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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


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 ...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, 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 / 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. / 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: To Be Published
Title: De Novo Design of Potent Inhibitors of Clostridial Family Toxins
Authors: Rogotte, R.J. / Tam, J. / Liang, H. / Miletic, S. / Palou, R. / Weidle, C. / Li, Z. / Glogl, M. / Beilhartz, G.L. / Carr, K.D. / Borst, A.J. / Coventry, B. / Wang, X. / Rubinstein, J.L. / ...Authors: Rogotte, R.J. / Tam, J. / Liang, H. / Miletic, S. / Palou, R. / Weidle, C. / Li, Z. / Glogl, M. / Beilhartz, G.L. / Carr, K.D. / Borst, A.J. / Coventry, B. / Wang, X. / Rubinstein, J.L. / Schramek, D. / Tyers, M. / Melnyk, R.A. / Baker, D.
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

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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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