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- PDB-8ekl: Clostridioides difficile binary toxin translocase CDTb wild-type ... -

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Basic information

Entry
Database: PDB / ID: 8ekl
TitleClostridioides difficile binary toxin translocase CDTb wild-type after calcium depletion from receptor binding domain 1 (RBD1) - Class 1
ComponentsADP-ribosyltransferase binding componentPoly (ADP-ribose) polymerase
KeywordsTOXIN / translocase / binary toxin / pore-forming
Function / homology
Function and homology information


protein homooligomerization / transferase activity / extracellular region / metal ion binding
Similarity search - Function
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
Similarity search - Component
Biological speciesClostridioides difficile (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.06 Å
AuthorsAbeyawardhane, D.L. / Pozharski, E.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R56AI152397 United States
CitationJournal: To Be Published
Title: Calcium-mediated Pore Formation of Clostridioides difficile Binary Toxin
Authors: Abeyawardhane, D.L. / Adipietro, K.A. / Ruiz, R.G. / Varney, K.M. / Rustandi, R.R. / Silin, V.I. / Pozharski, E. / Weber, D.J.
History
DepositionSep 21, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 27, 2023Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: ADP-ribosyltransferase binding component
B: ADP-ribosyltransferase binding component
C: ADP-ribosyltransferase binding component
D: ADP-ribosyltransferase binding component
E: ADP-ribosyltransferase binding component
F: ADP-ribosyltransferase binding component
G: ADP-ribosyltransferase binding component
hetero molecules


Theoretical massNumber of molelcules
Total (without water)523,31521
Polymers522,7547
Non-polymers56114
Water0
1


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

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Components

#1: Protein
ADP-ribosyltransferase binding component / Poly (ADP-ribose) polymerase


Mass: 74679.086 Da / Num. of mol.: 7
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Clostridioides difficile (bacteria) / Strain: R20291 / Gene: cdtB / Cell line (production host): expresSF+ / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: O32739
#2: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 14 / 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: Single heptamer (class 1) of Clostridioides difficile binary toxin translocase CDTb wild-type after depletion of calcium from RBD1
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Clostridioides difficile (bacteria) / Strain: R20291
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7
Buffer component
IDConc.NameFormulaBuffer-ID
115 mMHEPESC8H18N2O4S1
2150 mMsodium chlorideNaClSodium chloride1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid type: Quantifoil
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 %

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Electron microscopy imaging

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 45000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 700 nm / Calibrated defocus min: 500 nm / Calibrated defocus max: 2700 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.20.1_4487: / Classification: refinement
EM software
IDNameVersionCategory
1cryoSPARC3.3.2particle selection
2SerialEMimage acquisition
4cryoSPARC3.3.2CTF correction
7UCSF Chimeramodel fitting
9Cootmodel refinement
10PHENIXmodel refinement
11cryoSPARC3.3.2initial Euler assignment
12cryoSPARC3.3.2final Euler assignment
13cryoSPARC3.3.2classification
14cryoSPARC3.3.23D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1109860
SymmetryPoint symmetry: C7 (7 fold cyclic)
3D reconstructionResolution: 3.06 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 33531 / Symmetry type: POINT
Atomic model building
IDProtocol
1AB INITIO MODEL
2AB INITIO MODEL
3AB INITIO MODEL
4AB INITIO MODEL
5AB INITIO MODEL
6AB INITIO MODEL
7AB INITIO MODEL
Atomic model building

Accession code: 6UWR / Initial refinement model-ID: 1 / PDB-ID: 6UWR

6uwr

/ Source name: PDB / Type: experimental model

IDPdb chain-ID 3D fitting-ID
1A1
2B2
3C3
4D4
5E5
6F6
7G7
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00319628
ELECTRON MICROSCOPYf_angle_d0.45826600
ELECTRON MICROSCOPYf_dihedral_angle_d4.1542646
ELECTRON MICROSCOPYf_chiral_restr0.0433024
ELECTRON MICROSCOPYf_plane_restr0.0033472

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