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- PDB-6sue: Structure of Photorhabdus luminescens Tc holotoxin pore, Mutation... -

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Basic information

Entry
Database: PDB / ID: 6sue
TitleStructure of Photorhabdus luminescens Tc holotoxin pore, Mutation TccC3-D651A
Components
  • TcdA1
  • TcdB2,TccC3
KeywordsTOXIN / Complex / Holotoxin / Photorhabdus / Insecticidal
Function / homology
Function and homology information


extracellular region / identical protein binding / cytoplasm
Similarity search - Function
Insecticide toxin TcdB middle/C-terminal / Insecticide toxin TcdB middle/N-terminal / Insecticide toxin TcdB middle/C-terminal region / Insecticide toxin TcdB middle/N-terminal region / Salmonella virulence plasmid 65kDa B protein / Salmonella virulence plasmid 65kDa B protein / Toxin complex C-like repeat / Tripartite Tc toxins repeat / ABC toxin, N-terminal domain / ABC toxin N-terminal region ...Insecticide toxin TcdB middle/C-terminal / Insecticide toxin TcdB middle/N-terminal / Insecticide toxin TcdB middle/C-terminal region / Insecticide toxin TcdB middle/N-terminal region / Salmonella virulence plasmid 65kDa B protein / Salmonella virulence plasmid 65kDa B protein / Toxin complex C-like repeat / Tripartite Tc toxins repeat / ABC toxin, N-terminal domain / ABC toxin N-terminal region / TcA 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 / Rhs repeat-associated core / Integrin alpha, N-terminal
Similarity search - Domain/homology
TccC3 / TcdB2 / TcdA1
Similarity search - Component
Biological speciesPhotorhabdus luminescens (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsRoderer, D. / Raunser, S.
Funding support Germany, 1items
OrganizationGrant numberCountry
European Research Council615984 Germany
Citation
Journal: Proc Natl Acad Sci U S A / Year: 2019
Title: Structure of a Tc holotoxin pore provides insights into the translocation mechanism.
Authors: Daniel Roderer / Oliver Hofnagel / Roland Benz / Stefan Raunser /
Abstract: Tc toxins are modular toxin systems of insect and human pathogenic bacteria. They are composed of a 1.4-MDa pentameric membrane translocator (TcA) and a 250-kDa cocoon (TcB and TcC) encapsulating the ...Tc toxins are modular toxin systems of insect and human pathogenic bacteria. They are composed of a 1.4-MDa pentameric membrane translocator (TcA) and a 250-kDa cocoon (TcB and TcC) encapsulating the 30-kDa toxic enzyme (C terminus of TcC). Binding of Tc toxins to target cells and a pH shift trigger the conformational transition from the soluble prepore state to the membrane-embedded pore. Subsequently, the toxic enzyme is translocated and released into the cytoplasm. A high-resolution structure of a holotoxin embedded in membranes is missing, leaving open the question of whether TcB-TcC has an influence on the conformational transition of TcA. Here we show in atomic detail a fully assembled 1.7-MDa Tc holotoxin complex from in the membrane. We find that the 5 TcA protomers conformationally adapt to fit around the cocoon during the prepore-to-pore transition. The architecture of the Tc toxin complex allows TcB-TcC to bind to an already membrane-embedded TcA pore to form a holotoxin. Importantly, assembly of the holotoxin at the membrane results in spontaneous translocation of the toxic enzyme, indicating that this process is not driven by a proton gradient or other energy source. Mammalian lipids with zwitterionic head groups are preferred over other lipids for the integration of Tc toxins. In a nontoxic Tc toxin variant, we can visualize part of the translocating toxic enzyme, which transiently interacts with alternating negative charges and hydrophobic stretches of the translocation channel, providing insights into the mechanism of action of Tc toxins.
#1: Journal: Biorxiv
Title: Structure of a Tc holotoxin pore provides insights into the translocation mechanism
Authors: Roderer, D. / Hofnagel, O. / Benz, R. / Raunser, S.
History
DepositionSep 13, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 6, 2019Provider: repository / Type: Initial release
Revision 1.1Nov 13, 2019Group: Data collection / Database references / Category: citation / Item: _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Nov 20, 2019Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID

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Structure visualization

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Assembly

Deposited unit
A: TcdA1
B: TcdA1
C: TcdA1
D: TcdA1
E: TcdA1
F: TcdB2,TccC3


Theoretical massNumber of molelcules
Total (without water)1,690,2966
Polymers1,690,2966
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area76640 Å2
ΔGint-410 kcal/mol
Surface area537710 Å2
MethodPISA

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Components

#1: Protein
TcdA1 / Toxin A


Mass: 283230.375 Da / Num. of mol.: 5
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Photorhabdus luminescens (bacteria) / Gene: tcdA, tcdA1 / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): RIPL / References: UniProt: Q9RN43
#2: Protein TcdB2,TccC3


Mass: 274144.250 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Photorhabdus luminescens (bacteria) / Gene: tcdB2, TccC3 / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): RIPL / References: UniProt: Q8GF99, UniProt: Q8GF97

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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: Tc holotoxin composed of the TcdA1 pentamer and TcdB2-TccC3
Type: COMPLEX / Details: Mutation D651A in TccC3 / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 1.7 MDa / Experimental value: NO
Source (natural)Organism: Photorhabdus luminescens (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 8
SpecimenConc.: 0.1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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 FIELDBright-field microscopy
Image recordingElectron dose: 100 e/Å2 / Detector mode: INTEGRATING / Film or detector model: FEI FALCON III (4k x 4k)

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Processing

EM softwareName: SPHIRE / Category: 3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 337823 / Symmetry type: POINT

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