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- PDB-9mf4: De novo designed minibinder complexed with Clostridioides diffici... -

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Basic information

Entry
Database: PDB / ID: 9mf4
TitleDe novo designed minibinder complexed with Clostridioides difficile Toxin B
Components
  • De novo designed cspg67 minibinder
  • Toxin B
KeywordsTOXIN / TcdB / minibinder / de novo / inhibitor
Function / homology
Function and homology information


symbiont-mediated perturbation of host actin cytoskeleton via filamentous actin depolymerization / glucosyltransferase activity / host cell cytosol / Transferases; Glycosyltransferases; Hexosyltransferases / 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 / host cell cytosol / Transferases; Glycosyltransferases; Hexosyltransferases / 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)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.02 Å
AuthorsMiletic, S. / Li, Z. / Ragotte, R.J. / Melnyk, R.
Funding support Canada, 1items
OrganizationGrant numberCountry
Canadian Institutes of Health Research (CIHR)452580 Canada
CitationJournal: Proc Natl Acad Sci U S A / Year: 2025
Title: De novo design of potent inhibitors of clostridial family toxins.
Authors: Robert J Ragotte / Huazhu Liang / John Tam / Sean Miletic / Jacob M Berman / Roger Palou / Connor Weidle / Zhijie Li / Matthias Glögl / Greg L Beilhartz / Kenneth D Carr / Andrew J Borst / ...Authors: Robert J Ragotte / Huazhu Liang / John Tam / Sean Miletic / Jacob M Berman / Roger Palou / Connor Weidle / Zhijie Li / Matthias Glögl / Greg L Beilhartz / Kenneth D Carr / Andrew J Borst / Brian Coventry / Xinru Wang / John L Rubinstein / Mike Tyers / Daniel Schramek / Roman A Melnyk / David Baker /
Abstract: remains a leading cause of hospital-acquired infections, with its primary virulence factor, toxin B (TcdB), responsible for severe colitis and recurrent disease. The closely related toxin, TcsL, ... remains a leading cause of hospital-acquired infections, with its primary virulence factor, toxin B (TcdB), responsible for severe colitis and recurrent disease. The closely related toxin, TcsL, from , causes a rarer but often fatal toxic shock syndrome, particularly in gynecological and obstetric contexts. We report the de novo design of small protein minibinders that directly neutralize TcdB and TcsL by preventing their entry into host cells. Using deep learning and Rosetta-based approaches, we generated high-affinity minibinders that protect cells from intoxication with picomolar potency and, in the case of TcsL, prolonged survival following lethal toxin challenge in mice. The designed proteins against TcdB demonstrate exceptional stability in proteolytic and acidic environments, making them well-suited for oral delivery-a valuable feature for treating infections localized to the gastrointestinal tract. For TcsL, potent inhibitors were identified from 48 initial designs and 48 optimized designs, highlighting the potential of computational design for rapidly developing countermeasures against life-threatening bacterial toxins.
History
DepositionDec 9, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 17, 2025Provider: repository / Type: Initial release
Revision 1.0Sep 17, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Sep 17, 2025Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Sep 17, 2025Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Sep 17, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Sep 17, 2025Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1Oct 8, 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 / _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 B
B: De novo designed cspg67 minibinder


Theoretical massNumber of molelcules
Total (without water)277,4082
Polymers277,4082
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 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: Protein De novo designed cspg67 minibinder


Mass: 6640.785 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: De novo designed mini protein inhibitor (minibinder) targeting CSPG4 receptor binding site on TcdB
Source: (gene. exp.) synthetic construct (others) / Plasmid: LM627 / Details (production host): pET29b+ backbone / Production host: Escherichia coli BL21 (bacteria)
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: De novo designed minibinder complexed with Clostridioides difficile Toxin B
Type: COMPLEX / Entity ID: all / Source: MULTIPLE SOURCES
Molecular weightValue: 0.278 MDa / Experimental value: NO
Source (natural)Organism: Clostridioides difficile (bacteria) / Strain: VPI 10463
Source (recombinant)Organism: Priestia megaterium (bacteria) / Strain: WH320 / Plasmid: pHIS1522
Buffer solutionpH: 8
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 CSPG4 minibinder #67 at a 1:1 molar ratio 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: 42 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
9cryoSPARC4initial Euler assignment
10cryoSPARC4final Euler assignment
11cryoSPARC4classification
12cryoSPARC43D reconstruction
13PHENIX1.20.1-4487model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.02 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 179414 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingB value: 113 / Protocol: OTHER
Details: Predicted models were docked into the map using ChimeraX. The model was then manually with ISOLDE 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: 127.37 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.01149517
ELECTRON MICROSCOPYf_angle_d0.821812854
ELECTRON MICROSCOPYf_chiral_restr0.05761420
ELECTRON MICROSCOPYf_plane_restr0.00741669
ELECTRON MICROSCOPYf_dihedral_angle_d12.8983589

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