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- PDB-6rlr: Crystal structure of CD9 large extracellular loop -

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Basic information

Entry
Database: PDB / ID: 6rlr
TitleCrystal structure of CD9 large extracellular loop
ComponentsCD9 antigen
KeywordsIMMUNE SYSTEM / CD9 antigen / tetraspanin
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)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2 Å
AuthorsNeviani, V. / Kroon-Batenburg, L. / Lutz, M. / Pearce, N.M. / Pos, W. / Schotte, R. / Spits, H. / Gros, P.
Funding support Netherlands, 1items
OrganizationGrant numberCountry
Netherlands Organisation for Scientific Research024.002.009 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 2, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Sep 23, 2020Provider: repository / Type: Initial release
Revision 1.1Oct 14, 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 / struct_ncs_dom_lim
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_ncs_dom_lim.beg_auth_comp_id / _struct_ncs_dom_lim.beg_label_asym_id / _struct_ncs_dom_lim.beg_label_comp_id / _struct_ncs_dom_lim.beg_label_seq_id / _struct_ncs_dom_lim.end_auth_comp_id / _struct_ncs_dom_lim.end_label_asym_id / _struct_ncs_dom_lim.end_label_comp_id / _struct_ncs_dom_lim.end_label_seq_id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: CD9 antigen
B: CD9 antigen
C: CD9 antigen
D: CD9 antigen


Theoretical massNumber of molelcules
Total (without water)41,0434
Polymers41,0434
Non-polymers00
Water79344
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area7920 Å2
ΔGint-73 kcal/mol
Surface area20360 Å2
MethodPISA
Unit cell
Length a, b, c (Å)39.986, 39.998, 63.643
Angle α, β, γ (deg.)80.390, 76.290, 68.150
Int Tables number1
Space group name H-MP1
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11A
21B
12A
22C
13A
23D
14B
24C
15B
25D
16C
26D

NCS domain segments:

Component-ID: _ / Beg auth comp-ID: GLY / Beg label comp-ID: GLY / Refine code: _

Dom-IDEns-IDEnd auth comp-IDEnd label comp-IDAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
11PHEPHEAA112 - 1892 - 79
21PHEPHEBB112 - 1892 - 79
12ASNASNAA112 - 1912 - 81
22ASNASNCC112 - 1912 - 81
13ASNASNAA112 - 1912 - 81
23ASNASNDD112 - 1912 - 81
14PHEPHEBB112 - 1892 - 79
24PHEPHECC112 - 1892 - 79
15PHEPHEBB112 - 1892 - 79
25PHEPHEDD112 - 1892 - 79
16ASNASNCC112 - 1912 - 81
26ASNASNDD112 - 1912 - 81

NCS ensembles :
ID
1
2
3
4
5
6

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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: 10260.629 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CD9, MIC3, TSPAN29, GIG2 / Cell line (production host): HEK293 / Production host: Homo sapiens (human) / References: UniProt: P21926
#2: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 44 / 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: 2.52 Å3/Da / Density % sol: 51.25 %
Crystal growTemperature: 293.15 K / Method: vapor diffusion, sitting drop / pH: 6
Details: pentaerythritol propoxylate, potassium chloride, sodium citrate

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: Diamond / Beamline: I04 / Wavelength: 0.98 Å
DetectorType: DECTRIS EIGER X 16M / Detector: PIXEL / Date: Jan 21, 2019
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.98 Å / Relative weight: 1
ReflectionResolution: 2→29.017 Å / Num. obs: 22863 / % possible obs: 95.613 % / Redundancy: 3.407 % / CC1/2: 0.991 / Rpim(I) all: 0.065 / Rrim(I) all: 0.121 / Rsym value: 0.101 / Net I/σ(I): 4.82
Reflection shellResolution: 2→2.072 Å / Redundancy: 3.355 % / Mean I/σ(I) obs: 0.82 / Num. unique obs: 2180 / CC1/2: 0.322 / Rpim(I) all: 0.732 / Rrim(I) all: 1.364 / Rsym value: 1.145 / % possible all: 93.003

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Processing

Software
NameVersionClassification
EVAL15data reduction
EVAL15data scaling
PHASERphasing
Cootmodel building
REFMAC5.8.0238refinement
PDB_EXTRACT3.25data extraction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 6RLO

6rlo


Resolution: 2→29.017 Å / Cor.coef. Fo:Fc: 0.941 / Cor.coef. Fo:Fc free: 0.918 / SU B: 7.931 / SU ML: 0.202 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.234 / ESU R Free: 0.198
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS U VALUES : REFINED INDIVIDUALLY
RfactorNum. reflection% reflectionSelection details
Rfree0.2794 1148 5 %RANDOM
Rwork0.2391 ---
obs0.241 21714 95.51 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å
Displacement parametersBiso max: 110.51 Å2 / Biso mean: 38.814 Å2 / Biso min: 20.1 Å2
Baniso -1Baniso -2Baniso -3
1--0.97 Å21.27 Å2-0.32 Å2
2---0.18 Å2-0.66 Å2
3---1.85 Å2
Refinement stepCycle: final / Resolution: 2→29.017 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2525 0 0 44 2569
Biso mean---36.06 -
Num. residues----319
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0080.0132573
X-RAY DIFFRACTIONr_bond_other_d0.0010.0172405
X-RAY DIFFRACTIONr_angle_refined_deg1.3931.6563465
X-RAY DIFFRACTIONr_angle_other_deg1.3091.5965649
X-RAY DIFFRACTIONr_dihedral_angle_1_deg7.4295315
X-RAY DIFFRACTIONr_dihedral_angle_2_deg40.51326.066122
X-RAY DIFFRACTIONr_dihedral_angle_3_deg16.65815494
X-RAY DIFFRACTIONr_dihedral_angle_4_deg3.595154
X-RAY DIFFRACTIONr_chiral_restr0.0690.2343
X-RAY DIFFRACTIONr_gen_planes_refined0.0060.022801
X-RAY DIFFRACTIONr_gen_planes_other0.0010.02459
Refine LS restraints NCS

Refine-ID: X-RAY DIFFRACTION / Type: interatomic distance / Weight position: 0.05

Ens-IDDom-IDAuth asym-IDNumberRms dev position (Å)
11A21510.1
12B21510.1
21A22680.07
22C22680.07
31A22100.11
32D22100.11
41B21360.1
42C21360.1
51B21980.08
52D21980.08
61C22020.1
62D22020.1
LS refinement shellResolution: 1.999→2.051 Å / Rfactor Rfree error: 0 / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.421 76 -
Rwork0.408 1503 -
all-1579 -
obs--91.59 %

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