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- PDB-8u4v: Cryo-EM structure of human claudin-4 complex with Clostridium per... -

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Basic information

Entry
Database: PDB / ID: 8u4v
TitleCryo-EM structure of human claudin-4 complex with Clostridium perfringens enterotoxin C-terminal domain, sFab COP-1, and Nanobody
Components
  • Anti-fab nanobody
  • COP-1 sFab Heavy Chain
  • COP-1 sFab Light Chain
  • Claudin-4CLDN4
  • Heat-labile enterotoxin B chain
KeywordsMEMBRANE PROTEIN / Claudin / Fab / Toxin / Nanobody
Function / homology
Function and homology information


positive regulation of metallopeptidase activity / calcium-independent cell-cell adhesion via plasma membrane cell-adhesion molecules / Tight junction interactions / bicellular tight junction assembly / apicolateral plasma membrane / regulation of cell morphogenesis / tight junction / positive regulation of wound healing / renal absorption / chloride channel activity ...positive regulation of metallopeptidase activity / calcium-independent cell-cell adhesion via plasma membrane cell-adhesion molecules / Tight junction interactions / bicellular tight junction assembly / apicolateral plasma membrane / regulation of cell morphogenesis / tight junction / positive regulation of wound healing / renal absorption / chloride channel activity / chloride channel complex / lateral plasma membrane / bicellular tight junction / establishment of skin barrier / basal plasma membrane / response to progesterone / female pregnancy / circadian rhythm / transmembrane signaling receptor activity / cell-cell junction / toxin activity / cell adhesion / positive regulation of cell migration / apical plasma membrane / structural molecule activity / extracellular region / identical protein binding / plasma membrane
Similarity search - Function
Claudin-4 / Claudin / Claudin, conserved site / Claudin family signature. / Clostridium enterotoxin / Clostridium enterotoxin / PMP-22/EMP/MP20/Claudin family / PMP-22/EMP/MP20/Claudin superfamily
Similarity search - Domain/homology
Lauryl Maltose Neopentyl Glycol / Claudin-4 / Heat-labile enterotoxin B chain
Similarity search - Component
Biological speciesHomo sapiens (human)
Clostridium perfringens (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.99 Å
AuthorsVecchio, A.J.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM138368 United States
Citation
Journal: To be published
Title: Cryo-EM structures of a synthetic antibody against 22 kDa claudin-4 reveal its complex with Clostridium perfringens enterotoxin
Authors: Vecchio, A.J.
#1: Journal: bioRxiv / Year: 2023
Title: Cryo-EM structures of a synthetic antibody against 22 kDa claudin-4 reveal its complex with
Authors: Erramilli, S.K. / Dominik, P.K. / Ogbu, C.P. / Kossiakoff, A.A. / Vecchio, A.J.
History
DepositionSep 11, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 25, 2023Provider: repository / Type: Initial release
Revision 1.1Dec 13, 2023Group: Derived calculations / Structure summary / Category: chem_comp / entity / pdbx_entity_nonpoly
Item: _chem_comp.name / _chem_comp.pdbx_synonyms ..._chem_comp.name / _chem_comp.pdbx_synonyms / _entity.pdbx_description / _pdbx_entity_nonpoly.name

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Claudin-4
B: Heat-labile enterotoxin B chain
L: COP-1 sFab Light Chain
H: COP-1 sFab Heavy Chain
K: Anti-fab nanobody
hetero molecules


Theoretical massNumber of molelcules
Total (without water)102,1626
Polymers101,1575
Non-polymers1,0051
Water543
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 2 types, 2 molecules AB

#1: Protein Claudin-4 / CLDN4 / Clostridium perfringens enterotoxin receptor / CPE-receptor / Williams-Beuren syndrome chromosomal ...Clostridium perfringens enterotoxin receptor / CPE-receptor / Williams-Beuren syndrome chromosomal region 8 protein


Mass: 22613.852 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CLDN4, CPER, CPETR1, WBSCR8 / Plasmid: pFastBac1 / Cell line (production host): Tn5 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: O14493
#2: Protein Heat-labile enterotoxin B chain


Mass: 14044.594 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Clostridium perfringens (bacteria) / Gene: cpe / Plasmid: pFastBac1 / Cell line (production host): Tn5 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P01558

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Antibody , 3 types, 3 molecules LHK

#3: Antibody COP-1 sFab Light Chain


Mass: 23258.783 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli (E. coli)
#4: Antibody COP-1 sFab Heavy Chain


Mass: 28064.498 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli (E. coli)
#5: Antibody Anti-fab nanobody


Mass: 13175.438 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli (E. coli)

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Non-polymers , 2 types, 4 molecules

#6: Chemical ChemComp-AV0 / Lauryl Maltose Neopentyl Glycol / 2,2-didecylpropane-1,3-bis-b-D-maltopyranoside / 2-decyl-2-{[(4-O-alpha-D-glucopyranosyl-beta-D-glucopyranosyl)oxy]methyl}dodecyl4-O-alpha-D-glucopyranosyl-beta-D-glucopyranoside


Mass: 1005.188 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C47H88O22
#7: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 3 / 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: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Human claudin-4 complex with Clostridium perfringens enterotoxin C-terminal domain, sFab COP-1, and Nanobody
Type: COMPLEX
Details: Assembled complex of 5 proteins (Fab is 2 protein) expressed from insect cells and E coli
Entity ID: #1-#5 / Source: MULTIPLE SOURCES
Molecular weightValue: 0.1 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Trichoplusia ni (cabbage looper)
Buffer solutionpH: 7.4 / Details: 20 mM Hepes pH 7.4, 100 mM NaCl, and 0.003% LMNG
SpecimenConc.: 7 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 278 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 81000 X / Nominal defocus max: 2100 nm / Nominal defocus min: 900 nm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 5039

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Processing

EM software
IDNameVersionCategory
1cryoSPARCparticle selection
2EPUimage acquisition
4cryoSPARCCTF correction
7PHENIXmodel fitting
9PHENIX1.20.1_4487:model refinement
10cryoSPARCinitial Euler assignment
11cryoSPARCfinal Euler assignment
12cryoSPARCclassification
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.99 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 1126378 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 7kp4

7kp4


Accession code: 7kp4 / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0076796
ELECTRON MICROSCOPYf_angle_d1.1089249
ELECTRON MICROSCOPYf_dihedral_angle_d4.25985
ELECTRON MICROSCOPYf_chiral_restr0.1481059
ELECTRON MICROSCOPYf_plane_restr0.0051157

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