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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


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 / chloride channel activity / renal absorption ...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 / chloride channel activity / renal absorption / 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)
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: To Be Published
Title: Cryo-EM Structures of Clostridium perfringens Enterotoxin Bound to its Human Receptor, Claudin-4
Authors: Vecchio, A.J. / Kossiakoff, A.A. / Erramilli, S.K. / Rathnayake, S.S.
History
DepositionJul 15, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 7, 2024Provider: repository / Type: Initial release

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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

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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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