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- PDB-8qeo: cryo-EM structure complex of Frizzled-7 and Clostridioides diffic... -

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Basic information

Entry
Database: PDB / ID: 8qeo
Titlecryo-EM structure complex of Frizzled-7 and Clostridioides difficile toxin B
Components
  • Frizzled-7
  • Toxin B
KeywordsTOXIN / micriobiology / class F G protein-coupled receptors / CROP dynamics
Function / homology
Function and homology information


negative regulation of ectodermal cell fate specification / negative regulation of cardiac muscle cell differentiation / symbiont-mediated perturbation of host actin cytoskeleton via filamentous actin depolymerization / somatic stem cell division / skeletal muscle satellite cell maintenance involved in skeletal muscle regeneration / Wnt receptor activity / non-canonical Wnt signaling pathway / mesenchymal to epithelial transition / positive regulation of epithelial cell proliferation involved in wound healing / Wnt-protein binding ...negative regulation of ectodermal cell fate specification / negative regulation of cardiac muscle cell differentiation / symbiont-mediated perturbation of host actin cytoskeleton via filamentous actin depolymerization / somatic stem cell division / skeletal muscle satellite cell maintenance involved in skeletal muscle regeneration / Wnt receptor activity / non-canonical Wnt signaling pathway / mesenchymal to epithelial transition / positive regulation of epithelial cell proliferation involved in wound healing / Wnt-protein binding / glucosyltransferase activity / WNT5:FZD7-mediated leishmania damping / frizzled binding / PCP/CE pathway / Class B/2 (Secretin family receptors) / regulation of canonical Wnt signaling pathway / Wnt signaling pathway, planar cell polarity pathway / Transferases; Glycosyltransferases; Hexosyltransferases / host cell cytosol / stem cell population maintenance / positive regulation of phosphorylation / negative regulation of cell-substrate adhesion / canonical Wnt signaling pathway / cellular response to retinoic acid / phosphatidylinositol-4,5-bisphosphate binding / cysteine-type peptidase activity / substrate adhesion-dependent cell spreading / host cell endosome membrane / Asymmetric localization of PCP proteins / positive regulation of JNK cascade / PDZ domain binding / G protein-coupled receptor activity / recycling endosome membrane / neuron differentiation / T cell differentiation in thymus / toxin activity / Hydrolases; Acting on peptide bonds (peptidases); Cysteine endopeptidases / positive regulation of MAPK cascade / intracellular membrane-bounded organelle / lipid binding / regulation of DNA-templated transcription / positive regulation of DNA-templated transcription / host cell plasma membrane / proteolysis / extracellular region / metal ion binding / membrane / plasma membrane
Similarity search - Function
Frizzled-7, cysteine-rich Wnt-binding domain / Frizzled/Smoothened, transmembrane domain / Frizzled/Smoothened family membrane region / Frizzled/Smoothened family membrane region / Frizzled/secreted frizzled-related protein / TcdA/TcdB toxin, pore forming domain / TcdA/TcdB pore forming domain / Frizzled / CGT/MARTX, cysteine protease (CPD) domain / CGT/MARTX, cysteine protease (CPD) domain superfamily ...Frizzled-7, cysteine-rich Wnt-binding domain / Frizzled/Smoothened, transmembrane domain / Frizzled/Smoothened family membrane region / Frizzled/Smoothened family membrane region / Frizzled/secreted frizzled-related protein / TcdA/TcdB toxin, pore forming domain / TcdA/TcdB pore forming domain / Frizzled / 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 / Frizzled domain / Frizzled cysteine-rich domain superfamily / Fz domain / Frizzled (fz) domain profile. / Choline-binding repeat / Putative cell wall binding repeat / Cell wall/choline-binding repeat / Cell wall-binding repeat profile. / GPCR, family 2-like / G-protein coupled receptors family 2 profile 2. / Nucleotide-diphospho-sugar transferases
Similarity search - Domain/homology
Frizzled-7 / Toxin B
Similarity search - Component
Biological speciesClostridioides difficile (bacteria)
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.26 Å
AuthorsKinsolving, J. / Bous, J.
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 government2018-01562
Swedish Research Council2022-03681 Sweden
Swedish Research Council2018-03406 Sweden
Cancerfonden20 1287 PjF Sweden
Swedish Research Council2022-01398 Sweden
Cancerfonden20 0264 PjF Sweden
Other government2021-00430 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
Revision 1.1Nov 6, 2024Group: Data collection / Structure summary
Category: em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Toxin B
B: Frizzled-7
hetero molecules


Theoretical massNumber of molelcules
Total (without water)341,0783
Polymers341,0122
Non-polymers651
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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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: pHis22 / Production host: Priestia megaterium DSM 319 (bacteria) / References: UniProt: P18177
#2: Protein Frizzled-7 / Fz-7 / hFz7 / FzE3


Mass: 67460.859 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: FZD7 / Plasmid: pFastBac1 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: O75084
#3: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY
Has protein modificationY

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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
1Complex of Frizzled-7 and Clostridioides difficile toxin BCOMPLEX#1-#20RECOMBINANT
2masked region--CROP domainORGANELLE OR CELLULAR COMPONENT#11RECOMBINANT
3masked region-core domainORGANELLE OR CELLULAR COMPONENT#11RECOMBINANT
4masked region-FZD7 CRD and leg of TcdBORGANELLE OR CELLULAR COMPONENT#1-#21RECOMBINANT
Molecular weight
IDEntity assembly-IDExperimental value
11NO
21NO
31NO
41NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Spodoptera frugiperda (fall armyworm)7108
32Priestia megaterium DSM 319 (bacteria)592022
43Priestia megaterium DSM 319 (bacteria)592022
54Priestia megaterium DSM 319 (bacteria)592022
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
21Spodoptera frugiperda (fall armyworm)7108
32Priestia megaterium DSM 319 (bacteria)592022
43Priestia megaterium DSM 319 (bacteria)592022
54Priestia 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-HCl1
20.2 Msodium chlorideNaCl1
30.002 %lauryl maltose neopentyl glycolLMNG1
40.0002 %cholesteryl hemisuccinate tris saltCHS1
50.0002 %glyco diosgeninGDN1
SpecimenConc.: 0.272 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 FIELD / 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
3EPU2.14.0image acquisition
5cryoSPARC4.2.1CTF correction
8Coot0.9model fitting
12cryoSPARC4.2.1initial Euler assignment
14cryoSPARC4.2.1final Euler assignment
16cryoSPARC4.2.1classification
18cryoSPARC4.2.13D reconstruction
20Rosettamodel refinement
21PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 5240176
3D reconstructionResolution: 3.26 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 151385 / 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_d0.00218532
ELECTRON MICROSCOPYf_angle_d0.4725087
ELECTRON MICROSCOPYf_dihedral_angle_d11.3086824
ELECTRON MICROSCOPYf_chiral_restr0.0422794
ELECTRON MICROSCOPYf_plane_restr0.0033263

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