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- PDB-8cp6: Type six secretion system exported effector 5 (Tse5) -

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Basic information

Entry
Database: PDB / ID: 8cp6
TitleType six secretion system exported effector 5 (Tse5)
Components(Toxin protein Tse5) x 3
KeywordsTOXIN / Pore-forming protein / P.aeruginosa / effector / Bacterial Rearrangement hot spot protein / ion channel / type VI secretion system
Function / homology
Function and homology information


protein secretion by the type VI secretion system / toxin sequestering activity
Similarity search - Function
Domain of unknown function DUF6531 / Domain of unknown function (DUF6531) / RHS protein / RHS protein / RHS repeat / RHS Repeat / YD repeat / Rhs repeat-associated core
Similarity search - Domain/homology
Biological speciesPseudomonas aeruginosa (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.45 Å
AuthorsGonzalez-Magana, A. / Tascon, I. / Ubarretxena-Belandia, I. / Albesa-Jove, D.
Funding support Spain, 2items
OrganizationGrant numberCountry
Other governmentIT1745-22
Spanish Ministry of Science, Innovation, and UniversitiesPID2021-127816NB-I00 Spain
CitationJournal: Nat Commun / Year: 2023
Title: Structural and functional insights into the delivery of a bacterial Rhs pore-forming toxin to the membrane.
Authors: Amaia González-Magaña / Igor Tascón / Jon Altuna-Alvarez / María Queralt-Martín / Jake Colautti / Carmen Velázquez / Maialen Zabala / Jessica Rojas-Palomino / Marité Cárdenas / ...Authors: Amaia González-Magaña / Igor Tascón / Jon Altuna-Alvarez / María Queralt-Martín / Jake Colautti / Carmen Velázquez / Maialen Zabala / Jessica Rojas-Palomino / Marité Cárdenas / Antonio Alcaraz / John C Whitney / Iban Ubarretxena-Belandia / David Albesa-Jové /
Abstract: Bacterial competition is a significant driver of toxin polymorphism, which allows continual compensatory evolution between toxins and the resistance developed to overcome their activity. Bacterial ...Bacterial competition is a significant driver of toxin polymorphism, which allows continual compensatory evolution between toxins and the resistance developed to overcome their activity. Bacterial Rearrangement hot spot (Rhs) proteins represent a widespread example of toxin polymorphism. Here, we present the 2.45 Å cryo-electron microscopy structure of Tse5, an Rhs protein central to Pseudomonas aeruginosa type VI secretion system-mediated bacterial competition. This structural insight, coupled with an extensive array of biophysical and genetic investigations, unravels the multifaceted functional mechanisms of Tse5. The data suggest that interfacial Tse5-membrane binding delivers its encapsulated pore-forming toxin fragment to the target bacterial membrane, where it assembles pores that cause cell depolarisation and, ultimately, bacterial death.
History
DepositionMar 1, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 6, 2023Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Toxin protein Tse5
B: Toxin protein Tse5
C: Toxin protein Tse5


Theoretical massNumber of molelcules
Total (without water)149,0193
Polymers149,0193
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area4320 Å2
ΔGint-29 kcal/mol
Surface area48510 Å2

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Components

#1: Protein Toxin protein Tse5


Mass: 7456.322 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas aeruginosa (bacteria) / Gene: tse5, PA2684 / Plasmid: pet29a / Details (production host): Kanamycin resistance / Production host: Escherichia coli (E. coli) / Strain (production host): Lemo 21 / References: UniProt: Q9I0F4
#2: Protein Toxin protein Tse5


Mass: 125970.461 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas aeruginosa (bacteria) / Gene: tse5, PA2684 / Plasmid: pert29a / Details (production host): Kanamycin resistance / Production host: Escherichia coli (E. coli) / Strain (production host): Lemo 21 / References: UniProt: Q9I0F4
#3: Protein Toxin protein Tse5


Mass: 15592.012 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas aeruginosa (bacteria) / Gene: tse5, PA2684 / Plasmid: pet29a / Details (production host): Kan resistance / Production host: Escherichia coli (E. coli) / Strain (production host): Lemo 21 / References: UniProt: Q9I0F4

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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: Tse5 / Type: ORGANELLE OR CELLULAR COMPONENT
Details: Pseudomonas aeruginosa type VI secretion system (T6SS) exported effector Tse5. The three chains result from the auto-cleavage of Tse5
Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.146 MDa / Experimental value: YES
Source (natural)Organism: Pseudomonas (RNA similarity group I)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: Lemo 21
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTris HClC4H11NO31
2150 mMNaClNaCl1
32 mMDTTC4H10O2S21
SpecimenConc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 94 % / Chamber temperature: 289 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm / Alignment procedure: COMA FREE
Image recordingAverage exposure time: 10.84 sec. / Electron dose: 60 e/Å2 / Film or detector model: TFS FALCON 4i (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 10244

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4particle selection
2EPU2.14image acquisition
4cryoSPARC4CTF correction
7Coot0.9.8.91model fitting
9cryoSPARC4initial Euler assignment
10cryoSPARC4final Euler assignment
11cryoSPARC4classification
12cryoSPARC43D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 4866119
3D reconstructionResolution: 2.45 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 323963 / Num. of class averages: 3 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingDetails: Buccanner / Source name: Other / Type: in silico model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0019105
ELECTRON MICROSCOPYf_angle_d0.38912338
ELECTRON MICROSCOPYf_dihedral_angle_d9.6953377
ELECTRON MICROSCOPYf_chiral_restr0.0381249
ELECTRON MICROSCOPYf_plane_restr0.0031678

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