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- PDB-7q97: Structure of the bacterial type VI secretion system effector RhsA. -

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Basic information

Entry
Database: PDB / ID: 7q97
TitleStructure of the bacterial type VI secretion system effector RhsA.
ComponentsRhs family protein
KeywordsTOXIN / bacterial type VI secretion system / effector / Rhs repeats
Function / homology
Function and homology information


Domain of unknown function DUF6531 / RHS protein / Domain of unknown function (DUF6531) / RHS protein / PAAR motif / PAAR motif / RHS repeat / RHS Repeat / YD repeat / Rhs repeat-associated core / :
Similarity search - Domain/homology
Biological speciesPseudomonas protegens Pf-5 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsGuenther, P. / Quentin, D. / Ahmad, S. / Sachar, K. / Gatsogiannis, C. / Whitney, J.C. / Raunser, S.
Funding support3items
OrganizationGrant numberCountry
Max Planck Society
Natural Sciences and Engineering Research Council (NSERC, Canada)RGPIN 2017 05350
Other private
CitationJournal: PLoS Pathog / Year: 2022
Title: Structure of a bacterial Rhs effector exported by the type VI secretion system.
Authors: Patrick Günther / Dennis Quentin / Shehryar Ahmad / Kartik Sachar / Christos Gatsogiannis / John C Whitney / Stefan Raunser /
Abstract: The type VI secretion system (T6SS) is a widespread protein export apparatus found in Gram-negative bacteria. The majority of T6SSs deliver toxic effector proteins into competitor bacteria. Yet, the ...The type VI secretion system (T6SS) is a widespread protein export apparatus found in Gram-negative bacteria. The majority of T6SSs deliver toxic effector proteins into competitor bacteria. Yet, the structure, function, and activation of many of these effectors remains poorly understood. Here, we present the structures of the T6SS effector RhsA from Pseudomonas protegens and its cognate T6SS spike protein, VgrG1, at 3.3 Å resolution. The structures reveal that the rearrangement hotspot (Rhs) repeats of RhsA assemble into a closed anticlockwise β-barrel spiral similar to that found in bacterial insecticidal Tc toxins and in metazoan teneurin proteins. We find that the C-terminal toxin domain of RhsA is autoproteolytically cleaved but remains inside the Rhs 'cocoon' where, with the exception of three ordered structural elements, most of the toxin is disordered. The N-terminal 'plug' domain is unique to T6SS Rhs proteins and resembles a champagne cork that seals the Rhs cocoon at one end while also mediating interactions with VgrG1. Interestingly, this domain is also autoproteolytically cleaved inside the cocoon but remains associated with it. We propose that mechanical force is required to remove the cleaved part of the plug, resulting in the release of the toxin domain as it is delivered into a susceptible bacterial cell by the T6SS.
History
DepositionNov 12, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 22, 2021Provider: repository / Type: Initial release
Revision 1.1Feb 2, 2022Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.name
Revision 1.2Jul 17, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond / em_admin / Item: _em_admin.last_update

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Structure visualization

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Assembly

Deposited unit
A: Rhs family protein
B: Rhs family protein


Theoretical massNumber of molelcules
Total (without water)319,4392
Polymers319,4392
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area2670 Å2
ΔGint-3 kcal/mol
Surface area99010 Å2

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Components

#1: Protein Rhs family protein


Mass: 159719.453 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas protegens Pf-5 (bacteria) / Strain: ATCC BAA-477 / NRRL B-23932 / Pf-5 / Gene: PFL_6096 / Plasmid: pETDuet-1 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q4K3M9

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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: Structure of RhsA. / Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.16491297 MDa / Experimental value: NO
Source (natural)Organism: Pseudomonas protegens Pf-5 (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 8
SpecimenConc.: 4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: The sample was monodisperse.
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 281 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: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 105000 X / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 70 K / Temperature (min): 70 K
Image recordingAverage exposure time: 2 sec. / Electron dose: 61 e/Å2 / Film or detector model: GATAN K2 (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 13090
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scansWidth: 5760 / Height: 4092

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
EM software
IDNameVersionCategory
1crYOLO1.7particle selection
2EPU1.9image acquisition
4CTFFIND4.1CTF correction
7UCSF ChimeraX1.2model fitting
9SPHIRE1.5initial Euler assignment
10SPHIRE1.5final Euler assignment
11RELION3.1classification
12SPHIRE1.53D reconstruction
19PHENIX1.19.2model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 454740 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 50 / Protocol: AB INITIO MODEL / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00917440
ELECTRON MICROSCOPYf_angle_d0.64823610
ELECTRON MICROSCOPYf_dihedral_angle_d10.0476448
ELECTRON MICROSCOPYf_chiral_restr0.0452416
ELECTRON MICROSCOPYf_plane_restr0.0053164

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