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- PDB-9ihc: CryoEM structure of a synthetic antibody, COP-2, in complex with ... -

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Basic information

Entry
Database: PDB / ID: 9ihc
TitleCryoEM structure of a synthetic antibody, COP-2, in complex with the C-terminal domain of Clostridium perfringens Enterotoxin
Components
  • COP-2 antibody heavy chain
  • COP-2 antibody light chain
  • Heat-labile enterotoxin B chain
KeywordsTOXIN / clostridium / COP-2
Function / homologyClostridium enterotoxin / Clostridium enterotoxin / toxin activity / extracellular region / Heat-labile enterotoxin B chain
Function and homology information
Biological speciesClostridium perfringens (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.95 Å
AuthorsPacesa, M. / Goldbach, N. / Vecchio, A.J. / Correia, B.E.
Funding support Switzerland, 1items
OrganizationGrant numberCountry
Swiss National Science Foundation Switzerland
CitationJournal: Protein Sci / Year: 2025
Title: Structures of a synthetic antibody selected against and bound to the C-terminal domain of Clostridium perfringens enterotoxin.
Authors: Chinemerem P Ogbu / Nicolas Manuel Goldbach / Martin Pacesa / Srajan Kapoor / Bruno E Correia / Alex J Vecchio /
Abstract: Clostridium perfringens enterotoxin (CpE) causes cytotoxic gastrointestinal disease in mammalian epithelium by binding membrane protein receptors called claudins. Claudins direct the formation of ...Clostridium perfringens enterotoxin (CpE) causes cytotoxic gastrointestinal disease in mammalian epithelium by binding membrane protein receptors called claudins. Claudins direct the formation of cell/cell tight junctions through oligomerization and govern the transport of molecules between individual cells. CpE binds claudins through its C-terminal domain (cCpE) and induces cytotoxicity through its N-terminal domain. The non-toxic cCpE is a useful tool to study claudins, tight junctions, and for translational applications, such as increasing the permeability of restrictive tissues like the blood-brain barrier or selective targeting of claudin overexpressing cancers. Conversely, there are no specialized molecular tools to study CpE or cCpE, or to modulate or inhibit their functions. We previously reported the development of synthetic antigen-binding fragments (sFabs) that bind cCpE, and low-resolution structures of them bound to claudin/cCpE complexes. Here, we determine high-resolution structures of sFab COP-2 bound to cCpE using X-ray crystallography and cryogenic electron microscopy. The structures and biophysical findings provide the mechanism of COP-2 binding to cCpE and the molecular determinants driving their interactions. These insights can advance the design of new antibody-based tools from our COP-2 scaffold to study or alter cCpE function and give rise to a "Trojan horse" strategy that exploits cCpE's tight junction barrier disrupting function to selectively deliver conjugated therapeutics through normally impermeable tissues.
History
DepositionFeb 21, 2025Deposition site: PDBE / Processing site: PDBE
Revision 1.0Mar 12, 2025Provider: repository / Type: Initial release
Revision 1.0Mar 12, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Mar 12, 2025Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Mar 12, 2025Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Mar 12, 2025Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Mar 12, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Mar 12, 2025Data content type: Mask / Part number: 1 / Data content type: Mask / Provider: repository / Type: Initial release
Revision 1.0Mar 12, 2025Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1Sep 24, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _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

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
B: Heat-labile enterotoxin B chain
H: COP-2 antibody heavy chain
L: COP-2 antibody light chain


Theoretical massNumber of molelcules
Total (without water)68,9673
Polymers68,9673
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein Heat-labile enterotoxin B chain


Mass: 15114.945 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Clostridium perfringens (bacteria) / Gene: cpe / Production host: Escherichia coli (E. coli) / References: UniProt: P01558
#2: Antibody COP-2 antibody heavy chain


Mass: 27799.080 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Homo sapiens (human)
#3: Antibody COP-2 antibody light chain


Mass: 26053.135 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Homo sapiens (human)
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: Complex of synthetic antibody COP-2 and the C-terminal domain of Clostridium perfringens Enterotoxin
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Clostridium perfringens (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm
Image recordingElectron dose: 40 e/Å2 / Film or detector model: TFS FALCON 4i (4k x 4k)

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Processing

EM softwareName: PHENIX / Version: dev_5316 / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.95 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 87280 / Symmetry type: POINT
RefinementHighest resolution: 2.95 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)

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