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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: To Be Published
Title: De novo design of potent inhibitors of Clostridioides difficile toxin B
Authors: Ragotte, R.J. / Tam, T. / Miletic, S. / Palou, R. / Weidle, C. / Li, Z. / Gloegl, M. / Beilhartz, G.L. / Liang, H. / Carr, K.D. / Borst, A.J. / Coventry, B. / Wang, X. / Rubinstein, J.L. / ...Authors: Ragotte, R.J. / Tam, T. / Miletic, S. / Palou, R. / Weidle, C. / Li, Z. / Gloegl, M. / Beilhartz, G.L. / Liang, H. / Carr, K.D. / Borst, A.J. / Coventry, B. / Wang, X. / Rubinstein, J.L. / Tyers, M. / Melnyk, R.A. / Baker, D.
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

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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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