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- PDB-6z1z: Structure of the anti-CD9 nanobody 4C8 -

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Basic information

Entry
Database: PDB / ID: 6z1z
TitleStructure of the anti-CD9 nanobody 4C8
ComponentsNanobody 4C8
KeywordsIMMUNE SYSTEM / nanobody / CD9-binding
Biological speciesLama glama (llama)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.7 Å
AuthorsNeviani, N. / Oosterheert, W. / Pearce, N.M. / Lutz, M. / Kroon-Batenburg, L.M.J. / 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

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Assembly

Deposited unit
A: Nanobody 4C8
B: Nanobody 4C8


Theoretical massNumber of molelcules
Total (without water)28,1812
Polymers28,1812
Non-polymers00
Water2,522140
1
A: Nanobody 4C8


Theoretical massNumber of molelcules
Total (without water)14,0911
Polymers14,0911
Non-polymers00
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
B: Nanobody 4C8


Theoretical massNumber of molelcules
Total (without water)14,0911
Polymers14,0911
Non-polymers00
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)48.901, 41.417, 56.853
Angle α, β, γ (deg.)90.000, 89.691, 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 Nanobody 4C8


Mass: 14090.641 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lama glama (llama) / Plasmid: pHEN6 / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): Codon Plus (RIL)
#2: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 140 / 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: 2.04 Å3/Da / Density % sol: 39.79 % / Description: Needle
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop / pH: 7.5
Details: 0.1 M HEPES pH 7.5, 20% (w/v) PEG 8,000 The crystal was cryoprotected by soaking in reservoir solution supplemented with 30% (v/v) PEG 400
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: ESRF / Beamline: ID23-2 / Wavelength: 0.8731 Å
DetectorType: DECTRIS PILATUS3 X 2M / Detector: PIXEL / Date: Jun 16, 2018
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.8731 Å / Relative weight: 1
ReflectionResolution: 1.7→24.64 Å / Num. obs: 23719 / % possible obs: 94.1 % / Redundancy: 3.4 % / CC1/2: 0.993 / Rmerge(I) obs: 0.129 / Rpim(I) all: 0.083 / Net I/σ(I): 4.1
Reflection shellResolution: 1.7→1.76 Å / Redundancy: 3.4 % / Rmerge(I) obs: 1.057 / Mean I/σ(I) obs: 0.8 / Num. unique obs: 2338 / CC1/2: 0.429 / Rpim(I) all: 0.677 / % possible all: 93.4

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Processing

Software
NameVersionClassification
REFMAC5refinement
PHENIX1.14_3260refinement
EVAL15data reduction
EVAL15data scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 5IMK

5imk


Resolution: 1.7→24.63 Å / SU ML: 0.229 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 25.3116 / Stereochemistry target values: CDL v1.2
RfactorNum. reflection% reflectionSelection details
Rfree0.231 1170 4.93 %random 5% of data
Rwork0.2011 22543 --
obs0.2027 23713 93.69 %-
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 25.27 Å2
Refinement stepCycle: LAST / Resolution: 1.7→24.63 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1772 0 0 140 1912
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00231877
X-RAY DIFFRACTIONf_angle_d0.55072559
X-RAY DIFFRACTIONf_chiral_restr0.0434269
X-RAY DIFFRACTIONf_plane_restr0.0027331
X-RAY DIFFRACTIONf_dihedral_angle_d15.56361062
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.7-1.780.30151450.32342787X-RAY DIFFRACTION93.29
1.78-1.870.30211390.26652797X-RAY DIFFRACTION93.71
1.87-1.990.2791460.23212801X-RAY DIFFRACTION93.85
1.99-2.140.24371580.21662827X-RAY DIFFRACTION94.37
2.14-2.360.20671230.19012787X-RAY DIFFRACTION93.03
2.36-2.70.2711440.19392848X-RAY DIFFRACTION94.5
2.7-3.40.22061500.18932858X-RAY DIFFRACTION94.77
3.4-24.630.19381650.17412838X-RAY DIFFRACTION92.12
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
11.59625212145-0.484816549043-0.0202135652141.23918430394-0.05874245238471.48191085490.04585927864980.116283992871-0.0739243924981-0.154270380471-0.10285072816-0.0346393398559-0.013341831777-0.0850469989348-0.02371642169030.118454616034-0.000340747667375-0.007613872722040.14284037776-0.01807361307380.13948354361812.0107785784-2.311163051250.964914243534
21.740592762840.290275487501-0.5462610666220.716921672831-0.05043087218652.352677421630.0523780362671-0.134554771420.1299636389860.0857386645128-0.07303334759560.069860574554-0.0191929562690.03926135985670.008429917456230.1348625969520.00396197610384-0.009031980052350.145336985536-0.01709281708040.14934820846812.111028108310.8400508743-26.7782902004
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection details
1X-RAY DIFFRACTION1(chain A and resseq 1:121)
2X-RAY DIFFRACTION2(chain B and resseq 1:121)

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