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- PDB-9cm5: CryoEM Strucuture of TcdB in complex with De Novo Minibinder fzd48 -

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Basic information

Entry
Database: PDB / ID: 9cm5
TitleCryoEM Strucuture of TcdB in complex with De Novo Minibinder fzd48
Components
  • De Novo Minibinder fzd48
  • Toxin B
KeywordsTOXIN / TcdB / Clostridium difficile / C. Diff. / fzd48 / De Novo Minibinder
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 / 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 speciessynthetic construct (others)
Clostridioides difficile (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.61 Å
AuthorsWeidle, C. / Carr, K.D. / Borst, A.J.
Funding support United States, 1items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
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
DepositionJul 12, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 21, 2025Provider: repository / Type: Initial release
Revision 1.0May 21, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0May 21, 2025Data content type: Additional map / Part number: 1 / Data content type: Additional map / Provider: repository / Type: Initial release
Revision 1.0May 21, 2025Data content type: Additional map / Part number: 2 / Data content type: Additional map / Provider: repository / Type: Initial release
Revision 1.0May 21, 2025Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0May 21, 2025Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0May 21, 2025Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0May 21, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0May 21, 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
B: De Novo Minibinder fzd48
A: Toxin B


Theoretical massNumber of molelcules
Total (without water)250,1562
Polymers250,1562
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein De Novo Minibinder fzd48


Mass: 8926.099 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others)
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
#2: Protein Toxin B


Mass: 241229.938 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Clostridioides difficile (bacteria) / Gene: tcdB, toxB
Production host: Priestia megaterium NBRC 15308 = ATCC 14581 (bacteria)
References: UniProt: P18177, Hydrolases; Acting on peptide bonds (peptidases); Cysteine endopeptidases
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: tcdB with de novo minibinder fzd48 / Type: COMPLEX
Details: tcdB and minibinder were mixed at 1:3 molar ratio 10 minutes before freezing.
Entity ID: all / Source: MULTIPLE SOURCES
Molecular weightValue: 0.24989658 MDa / Experimental value: NO
Source (natural)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenConc.: 0.81 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Sample was monodisperse, screened by negative stain prior to freezing
Specimen supportDetails: - 15mA / Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: C-flat-2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 295.15 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 1800 nm / Nominal defocus min: 800 nm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 42 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 6766

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Processing

EM software
IDNameCategoryDetails
1cryoSPARCparticle selectionblobpicker
2cryoSPARCparticle selectiontopaz
5cryoSPARCCTF correctionPatchCTF
9PHENIXmodel refinement
11cryoSPARCinitial Euler assignment
12cryoSPARCfinal Euler assignment
13cryoSPARCclassification
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 4.61 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 108076 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.019681
ELECTRON MICROSCOPYf_angle_d2.17813525
ELECTRON MICROSCOPYf_dihedral_angle_d9.042064
ELECTRON MICROSCOPYf_chiral_restr0.0861836
ELECTRON MICROSCOPYf_plane_restr0.0081971

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