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- PDB-9cmi: Cryo-EM structure of human claudin-4 complex with Clostridium per... -

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Basic information

Entry
Database: PDB / ID: 9cmi
TitleCryo-EM structure of human claudin-4 complex with Clostridium perfringens enterotoxin, sFab COP-1, and Nanobody
Components
  • Anti-Fab Nanobody
  • COP-1 sFab Heavy Chain
  • COP-1 sFab Light Chain
  • Claudin-4
  • Heat-labile enterotoxin B chain
KeywordsMEMBRANE PROTEIN / claudin / Fab / enterotoxin / nanobody
Function / homology
Function and homology information


paracellular transport / calcium-independent cell-cell adhesion / 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 ...paracellular transport / calcium-independent cell-cell adhesion / 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 / lateral plasma membrane / bicellular tight junction / establishment of skin barrier / chloride channel complex / response to progesterone / basal plasma membrane / female pregnancy / circadian rhythm / cell-cell junction / transmembrane signaling receptor activity / 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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.83 Å
AuthorsVecchio, A.J.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM138368 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM117372 United States
CitationJournal: Structure / Year: 2024
Title: Cryo-EM structures of Clostridium perfringens enterotoxin bound to its human receptor, claudin-4.
Authors: Sewwandi S Rathnayake / Satchal K Erramilli / Anthony A Kossiakoff / Alex J Vecchio /
Abstract: Clostridium perfringens enterotoxin (CpE) causes prevalent and deadly gastrointestinal disorders. CpE binds to receptors called claudins on the apical surfaces of small intestinal epithelium. ...Clostridium perfringens enterotoxin (CpE) causes prevalent and deadly gastrointestinal disorders. CpE binds to receptors called claudins on the apical surfaces of small intestinal epithelium. Claudins normally regulate paracellular transport but are hijacked from doing so by CpE and are instead led to form claudin/CpE complexes. Claudin/CpE complexes are the building blocks of oligomeric β-barrel pores that penetrate the plasma membrane and induce gut cytotoxicity. Here, we present the structures of CpE in complex with its native claudin receptor in humans, claudin-4, using cryogenic electron microscopy. The structures reveal the architecture of the claudin/CpE complex, the residues used in binding, the orientation of CpE relative to the membrane, and CpE-induced changes to claudin-4. Further, structures and modeling allude to the biophysical procession from claudin/CpE complexes to cytotoxic β-barrel pores during pathogenesis. In full, this work proposes a model of claudin/CpE assembly and provides strategies to obstruct its formation to treat CpE diseases.
History
DepositionJul 15, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 7, 2024Provider: repository / Type: Initial release
Revision 1.1Oct 30, 2024Group: Data collection / Database references / Structure summary
Category: citation / citation_author ...citation / citation_author / em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name / _em_admin.last_update / _pdbx_entry_details.has_protein_modification
Revision 1.2Nov 13, 2024Group: Data collection / Category: em_admin / Item: _em_admin.last_update
Revision 1.3Nov 27, 2024Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.journal_volume / _citation.page_first / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)123,2756
Polymers122,2705
Non-polymers1,0051
Water362
1


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

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Components

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Protein , 2 types, 2 molecules AB

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


Mass: 22234.332 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CLDN4, CPER, CPETR1, WBSCR8 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: O14493
#2: Protein Heat-labile enterotoxin B chain


Mass: 33000.582 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Trypsin treated CpE / Source: (gene. exp.) Clostridium perfringens (bacteria) / Gene: cpe / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P01558

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Antibody , 3 types, 3 molecules HKL

#3: Antibody COP-1 sFab Heavy Chain


Mass: 28064.498 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)
#4: Antibody Anti-Fab Nanobody


Mass: 13175.438 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)
#5: Antibody COP-1 sFab Light Chain


Mass: 25794.859 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)

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Non-polymers , 2 types, 3 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


Mass: 18.015 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestN
Has protein modificationY

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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, sFab COP-1, and Nanobody
Type: COMPLEX / Details: 5-protein complex / Entity ID: #1-#5 / Source: MULTIPLE SOURCES
Molecular weightValue: 0.12 MDa / Experimental value: NO
Source (natural)Organism: synthetic construct (others)
Source (recombinant)Organism: synthetic construct (others)
Buffer solutionpH: 7.4 / Details: 20 mM Hepes pH 7.4, 100 mM NaCl, and 0.003% LMNG
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHepes pH 7.41
2100 mMNaClNaCl1
30.003 %LMNG1
SpecimenConc.: 5.6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: glow-discharged for 30 seconds at 20 W using a Solarus 950 (Gatan) plasma cleaner
Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 281 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 FIELD / Nominal magnification: 105000 X / Nominal defocus max: 2100 nm / Nominal defocus min: 900 nm / Alignment procedure: BASIC
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 70 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 6774
EM imaging opticsEnergyfilter name: GIF Bioquantum

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.4particle selection
2EPUimage acquisition
4cryoSPARC4.4CTF correction
7UCSF Chimeramodel fitting
9cryoSPARC4.4initial Euler assignment
10cryoSPARC4.4final Euler assignment
11cryoSPARC4.4classification
12cryoSPARC4.43D reconstruction
13PHENIX1.20.1_4487:model refinement
CTF correctionType: NONE
Particle selectionNum. of particles selected: 5766325
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.83 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 431680 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model building
IDPDB-ID 3D fitting-IDAccession codeInitial refinement model-IDSource nameType
184uv

84uv

184uv1PDBexperimental model
28u5f

8u5f

18u5f2PDBexperimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0038051
ELECTRON MICROSCOPYf_angle_d0.78210950
ELECTRON MICROSCOPYf_dihedral_angle_d6.0021125
ELECTRON MICROSCOPYf_chiral_restr0.0421254
ELECTRON MICROSCOPYf_plane_restr0.0041378

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