+1/2
#8: -x+1/2,-y+1,z+1/2

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Components

#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.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CD9, MIC3, TSPAN29, GIG2 / Plasmid: 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 4C8


Mass: 14090.641 Da / Num. of mol.: 2
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 (RIL)
#3: Chemical ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C3H8O3
#4: Chemical ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Cl
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 6 / 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: 3.06 Å3/Da / Density % sol: 59.85 % / Description: needle
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop / pH: 5.6
Details: 0.095 M sodium citrate pH 5.6, 5% (v/v) glycerol, 19% (v/v) isopropanol, 20% (w/v) PEG 4,000. The crystal was cryoprotected by soaking in reservoir solution supplemented with 25% glycerol (final concentration).
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: I03 / Wavelength: 0.9763 Å
DetectorType: DECTRIS EIGER2 XE 16M / Detector: PIXEL / Date: May 17, 2019
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9763 Å / Relative weight: 1
ReflectionResolution: 2.68→88.49 Å / Num. obs: 14256 / % possible obs: 93.9 % / Redundancy: 6.7 % / Biso Wilson estimate: 66.57 Å2 / CC1/2: 0.997 / Rmerge(I) obs: 0.13 / Rpim(I) all: 0.05 / Net I/σ(I): 9.5
Reflection shellResolution: 2.68→2.83 Å / Redundancy: 7.3 % / Rmerge(I) obs: 1.78 / Mean I/σ(I) obs: 1.1 / Num. unique obs: 750 / CC1/2: 0.507 / Rpim(I) all: 0.71 / % possible all: 55.8

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Processing

Software
NameVersionClassification
REFMAC5refinement
PHENIX1.14_3260refinement
DIALSdata reduction
STARANISOdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 6RLO

6rlo


Resolution: 2.68→60.71 Å / SU ML: 0.3054 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 29.879 / Stereochemistry target values: CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2483 734 5.15 %
Rwork0.2061 13509 -
obs0.2084 14243 83.45 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 68.91 Å2
Refinement stepCycle: LAST / Resolution: 2.68→60.71 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3104 0 13 6 3123
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00343182
X-RAY DIFFRACTIONf_angle_d0.49544293
X-RAY DIFFRACTIONf_chiral_restr0.0405457
X-RAY DIFFRACTIONf_plane_restr0.0027550
X-RAY DIFFRACTIONf_dihedral_angle_d17.38851887
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.68-2.890.37390.3112998X-RAY DIFFRACTION31.01
2.89-3.180.32421470.28632724X-RAY DIFFRACTION85.07
3.18-3.640.28811880.22373179X-RAY DIFFRACTION99.94
3.64-4.580.22421770.18493249X-RAY DIFFRACTION99.94
4.58-60.710.22641830.1913359X-RAY DIFFRACTION99.75
Refinement TLS params.Method: refined / Origin x: 20.6220696969 Å / Origin y: 11.8145232372 Å / Origin z: -22.5004079045 Å
111213212223313233
T0.355199721333 Å20.130851009426 Å2-0.0450834836326 Å2-0.418177769218 Å2-0.0549746343289 Å2--0.350062340547 Å2
L1.10128655091 °2-0.220827650013 °20.167161151285 °2-1.99211920602 °2-0.607978535594 °2--1.14716072606 °2
S-0.259562451676 Å °-0.134223096192 Å °0.221670821778 Å °0.367890917569 Å °0.443627876284 Å °0.235217896315 Å °-0.170395812173 Å °-0.181276669371 Å °0.0362726526662 Å °
Refinement TLS groupSelection details: all

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