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- PDB-7vnn: Complex structure of Clostridioides difficile enzymatic component... -

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Basic information

Entry
Database: PDB / ID: 7vnn
TitleComplex structure of Clostridioides difficile enzymatic component (CDTa) and binding component (CDTb) pore with long stem
Components
  • ADP-ribosylating binary toxin binding subunit CdtB
  • CdtA
KeywordsTOXIN / Complex / Translocation / Oligomer / Unfoldase
Function / homology
Function and homology information


protein homooligomerization / transferase activity / extracellular region / identical protein binding / metal ion binding
Similarity search - Function
Binary exotoxin A, clostridial type / 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 / ADP ribosyltransferase ...Binary exotoxin A, clostridial type / 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 / ADP ribosyltransferase / ADP-ribosyltransferase exoenzyme / Toxin-related mono-ADP-ribosyltransferase (TR mART) core domain profile. / PA14 domain / PA14 / PA14 domain
Similarity search - Domain/homology
ADP-ribosyltransferase binding component / CdtA
Similarity search - Component
Biological speciesClostridioides difficile (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.64 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
DepositionOct 11, 2021Deposition 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 1.2Jun 19, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / 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

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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: 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 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: F5B5W8
#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

Component
IDNameTypeEntity IDParent-IDSource
1CDTa-bound CDTb-pore (long)COMPLEX#1-#20RECOMBINANT
2CDTb-poreCOMPLEX#11RECOMBINANT
4CdtACOMPLEX#21RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Clostridioides difficile (bacteria)1496
32Clostridioides difficile (bacteria)1496
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDStrain
21Escherichia coli BL21(DE3) (bacteria)469008BL21(DE3)
32Escherichia coli BL21(DE3) (bacteria)469008BL21(DE3)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
110 mM2-[4-(2-Hydroxyethyl)-1-piperazinyl]ethanesulfonic acidC8H18N2O4S1
21 mMCalcium chlorideCaCl21
30.003 % (w/v)Lauryl Maltose Neopentyl GlycolC47H88O221
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
4GctfCTF correction
7UCSF Chimeramodel fitting
9PHENIXmodel refinement
10Cootmodel refinement
11cryoSPARC2initial Euler assignment
12RELION3.1final Euler assignment
14RELION3.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 735102
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.64 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 83061 / Symmetry type: POINT
Atomic model building
IDPDB-ID 3D fitting-IDAccession codeInitial refinement model-IDSource nameType
16UWR

6uwr

16UWR1PDBexperimental model
26V1S

6v1s

16V1S2PDBexperimental model

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