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- PDB-6yew: Morganella morganii TcdA4 in complex with porcine mucosa heparin -

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Basic information

Entry
Database: PDB / ID: 6yew
TitleMorganella morganii TcdA4 in complex with porcine mucosa heparin
ComponentsInsecticidal toxin protein
KeywordsTOXIN / complex / glycan
Biological speciesMorganella morganii (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 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
Revision 1.2May 22, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _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

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Assembly

Deposited unit
A: Insecticidal toxin protein
B: Insecticidal toxin protein
C: Insecticidal toxin protein
D: Insecticidal toxin protein
E: Insecticidal toxin protein


Theoretical massNumber of molelcules
Total (without water)1,378,2925
Polymers1,378,2925
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area109380 Å2
ΔGint-511 kcal/mol
Surface area443640 Å2
MethodPISA

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Components

#1: Protein
Insecticidal toxin protein


Mass: 275658.312 Da / Num. of mol.: 5
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Morganella morganii (bacteria) / Gene: CRX48_03350 / Production host: Escherichia coli (E. coli)

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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: Morganella morganii TcdA4 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: Morganella morganii (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8
SpecimenConc.: 0.1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: 0.10 mg/ml TcdA4 were pre-applied on the grid for 20s. Subsequently, the grid was manually blotted and 0.30 mg/ml porcine mucosa heparin was applied and incubated for 2 min.
Specimen supportGrid material: COPPER / Grid type: Quantifoil R2/1
VitrificationInstrument: GATAN CRYOPLUNGE 3 / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 293 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: SPOT SCAN
Electron lensMode: BRIGHT FIELD / Alignment procedure: ZEMLIN TABLEAU
Image recordingElectron dose: 100 e/Å2 / Detector mode: INTEGRATING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of real images: 5549

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Processing

SoftwareName: PHENIX / Version: 1.15.2_3472: / Classification: refinement
EM software
IDNameCategory
1Gautomatchparticle selection
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: 477602
SymmetryPoint symmetry: C5 (5 fold cyclic)
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 182506 / Symmetry type: POINT
Atomic model buildingB value: 127.6 / Protocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 6RW9

6rw9


Accession code: 6RW9 / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.01190390
ELECTRON MICROSCOPYf_angle_d0.85122855
ELECTRON MICROSCOPYf_dihedral_angle_d13.22954770
ELECTRON MICROSCOPYf_chiral_restr0.0514085
ELECTRON MICROSCOPYf_plane_restr0.00415950

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