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- PDB-8qen: cryo-EM structure of apo Clostridioides difficile toxin B -

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Basic information

Entry
Database: PDB / ID: 8qen
Titlecryo-EM structure of apo Clostridioides difficile toxin B
ComponentsToxin B
KeywordsTOXIN / microbiology / pore forming region / CROP dynamics / Structural Genomics / Structural Genomics Consortium / SGC
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 / membrane / metal ion binding
Similarity search - Function
Dermonecrotic/RTX toxin, membrane localization domain / TcdA/TcdB toxin, N-terminal helical domain / TcdB toxin N-terminal helical domain / Membrane Localization 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 ...Dermonecrotic/RTX toxin, membrane localization domain / TcdA/TcdB toxin, N-terminal helical domain / TcdB toxin N-terminal helical domain / Membrane Localization 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 speciesClostridioides difficile (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsKinsolving, J. / Bous, J. / Structural Genomics Consortium (SGC)
Funding support Sweden, Denmark, 11items
OrganizationGrant numberCountry
Swedish Research Council2019-01190 Sweden
Cancerfonden20 1102 PjF Sweden
Novo Nordisk FoundationNNF21OC0070008 Denmark
Novo Nordisk FoundationNNF22OC0078104 Denmark
Other government2021-00430
Other government2018-01562
Swedish Research Council2022-03681 Sweden
Swedish Research Council2018-03406 Sweden
Cancerfonden20 1287 PjF Sweden
Cancerfonden202 0264 PjF Sweden
Swedish Research Council2022-01398 Sweden
CitationJournal: Cell Rep / Year: 2024
Title: Structural and functional insight into the interaction of Clostridioides difficile toxin B and FZD.
Authors: Julia Kinsolving / Julien Bous / Pawel Kozielewicz / Sara Košenina / Rawan Shekhani / Lukas Grätz / Geoffrey Masuyer / Yuankai Wang / Pål Stenmark / Min Dong / Gunnar Schulte /
Abstract: The G protein-coupled receptors of the Frizzled (FZD) family, in particular FZD, are receptors that are exploited by Clostridioides difficile toxin B (TcdB), the major virulence factor responsible ...The G protein-coupled receptors of the Frizzled (FZD) family, in particular FZD, are receptors that are exploited by Clostridioides difficile toxin B (TcdB), the major virulence factor responsible for pathogenesis associated with Clostridioides difficile infection. We employ a live-cell assay examining the affinity between full-length FZDs and TcdB. Moreover, we present cryoelectron microscopy structures of TcdB alone and in complex with full-length FZD, which reveal that large structural rearrangements of the combined repetitive polypeptide domain are required for interaction with FZDs and other TcdB receptors, constituting a first step for receptor recognition. Furthermore, we show that bezlotoxumab, an FDA-approved monoclonal antibody to treat Clostridioides difficile infection, favors the apo-TcdB structure and thus disrupts binding with FZD. The dynamic transition between the two conformations of TcdB also governs the stability of the pore-forming region. Thus, our work provides structural and functional insight into how conformational dynamics of TcdB determine receptor binding.
History
DepositionSep 1, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Mar 20, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Toxin B
hetero molecules


Theoretical massNumber of molelcules
Total (without water)273,6172
Polymers273,5521
Non-polymers651
Water0
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 Toxin B


Mass: 273551.562 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Clostridioides difficile (bacteria) / Gene: tcdB / Plasmid: pHis1522 / Production host: Priestia megaterium DSM 319 (bacteria) / References: UniProt: P18177
#2: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn
Has ligand of interestN

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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
1apo-structure of Clostridioides difficile toxin BORGANELLE OR CELLULAR COMPONENT#10RECOMBINANT
2Masked region 1ORGANELLE OR CELLULAR COMPONENT#11RECOMBINANT
3Masked region 2ORGANELLE OR CELLULAR COMPONENT#11RECOMBINANT
Molecular weight
IDEntity assembly-IDExperimental value
11NO
21NO
31NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Priestia megaterium DSM 319 (bacteria)592022
32Priestia megaterium DSM 319 (bacteria)592022
43Priestia megaterium DSM 319 (bacteria)592022
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
21Priestia megaterium DSM 319 (bacteria)592022
32Priestia megaterium DSM 319 (bacteria)592022
43Priestia megaterium DSM 319 (bacteria)592022
Buffer solutionpH: 7.5
Details: 100 mM TRIS-HCl pH 7.5 200 mM NaCl 0.002% LMNG 0.0002% CHS 0.0002% GDN
Buffer component
IDConc.NameFormulaBuffer-ID
10.1 Mtris hydrochlorideTRIS-HClTris1
20.2 Msodium chlorideNaClSodium chloride1
30.002 %lauryl maltose neopentyl glycolLMNG1
40.0002 %cholesteryl hemisuccinate tris saltCHS1
50.0002 %glyco diosgeninGDN1
SpecimenConc.: 0.63 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
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen 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 FIELDBright-field microscopy / Calibrated magnification: 105000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 600 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 3 sec. / Electron dose: 50.453 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 17269
Details: Images collected in super-resolution mode, faster acquisition mode, with 4 exposures per hole
EM imaging opticsEnergyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategory
1Topaz0.2.5aparticle selection
2EPU2.14.0image acquisition
4cryoSPARC4.2.1CTF correction
7Coot0.9model fitting
9cryoSPARC4.2.1initial Euler assignment
10cryoSPARC4.2.1final Euler assignment
11cryoSPARC4.2.1classification
12cryoSPARC4.2.13D reconstruction
25Rosettamodel refinement
26PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 5240176
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 41523 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model buildingSource name: AlphaFold / Type: in silico model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00319379
ELECTRON MICROSCOPYf_angle_d0.51626235
ELECTRON MICROSCOPYf_dihedral_angle_d10.6387126
ELECTRON MICROSCOPYf_chiral_restr0.0452905
ELECTRON MICROSCOPYf_plane_restr0.0033413

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