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- PDB-9ohd: CryoEM structure of Toxin B (TcdB) from clostridioides difficile ... -

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Basic information

Entry
Database: PDB / ID: 9ohd
TitleCryoEM structure of Toxin B (TcdB) from clostridioides difficile complexed with methyl cholate
ComponentsToxin B
KeywordsTOXIN / clostridioides difficile / Toxin B / TcdB / Bile acid / methyl cholate
Function / homology
Function and homology information


symbiont-mediated perturbation of host actin cytoskeleton via filamentous actin depolymerization / glucosyltransferase activity / Transferases; Glycosyltransferases; Hexosyltransferases / host cell cytosol / cysteine-type peptidase activity / host cell endosome membrane / toxin activity / Hydrolases; Acting on peptide bonds (peptidases); Cysteine endopeptidases / lipid binding / host cell plasma membrane ...symbiont-mediated perturbation of host actin cytoskeleton via filamentous actin depolymerization / glucosyltransferase activity / Transferases; Glycosyltransferases; Hexosyltransferases / host cell cytosol / 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
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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.87 Å
AuthorsMiletic, S. / Li, Z. / Melnyk, R.A.
Funding support Canada, 1items
OrganizationGrant numberCountry
The Hospital For Sick Children Foundation Canada
CitationJournal: Nat Microbiol / Year: 2025
Title: Structure-guided design of a synthetic bile acid that inhibits Clostridioides difficile TcdB toxin.
Authors: Sean Miletic / Simoun Icho / Zhijie Li / John Tam / Elizabeth C Rose / Cypress E Perkins / Ali Nakhi / Xinglin Yang / Howard C Hang / John L Rubinstein / Peter I Dosa / Casey M Theriot / Roman A Melnyk /
Abstract: Intestinal bile acids are a family of host and microbiota metabolites that can directly inhibit toxin B (TcdB), the primary virulence factor of Clostridioides difficile that causes infectious ...Intestinal bile acids are a family of host and microbiota metabolites that can directly inhibit toxin B (TcdB), the primary virulence factor of Clostridioides difficile that causes infectious diarrhoea and colitis. However, the mechanism underlying the inhibition is unclear. Here we used cryogenic electron microscopy and determined the structure of TcdB bound to inhibitory bile acids cholic acid (methyl ester) and taurochenodeoxycholic acid at 2.9 Å and 3.3 Å resolution, respectively. These structures revealed that bile acids lock the C-terminal CROP domain of TcdB in a conformation that allosterically masks the two receptor-binding sites and prevents target cell recognition. Guided by the structure, we synthesized gut-restricted bile acid derivatives, designed to evade the bile acid reuptake transporters within the gut. One of the derivatives, sBA-2, was retained within the gut upon oral dosing and protected mice from toxin-induced C. difficile disease pathology. Our study uncovers the structural basis of inhibition of TcdB by bile acids and its analogues, paving the way for the development of orally deliverable therapeutics against C. difficile.
History
DepositionMay 4, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 11, 2026Provider: repository / Type: Initial release
Revision 1.0Mar 11, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Mar 11, 2026Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Mar 11, 2026Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Mar 11, 2026Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Mar 11, 2026Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Toxin B
hetero molecules


Theoretical massNumber of molelcules
Total (without water)272,0354
Polymers270,7671
Non-polymers1,2683
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 B


Mass: 270767.281 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Clostridioides difficile (bacteria) / Strain: VPI 10463 / Gene: tcdB, toxB / Plasmid: pHIS1522
Details (production host): Expression plasmid for P. megaterium. Xylose-inducible expression. Tetracycline resistance.
Production host: Priestia megaterium (bacteria) / Strain (production host): WH320
References: UniProt: P18177, Hydrolases; Acting on peptide bonds (peptidases); Cysteine endopeptidases
#2: Chemical ChemComp-A1CBA / methyl cholate


Mass: 422.598 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C25H42O5 / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY
Has protein modificationN

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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 B (TcdB) complexed with the synthetic bile acid methyl cholate
Type: COMPLEX / Details: 200 micromolar methyl cholate / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.276 MDa / Experimental value: NO
Source (natural)Organism: Clostridioides difficile (bacteria) / Strain: VPI 10463
Source (recombinant)Organism: Priestia megaterium (bacteria) / Strain: WH320
Buffer solutionpH: 8
Details: 200 micromolar of methyl cholate was added before freezing.
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMsodium chlorideNaCl1
220 mMTris1
SpecimenConc.: 2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Purified TcdB was mixed with 200 micromolar of methyl cholate (4% DMSO final concentration) and incubated on ice until plunge freezing.
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Homemade
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 80 % / Chamber temperature: 277 K / Details: 30s preblot incubation on grid. 2-3s blot time.

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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: 75000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 40 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)

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Processing

EM software
IDNameVersionCategory
2EPU3.7image acquisition
7ISOLDEmodel fitting
8Coot09.8.91model fitting
10cryoSPARC4initial Euler assignment
11cryoSPARC4final Euler assignment
12cryoSPARC4classification
13cryoSPARC43D reconstruction
20PHENIX1.20.1-4487model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.87 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 339310 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL
Details: Predicted models were docked into the map using ChimeraX. The model was then manually built with ISOLDE and Coot and refined with phenix.real_space_refine.
Atomic model buildingAccession code: P18177 / Source name: AlphaFold / Type: in silico model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 161.18 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.003219501
ELECTRON MICROSCOPYf_angle_d0.502326421
ELECTRON MICROSCOPYf_chiral_restr0.04292941
ELECTRON MICROSCOPYf_plane_restr0.00383420
ELECTRON MICROSCOPYf_dihedral_angle_d12.97797211

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