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- PDB-6yey: Xenorhabdus nematophila XptA1 in complex with porcine mucosa heparin -

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Basic information

Entry
Database: PDB / ID: 6yey
TitleXenorhabdus nematophila XptA1 in complex with porcine mucosa heparin
ComponentsA component of insecticidal toxin complex (Tc)
KeywordsTOXIN / complex / glycan
Function / homologyTcA receptor binding domain / TcA receptor binding domain / Insecticidal toxin complex/plasmid virulence protein / Tc toxin complex TcA, C-terminal TcB-binding domain / Neuraminidase-like domain / Salmonella virulence plasmid 28.1kDa A protein / Tc toxin complex TcA C-terminal TcB-binding domain / Neuraminidase-like domain / A component of insecticidal toxin complex (Tc)
Function and homology information
Biological speciesXenorhabdus nematophila (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsRoderer, D. / Broecker, F. / Sitsel, O. / Kaplonek, P. / Leidreiter, F. / Seeberger, P.H. / Raunser, S.
Funding support Germany, 1items
OrganizationGrant numberCountry
European Research Council (ERC)615984 Germany
CitationJournal: Nat Commun / Year: 2020
Title: Glycan-dependent cell adhesion mechanism of Tc toxins.
Authors: Daniel Roderer / Felix Bröcker / Oleg Sitsel / Paulina Kaplonek / Franziska Leidreiter / Peter H Seeberger / Stefan Raunser /
Abstract: Toxin complex (Tc) toxins are virulence factors of pathogenic bacteria. Tcs are composed of three subunits: TcA, TcB and TcC. TcA facilitates receptor-toxin interaction and membrane permeation, TcB ...Toxin complex (Tc) toxins are virulence factors of pathogenic bacteria. Tcs are composed of three subunits: TcA, TcB and TcC. TcA facilitates receptor-toxin interaction and membrane permeation, TcB and TcC form a toxin-encapsulating cocoon. While the mechanisms of holotoxin assembly and pore formation have been described, little is known about receptor binding of TcAs. Here, we identify heparins/heparan sulfates and Lewis antigens as receptors for different TcAs from insect and human pathogens. Glycan array screening reveals that all tested TcAs bind negatively charged heparins. Cryo-EM structures of Morganella morganii TcdA4 and Xenorhabdus nematophila XptA1 reveal that heparins/heparan sulfates unexpectedly bind to different regions of the shell domain, including receptor-binding domains. In addition, Photorhabdus luminescens TcdA1 binds to Lewis antigens with micromolar affinity. Here, the glycan interacts with the receptor-binding domain D of the toxin. Our results suggest a glycan dependent association mechanism of Tc toxins on the host cell surface.
History
DepositionMar 25, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 8, 2020Provider: repository / Type: Initial release
Revision 1.1Jul 15, 2020Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name

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Structure visualization

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Assembly

Deposited unit
A: A component of insecticidal toxin complex (Tc)
B: A component of insecticidal toxin complex (Tc)
C: A component of insecticidal toxin complex (Tc)
D: A component of insecticidal toxin complex (Tc)
E: A component of insecticidal toxin complex (Tc)


Theoretical massNumber of molelcules
Total (without water)1,435,6045
Polymers1,435,6045
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area120350 Å2
ΔGint-607 kcal/mol
Surface area455120 Å2
MethodPISA

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Components

#1: Protein
A component of insecticidal toxin complex (Tc)


Mass: 287120.781 Da / Num. of mol.: 5
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Xenorhabdus nematophila (bacteria) / Gene: xptA, XNC2_2467 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A0R4FN93

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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: Xenorhabdus nematophila XptA1 pentamer in complex with porcine mucosa heparin
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 1.4 MDa / Experimental value: NO
Source (natural)Organism: Xenorhabdus nematophila (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenConc.: 0.1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: 0.10 mg/ml XptA1 were pre-applied on the grid for 30s. Subsequently, the grid was manually blotted and 0.30 mg/ml porcine mucosa heparin was applied and incubated for 4 min.
Specimen supportGrid material: GOLD / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 285 K

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELDBright-field microscopy / Alignment procedure: COMA FREE
Image recordingElectron dose: 52 e/Å2 / Detector mode: INTEGRATING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of real images: 1855

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Processing

SoftwareName: PHENIX / Version: 1.15.2_3472: / Classification: refinement
EM software
IDNameCategoryDetails
1SPHIREparticle selectionCRYOLO
2EPUimage acquisition
4SPHIRECTF correction
9SPHIREinitial Euler assignment
10SPHIREfinal Euler assignment
12SPHIRE3D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 346048
SymmetryPoint symmetry: C5 (5 fold cyclic)
3D reconstructionResolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 172596 / Symmetry type: POINT
Atomic model buildingB value: 65.8 / Protocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 6RW8

6rw8

Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00695500
ELECTRON MICROSCOPYf_angle_d0.768129690
ELECTRON MICROSCOPYf_dihedral_angle_d13.34257370
ELECTRON MICROSCOPYf_chiral_restr0.04814565
ELECTRON MICROSCOPYf_plane_restr0.00616710

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