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- PDB-7tdm: CryoEM Structure of sFab COP-2 Complex with human claudin-4 and C... -
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Open data
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Basic information
Entry | Database: PDB / ID: 7tdm | |||||||||
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Title | CryoEM Structure of sFab COP-2 Complex with human claudin-4 and Clostridium perfringens enterotoxin C-terminal domain | |||||||||
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Function / homology | ![]() 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 / ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() Similarity search - Function | |||||||||
Biological species | ![]() ![]() ![]() ![]() ![]() | |||||||||
Method | ![]() ![]() ![]() | |||||||||
![]() | Vecchio, A.J. | |||||||||
Funding support | ![]()
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![]() | ![]() Title: Development, structure, and mechanism of synthetic antibodies that target claudin and Clostridium perfringens enterotoxin complexes. Authors: Benjamin J Orlando / Pawel K Dominik / Sourav Roy / Chinemerem P Ogbu / Satchal K Erramilli / Anthony A Kossiakoff / Alex J Vecchio / ![]() Abstract: Strains of Clostridium perfringens produce a two-domain enterotoxin (CpE) that afflicts humans and domesticated animals, causing prevalent gastrointestinal illnesses. CpE's C-terminal domain (cCpE) ...Strains of Clostridium perfringens produce a two-domain enterotoxin (CpE) that afflicts humans and domesticated animals, causing prevalent gastrointestinal illnesses. CpE's C-terminal domain (cCpE) binds cell surface receptors, followed by a restructuring of its N-terminal domain to form a membrane-penetrating β-barrel pore, which is toxic to epithelial cells of the gut. The claudin family of membrane proteins are known receptors for CpE and also control the architecture and function of cell-cell contacts (tight junctions) that create barriers to intercellular molecular transport. CpE binding and assembly disables claudin barrier function and induces cytotoxicity via β-pore formation, disrupting gut homeostasis; however, a structural basis of this process and strategies to inhibit the claudin-CpE interactions that trigger it are both lacking. Here, we used a synthetic antigen-binding fragment (sFab) library to discover two sFabs that bind claudin-4 and cCpE complexes. We established these sFabs' mode of molecular recognition and binding properties and determined structures of each sFab bound to claudin-4-cCpE complexes using cryo-EM. The structures reveal that the sFabs bind a shared epitope, but conform distinctly, which explains their unique binding equilibria. Mutagenesis of antigen/sFab interfaces observed therein result in binding changes, validating the structures, and uncovering the sFab's targeting mechanism. From these insights, we generated a model for CpE's claudin-bound β-pore that predicted sFabs would not prevent cytotoxicity, which we then verified in vivo. Taken together, this work demonstrates the development and mechanism of claudin/cCpE-binding sFabs that provide a framework and strategy for obstructing claudin/CpE assembly to treat CpE-linked gastrointestinal diseases. | |||||||||
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Structure visualization
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Structure viewer | Molecule: ![]() ![]() |
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Downloads & links
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PDBx/mmCIF format | ![]() | 141.8 KB | Display | ![]() |
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PDB format | ![]() | 108.3 KB | Display | ![]() |
PDBx/mmJSON format | ![]() | Tree view | ![]() | |
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-Validation report
Arichive directory | ![]() ![]() | HTTPS FTP |
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-Related structure data
Related structure data | ![]() 25834MC ![]() 7tdnC M: map data used to model this data C: citing same article ( |
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Similar structure data |
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Links
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Assembly
Deposited unit | ![]()
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Components
#1: Protein | ![]() Mass: 22090.201 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() ![]() |
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#2: Protein | Mass: 15114.945 Da / Num. of mol.: 1 / Fragment: C-terminal domain (UNP residues 192-319) Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() ![]() ![]() |
#3: Antibody | Mass: 25263.010 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() ![]() ![]() |
#4: Antibody | Mass: 23517.057 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() ![]() ![]() |
-Experimental details
-Experiment
Experiment | Method: ![]() |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: ![]() |
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Sample preparation
Component | Name: Human claudin-4/cCpE/COP-2 Complex / Type: COMPLEX Details: COP-2 sFab fragment bound to Claudin-4/cCpE complex Entity ID: all / Source: MULTIPLE SOURCES |
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Molecular weight | Value: 85 kDa/nm / Experimental value: NO |
Buffer solution | pH: 7.4 |
Specimen | Conc.: 8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied![]() ![]() |
Specimen support | Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3 |
Vitrification![]() | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K |
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Electron microscopy imaging
Experimental equipment | ![]() Model: Talos Arctica / Image courtesy: FEI Company |
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Microscopy | Model: FEI TALOS ARCTICA |
Electron gun | Electron source![]() ![]() |
Electron lens | Mode: BRIGHT FIELD![]() |
Image recording | Electron dose: 32 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1946901 |
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Processing
Software | Name: PHENIX / Version: 1.19.2_4158: / Classification: refinement | ||||||||||||||||||||||||||||
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EM software |
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CTF correction![]() | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 90461 | ||||||||||||||||||||||||||||
Symmetry | Point symmetry![]() | ||||||||||||||||||||||||||||
3D reconstruction![]() | Resolution: 6.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 90461 / Symmetry type: POINT | ||||||||||||||||||||||||||||
Atomic model building | B value: 164.03 / Protocol: FLEXIBLE FIT / Space: REAL | ||||||||||||||||||||||||||||
Atomic model building | 3D fitting-ID: 1 / Accession code: 7KP4 / Initial refinement model-ID: 1 / PDB-ID: 7KP4 / Source name: PDB / Type: experimental model
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Refine LS restraints |
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