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- EMDB-52864: CryoEM structure of a synthetic antibody, COP-2, in complex with ... -

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Basic information

Entry
Database: EMDB / ID: EMD-52864
TitleCryoEM structure of a synthetic antibody, COP-2, in complex with the C-terminal domain of Clostridium perfringens Enterotoxin
Map data
Sample
  • Complex: Complex of synthetic antibody COP-2 and the C-terminal domain of Clostridium perfringens Enterotoxin
    • Protein or peptide: Heat-labile enterotoxin B chain
    • Protein or peptide: COP-2 antibody heavy chain
    • Protein or peptide: COP-2 antibody light chain
Keywordsclostridium / COP-2 / toxin
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.95 Å
AuthorsPacesa M / Goldbach N / Vecchio AJ / Correia BE
Funding support Switzerland, 1 items
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, 2025-
Header (metadata) releaseMar 12, 2025-
Map releaseMar 12, 2025-
UpdateSep 24, 2025-
Current statusSep 24, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_52864.png

Downloads & links

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Map

FileDownload / File: emd_52864.map.gz / Format: CCP4 / Size: 282.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

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AxesZ (Sec.)Y (Row.)X (Col.)
0.57 Å/pix.
x 420 pix.
= 239.4 Å		0.57 Å/pix.
x 420 pix.
= 239.4 Å		0.57 Å/pix.
x 420 pix.
= 239.4 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.57 Å
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Histogram (log scale)
Contour LevelBy AUTHOR: 0.0506
Minimum - Maximum-0.11950238 - 0.266405
Average (Standard dev.)0.000050643863 (±0.0051255617)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions420420420
Spacing420420420
CellA=B=C: 239.4 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_52864_msk_1.map
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Half map: #1

Fileemd_52864_half_map_1.map
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Half map: #2

Fileemd_52864_half_map_2.map
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Sample components

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Entire : Complex of synthetic antibody COP-2 and the C-terminal domain of ...

EntireName: Complex of synthetic antibody COP-2 and the C-terminal domain of Clostridium perfringens Enterotoxin
Components
  • Complex: Complex of synthetic antibody COP-2 and the C-terminal domain of Clostridium perfringens Enterotoxin
    • Protein or peptide: Heat-labile enterotoxin B chain
    • Protein or peptide: COP-2 antibody heavy chain
    • Protein or peptide: COP-2 antibody light chain

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Supramolecule #1: Complex of synthetic antibody COP-2 and the C-terminal domain of ...

SupramoleculeName: Complex of synthetic antibody COP-2 and the C-terminal domain of Clostridium perfringens Enterotoxin
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Clostridium perfringens (bacteria)

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Macromolecule #1: Heat-labile enterotoxin B chain

MacromoleculeName: Heat-labile enterotoxin B chain / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Clostridium perfringens (bacteria)
Molecular weightTheoretical: 15.114945 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MSTDIEKEIL DLAAATERLN LTDALNSNPA GNLYDWRSSN SYPWTQKLNL HLTITATGQK YRILASKIVD FNIYSNNFNN LVKLEQSLG DGVKDHYVDI SLDAGQYVLV MKANSSYSGN YPYSILFQKF GLVPR

UniProtKB: Heat-labile enterotoxin B chain

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Macromolecule #2: COP-2 antibody heavy chain

MacromoleculeName: COP-2 antibody heavy chain / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 27.79908 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MKKNIAFLLA SMFVFSIATN AYAEISEVQL VESGGGLVQP GGSLRLSCAA SGFNFSSSSI HWVRQAPGKG LEWVASISSY SGYTSYADS VKGRFTISAD TSKNTAYLQM NSLRAEDTAV YYCARYWSWY NSSHYIYSAL DYWGQGTLVT VSSASTKGPS V FPLAPSSK ...String:
MKKNIAFLLA SMFVFSIATN AYAEISEVQL VESGGGLVQP GGSLRLSCAA SGFNFSSSSI HWVRQAPGKG LEWVASISSY SGYTSYADS VKGRFTISAD TSKNTAYLQM NSLRAEDTAV YYCARYWSWY NSSHYIYSAL DYWGQGTLVT VSSASTKGPS V FPLAPSSK STSGGTAALG CLVKDYFPEP VTVSWNSGAL TSGVHTFPAV LQSSGLYSLS SVVTVPSSSL GTQTYICNVN HK PSNTKVD KKVEPKSCDK THT

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Macromolecule #3: COP-2 antibody light chain

MacromoleculeName: COP-2 antibody light chain / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 26.053135 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MKKNIAFLLA SMFVFSIATN AYASDIQMTQ SPSSLSASVG DRVTITCRAS QSVSSAVAWY QQKPGKAPKL LIYSASSLYS GVPSRFSGS RSGTDFTLTI SSLQPEDFAT YYCQQSYEWA PVTFGQGTKV EIKRTVAAPS VFIFPPSDSQ LKSGTASVVC L LNNFYPRE ...String:
MKKNIAFLLA SMFVFSIATN AYASDIQMTQ SPSSLSASVG DRVTITCRAS QSVSSAVAWY QQKPGKAPKL LIYSASSLYS GVPSRFSGS RSGTDFTLTI SSLQPEDFAT YYCQQSYEWA PVTFGQGTKV EIKRTVAAPS VFIFPPSDSQ LKSGTASVVC L LNNFYPRE AKVQWKVDNA LQSGNSQESV TEQDSKDSTY SLSSTLTLSK ADYEKHKVYA CEVTHQGLSS PVTKSFNRGE C

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.5
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: TFS FALCON 4i (4k x 4k) / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.95 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 87280
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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