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- PDB-6h6e: PTC3 holotoxin complex from Photorhabdus luminecens in prepore st... -

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Basic information

Entry
Database: PDB / ID: 6h6e
TitlePTC3 holotoxin complex from Photorhabdus luminecens in prepore state (TcdA1, TcdB2, TccC3)
Components
  • TcdA1
  • TcdB2,TccC3
KeywordsTOXIN / pore forming toxin / translocation / bacterial toxin / a-PFT / Photorhabdus / ABC
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: 3.95 Å
AuthorsGatsogiannis, C. / Merino, F. / Roderer, D. / Balchin, D. / Schubert, E. / Kuhlee, A. / Hayer-Hartl, M. / Raunser, S.
Funding support Germany, 2items
OrganizationGrant numberCountry
European Research Council615984 Germany
Max Planck Society Germany
CitationJournal: Nature / Year: 2018
Title: Tc toxin activation requires unfolding and refolding of a β-propeller.
Authors: Christos Gatsogiannis / Felipe Merino / Daniel Roderer / David Balchin / Evelyn Schubert / Anne Kuhlee / Manajit Hayer-Hartl / Stefan Raunser /
Abstract: Tc toxins secrete toxic enzymes into host cells using a unique syringe-like injection mechanism. They are composed of three subunits, TcA, TcB and TcC. TcA forms the translocation channel and the TcB- ...Tc toxins secrete toxic enzymes into host cells using a unique syringe-like injection mechanism. They are composed of three subunits, TcA, TcB and TcC. TcA forms the translocation channel and the TcB-TcC heterodimer functions as a cocoon that shields the toxic enzyme. Binding of the cocoon to the channel triggers opening of the cocoon and translocation of the toxic enzyme into the channel. Here we show in atomic detail how the assembly of the three components activates the toxin. We find that part of the cocoon completely unfolds and refolds into an alternative conformation upon binding. The presence of the toxic enzyme inside the cocoon is essential for its subnanomolar binding affinity for the TcA subunit. The enzyme passes through a narrow negatively charged constriction site inside the cocoon, probably acting as an extruder that releases the unfolded protein with its C terminus first into the translocation channel.
History
DepositionJul 27, 2018Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 3, 2018Provider: repository / Type: Initial release
Revision 1.1Nov 14, 2018Group: Data collection / Database references / Category: citation
Item: _citation.journal_id_ISSN / _citation.journal_volume ..._citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

Movie
  • Deposited structure unit
  • Imaged by Jmol
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  • Superimposition on EM map
  • EMDB-0149
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
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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)1,689,8266
Polymers1,689,8266
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area124280 Å2
ΔGint-549 kcal/mol
Surface area532170 Å2
MethodPISA

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Components

#1: Protein
TcdA1 / Toxin A


Mass: 283229.406 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 (E. coli) / References: UniProt: Q9RN43
#2: Protein TcdB2,TccC3


Mass: 273678.656 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 (E. coli) / 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

Component
IDNameTypeEntity IDParent-IDSourceDetails
1PTC3 holotoxin complex in prepore state (5 x TcdA1, 1 x TcdB2, 1 x TccC3)COMPLEXall0RECOMBINANT
2TcdA1COMPLEX#11RECOMBINANT5 TcdA1 subunits in the holotoxin complex
3TcdB2/TccC3COMPLEX#21RECOMBINANT1 TcdB2/TccC3 monomer in the holotoxin complex
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
111.7 NO
21NO
310.3 NO
41NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Photorhabdus luminescens (bacteria)29488
32Photorhabdus luminescens (bacteria)29488
43Photorhabdus luminescens (bacteria)29488
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
21Escherichia coli (E. coli)562
32Escherichia coli (E. coli)562
43Escherichia coli (E. coli)562
Buffer solutionpH: 8
Buffer component
IDConc.FormulaBuffer-ID
120 mMTris1
2150 mMNaClSodium chloride1
30.02 %Tween201
41
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: C-flat-2/1
VitrificationInstrument: GATAN CRYOPLUNGE 3 / Cryogen name: ETHANE / Humidity: 94 % / Details: blot 3 sec before plunging

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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: OTHER
Electron lensMode: BRIGHT FIELDBright-field microscopy
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 2.2 e/Å2 / Film or detector model: FEI FALCON II (4k x 4k) / Num. of real images: 3068

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Processing

EM software
IDNameVersionCategoryDetails
1EMAN2particle selection
2EPUimage acquisition
4SPHIRECTF correction
7UCSF Chimera1.12model fittingInitial fitting
8Rosettamodel fittingAb initio modeling of propeller
10SPHIREinitial Euler assignment
11SPHIREfinal Euler assignment
12SPHIREclassification
13SPHIRE3D reconstruction
14Rosettamodel refinementModel relaxation
15NAMD2.12model refinementMDFF as intermediate step
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 205336
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.95 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 89148 / Symmetry type: POINT
Atomic model buildingB value: 68.03 / Protocol: OTHER / Space: REAL / Target criteria: Model to Map FSC
Atomic model building
IDPDB-IDPdb chain-ID 3D fitting-ID
11VW1

1vw1
PDB Unreleased entry

A1
24O9X

4o9x

A1
31VW1

1vw1
PDB Unreleased entry

B1
41VW1

1vw1
PDB Unreleased entry

C1
51VW1

1vw1
PDB Unreleased entry

D1
61VW1

1vw1
PDB Unreleased entry

E1

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