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- PDB-9mx1: Clostridioides difficile Toxin A with mCDIFA-248-25 Fab -

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Basic information

Entry
Database: PDB / ID: 9mx1
TitleClostridioides difficile Toxin A with mCDIFA-248-25 Fab
Components
  • Toxin A
  • mCDIFA-248-25 Fab Heavy Chain
  • mCDIFA-248-25 Fab Light Chain
KeywordsTRANSFERASE / TOXIN/IMMUNE SYSTEM / Toxin A / TcdA / C.diff / Complex / TOXIN-IMMUNE SYSTEM complex
Function / homology
Function and homology information


host cell cytosol / Transferases; Glycosyltransferases; Hexosyltransferases / glycosyltransferase activity / cysteine-type peptidase activity / host cell endosome membrane / toxin activity / Hydrolases; Acting on peptide bonds (peptidases); Cysteine endopeptidases / lipid binding / host cell plasma membrane / proteolysis ...host cell cytosol / Transferases; Glycosyltransferases; Hexosyltransferases / glycosyltransferase activity / cysteine-type peptidase activity / host cell endosome membrane / toxin activity / Hydrolases; Acting on peptide bonds (peptidases); Cysteine endopeptidases / lipid binding / host cell plasma membrane / proteolysis / extracellular region / metal ion binding / membrane
Similarity search - Function
TcdA/TcdB toxin, pore forming domain / TcdA/TcdB pore forming domain / CGT/MARTX, cysteine protease (CPD) domain / CGT/MARTX, cysteine protease (CPD) domain superfamily / Peptidase C80 family / CGT/MARTX cysteine protease (CPD) domain profile. / TcdA/TcdB toxin, N-terminal helical domain / TcdB toxin N-terminal helical domain / TcdA/TcdB toxin, catalytic glycosyltransferase domain / TcdA/TcdB catalytic glycosyltransferase domain ...TcdA/TcdB toxin, pore forming domain / TcdA/TcdB pore forming domain / CGT/MARTX, cysteine protease (CPD) domain / CGT/MARTX, cysteine protease (CPD) domain superfamily / Peptidase C80 family / CGT/MARTX cysteine protease (CPD) domain profile. / TcdA/TcdB toxin, N-terminal helical domain / TcdB toxin N-terminal helical domain / TcdA/TcdB toxin, catalytic glycosyltransferase domain / TcdA/TcdB catalytic glycosyltransferase domain / Choline-binding repeat / Putative cell wall binding repeat / Cell wall/choline-binding repeat / Cell wall-binding repeat profile. / Nucleotide-diphospho-sugar transferases
Similarity search - Domain/homology
Biological speciesClostridioides difficile (bacteria)
Mus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsHuynh, K.W. / Ammirati, M. / Kroh, H.K. / Lacy, D.B. / Han, S.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: Infect Immun / Year: 2025
Title: Mouse monoclonal antibodies against toxins TcdA and TcdB target diverse epitopes for neutralization.
Authors: Heather K Kroh / Jaime L Jensen / Sabine Wellnitz / Jeong Jin Park / Alexandre Esadze / Kevin W Huynh / Mark Ammirati / Seungil Han / Annaliesa S Anderson / D Borden Lacy / Alexey Gribenko /
Abstract: is a spore-forming, Gram-positive bacterium that can cause infections in subjects with weakened immune system or following antibiotic treatment. These infections may lead to pseudomembranous colitis ... is a spore-forming, Gram-positive bacterium that can cause infections in subjects with weakened immune system or following antibiotic treatment. These infections may lead to pseudomembranous colitis and antibiotic-associated diarrhea in humans. As such, is a major cause of nosocomial illness worldwide. Major virulence factors of the bacterium are the large clostridium toxins A (TcdA) and B (TcdB)-high molecular mass proteins with intrinsic glucosyltransferase activity. Toxins bind to the intestinal epithelium and undergo endocytosis by the epithelial cells, followed by a conformational change triggered by the low pH of early endosomes. This conformational change leads to the exposure of hydrophobic segments, followed by membrane insertion, formation of pores, and translocation of the glucosyltransferase domain into the cellular cytoplasm. Once in the cytoplasm, the glucosyltransferase domain inactivates small GTPases of the Rho family of proteins, leading to the disruption of the cytoskeleton. In the current work, we describe the discovery and characterization of a panel of neutralizing mouse monoclonal antibodies capable of interfering with several steps of cellular intoxication by the toxins. The antibodies were produced using hybridoma technology. Neutralizing activity of the antibodies was confirmed using toxin neutralization assays, and functional assays were used to identify specific neutralization mechanisms. Binding epitopes of the antibodies were identified by hydrogen-deuterium exchange mass spectrometry and confirmed through negative-stain and cryo-electron microscopy. Together, our results show that full-length toxins and/or genetically- and chemically-modified toxoids can induce a wide spectrum of antibodies capable of neutralizing the toxins via a variety of mechanisms.
History
DepositionJan 17, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 9, 2025Provider: repository / Type: Initial release
Revision 1.0Jul 9, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Jul 9, 2025Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Jul 9, 2025Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Jul 9, 2025Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Jul 9, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Jul 9, 2025Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1Sep 3, 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_CSD / _citation.journal_id_ISSN / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Toxin A
B: mCDIFA-248-25 Fab Heavy Chain
C: mCDIFA-248-25 Fab Light Chain


Theoretical massNumber of molelcules
Total (without water)355,7123
Polymers355,7123
Non-polymers00
Water00
1


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

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Components

#1: Protein Toxin A


Mass: 308331.188 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Clostridioides difficile (bacteria) / Gene: tcdA, toxA / Production host: Clostridioides difficile (bacteria)
References: UniProt: P16154, Hydrolases; Acting on peptide bonds (peptidases); Cysteine endopeptidases
#2: Antibody mCDIFA-248-25 Fab Heavy Chain


Mass: 23654.594 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Mus musculus (house mouse)
#3: Antibody mCDIFA-248-25 Fab Light Chain


Mass: 23726.217 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Mus musculus (house mouse)
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: Clostridioides difficile Toxin A with mCDIFA-248-25 Fab
Type: COMPLEX / Entity ID: all / Source: MULTIPLE SOURCES
Molecular weightValue: 0.358 MDa / Experimental value: YES
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
125 mMTris1
2150 mMSodium ChlorideNaCl1
SpecimenConc.: 0.125 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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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 magnification: 165000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1500 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 12 sec. / Electron dose: 52.2 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2511

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Processing

EM software
IDNameVersionCategoryDetails
1Warp1.0.9particle selectionTemplate-Free Particle Picking
2RELION3.1.0particle selectionTemplate-Based Particle Picking
3SerialEMimage acquisition
5CTFFIND4CTF correction
10RELION3.1.0initial Euler assignmentInitial Refinement
11RELION3.1.0final Euler assignmentFinal Refinement
14PHENIX1.20_3594model refinementAtomic Model Refinement
CTF correctionType: NONE
Particle selectionNum. of particles selected: 527742
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 212265 / Symmetry type: POINT
RefinementHighest resolution: 3.2 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00421268
ELECTRON MICROSCOPYf_angle_d0.76128795
ELECTRON MICROSCOPYf_dihedral_angle_d5.1992800
ELECTRON MICROSCOPYf_chiral_restr0.0533191
ELECTRON MICROSCOPYf_plane_restr0.0053692

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