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- PDB-7yvq: Complex structure of Clostridioides difficile binary toxin folded... -

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Basic information

Entry
Database: PDB / ID: 7yvq
TitleComplex structure of Clostridioides difficile binary toxin folded CDTa-bound CDTb-pore (short).
Components
  • ADP-ribosylating binary toxin binding subunit CdtB
  • ADP-ribosylating binary toxin enzymatic subunit CdtA
KeywordsTOXIN / Complex / Translocation / Oligomer / Unfoldase
Function / homology
Function and homology information


Transferases; Glycosyltransferases; Pentosyltransferases / protein homooligomerization / transferase activity / nucleotide binding / extracellular region / metal ion binding / identical protein binding
Similarity search - Function
Binary exotoxin A, clostridial type / ADP ribosyltransferase / ADP-ribosyltransferase exoenzyme / Toxin-related mono-ADP-ribosyltransferase (TR mART) core domain profile. / Bacterial exotoxin B / Protective antigen, heptamerisation domain / Protective antigen, Ca-binding domain / Clostridial binary toxin B/anthrax toxin PA, domain 3 / Protective antigen, heptamerisation domain superfamily / Clostridial binary toxin B/anthrax toxin PA Ca-binding domain ...Binary exotoxin A, clostridial type / ADP ribosyltransferase / ADP-ribosyltransferase exoenzyme / Toxin-related mono-ADP-ribosyltransferase (TR mART) core domain profile. / Bacterial exotoxin B / Protective antigen, heptamerisation domain / Protective antigen, Ca-binding domain / Clostridial binary toxin B/anthrax toxin PA, domain 3 / Protective antigen, heptamerisation domain superfamily / Clostridial binary toxin B/anthrax toxin PA Ca-binding domain / Clostridial binary toxin B/anthrax toxin PA domain 2 / Clostridial binary toxin B/anthrax toxin PA domain 3 / PA14 domain / PA14 / PA14 domain
Similarity search - Domain/homology
ADP-ribosyltransferase binding component / ADP-ribosyltransferase enzymatic component
Similarity search - Component
Biological speciesClostridioides difficile (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.18 Å
AuthorsYamada, T. / Kawamoto, A. / Yoshida, T. / Sato, Y. / Kato, T. / Tsuge, H.
Funding support Japan, 2items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS) Japan
Japan Science and Technology Japan
CitationJournal: Nat Commun / Year: 2022
Title: Cryo-EM structures of the translocational binary toxin complex CDTa-bound CDTb-pore from Clostridioides difficile.
Authors: Akihiro Kawamoto / Tomohito Yamada / Toru Yoshida / Yusui Sato / Takayuki Kato / Hideaki Tsuge /
Abstract: Some bacteria express a binary toxin translocation system, consisting of an enzymatic subunit and translocation pore, that delivers enzymes into host cells through endocytosis. The most clinically ...Some bacteria express a binary toxin translocation system, consisting of an enzymatic subunit and translocation pore, that delivers enzymes into host cells through endocytosis. The most clinically important bacterium with such a system is Clostridioides difficile (formerly Clostridium). The CDTa and CDTb proteins from its system represent important therapeutic targets. CDTb has been proposed to be a di-heptamer, but its physiological heptameric structure has not yet been reported. Here, we report the cryo-EM structure of CDTa bound to the CDTb-pore, which reveals that CDTa binding induces partial unfolding and tilting of the first CDTa α-helix. In the CDTb-pore, an NSS-loop exists in 'in' and 'out' conformations, suggesting its involvement in substrate translocation. Finally, 3D variability analysis revealed CDTa movements from a folded to an unfolded state. These dynamic structural information provide insights into drug design against hypervirulent C. difficile strains.
History
DepositionAug 19, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Oct 26, 2022Provider: repository / Type: Initial release
Revision 1.1Nov 2, 2022Group: Database references / Category: citation
Item: _citation.page_first / _citation.page_last ..._citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Jul 3, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / em_admin / pdbx_initial_refinement_model
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: ADP-ribosylating binary toxin binding subunit CdtB
B: ADP-ribosylating binary toxin binding subunit CdtB
C: ADP-ribosylating binary toxin binding subunit CdtB
D: ADP-ribosylating binary toxin binding subunit CdtB
E: ADP-ribosylating binary toxin binding subunit CdtB
F: ADP-ribosylating binary toxin binding subunit CdtB
G: ADP-ribosylating binary toxin binding subunit CdtB
H: ADP-ribosylating binary toxin enzymatic subunit CdtA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)579,86229
Polymers579,0208
Non-polymers84221
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
ADP-ribosylating binary toxin binding subunit CdtB / ADP-ribosyltransferase binding component / CdtB


Mass: 75657.141 Da / Num. of mol.: 7
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Clostridioides difficile (bacteria) / Gene: cdtB / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: A8DS70
#2: Protein ADP-ribosylating binary toxin enzymatic subunit CdtA / ADP-ribosyltransferase enzymatic component / CdtA


Mass: 49420.469 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Clostridioides difficile (bacteria) / Gene: cdtA / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: Q9KH42
#3: Chemical...
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 21 / Source method: obtained synthetically / Formula: Ca / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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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 transferase complex / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Clostridioides difficile (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria) / Strain: BL21(DE3)
Buffer solutionpH: 7.5
Details: 10 mM HEPES (pH 7.5), 1 mM CaCl2, and 0.003% (w/v) LMNG
SpecimenConc.: 1.58 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationCryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 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: 2000 nm / Nominal defocus min: 800 nm
Image recordingAverage exposure time: 3.36 sec. / Electron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 11284

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Processing

EM software
IDNameVersionCategory
1RELION3.1particle selection
2cryoSPARCparticle selection
8UCSF Chimeramodel fitting
10cryoSPARC2initial Euler assignment
13cryoSPARC3D reconstruction
14PHENIXmodel refinement
15Cootmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 735102
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.18 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 24981 / Symmetry type: POINT
Atomic model building
IDPDB-ID 3D fitting-IDAccession codeInitial refinement model-IDSource nameType
17VNJ

7vnj

17VNJ1PDBexperimental model
22WN6

2wn6

12WN62PDBexperimental model

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