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- PDB-7pq5: Photorhabdus laumondii T6SS-associated Rhs protein carrying the T... -

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Basic information

Entry
Database: PDB / ID: 7pq5
TitlePhotorhabdus laumondii T6SS-associated Rhs protein carrying the Tre23 toxin domain
ComponentsTre23
KeywordsTOXIN / RHS / TVISS / T6SS
Function / homologyRHS repeat / RHS Repeat / PAAR motif / PAAR motif / YD repeat / Rhs repeat-associated core / membrane => GO:0016020 / Uncharacterized protein
Function and homology information
Biological speciesPhotorhabdus laumondii subsp. laumondii TTO1 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.17 Å
AuthorsJurenas, D. / Talachia Rosa, L. / Rey, M. / Chamot-Rooke, J. / Fronzes, R. / Cascales, E.
Funding support France, 1items
OrganizationGrant numberCountry
Centre National de la Recherche Scientifique (CNRS) France
CitationJournal: Nat Commun / Year: 2021
Title: Mounting, structure and autocleavage of a type VI secretion-associated Rhs polymorphic toxin.
Authors: Dukas Jurėnas / Leonardo Talachia Rosa / Martial Rey / Julia Chamot-Rooke / Rémi Fronzes / Eric Cascales /
Abstract: Bacteria have evolved toxins to outcompete other bacteria or to hijack host cell pathways. One broad family of bacterial polymorphic toxins gathers multidomain proteins with a modular organization, ...Bacteria have evolved toxins to outcompete other bacteria or to hijack host cell pathways. One broad family of bacterial polymorphic toxins gathers multidomain proteins with a modular organization, comprising a C-terminal toxin domain fused to a N-terminal domain that adapts to the delivery apparatus. Polymorphic toxins include bacteriocins, contact-dependent growth inhibition systems, and specialized Hcp, VgrG, PAAR or Rhs Type VI secretion (T6SS) components. We recently described and characterized Tre23, a toxin domain fused to a T6SS-associated Rhs protein in Photorhabdus laumondii, Rhs1. Here, we show that Rhs1 forms a complex with the T6SS spike protein VgrG and the EagR chaperone. Using truncation derivatives and cross-linking mass spectrometry, we demonstrate that VgrG-EagR-Rhs1 complex formation requires the VgrG C-terminal β-helix and the Rhs1 N-terminal region. We then report the cryo-electron-microscopy structure of the Rhs1-EagR complex, demonstrating that the Rhs1 central region forms a β-barrel cage-like structure that encapsulates the C-terminal toxin domain, and provide evidence for processing of the Rhs1 protein through aspartyl autoproteolysis. We propose a model for Rhs1 loading on the T6SS, transport and delivery into the target cell.
History
DepositionSep 16, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 1, 2021Provider: repository / Type: Initial release
Revision 1.1Mar 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

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Structure visualization

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Assembly

Deposited unit
L: Tre23


Theoretical massNumber of molelcules
Total (without water)165,5891
Polymers165,5891
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area0 Å2
ΔGint0 kcal/mol
Surface area50130 Å2
MethodPISA

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Components

#1: Protein Tre23


Mass: 165588.828 Da / Num. of mol.: 1 / Mutation: D1338N
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Photorhabdus laumondii subsp. laumondii TTO1 (bacteria)
Gene: plu0353 / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: Q7N9I0

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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: Tre23 / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Photorhabdus laumondii (bacteria)
Source (recombinant)Organism: Escherichia coli K-12 (bacteria)
Buffer solutionpH: 8.5
Details: 50 mM Tris-HCl pH8.5, 250 mM NaCl, 1 mM TCEP, cOmpleteTM protease inhibitor cocktail (Sigma-Aldrich)
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 2 mAu current / Grid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 45000 X / Nominal defocus max: 20000 nm / Nominal defocus min: 5000 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 3.7 sec. / Electron dose: 49.75 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 8550
Image scansMovie frames/image: 37 / Used frames/image: 1-37

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Processing

Software
NameVersionClassificationNB
phenix.real_space_refine1.17.1_3660refinement
PHENIX1.17.1_3660refinement
EM software
IDNameVersionCategory
2SerialEM3.8image acquisition
4RELION3.1CTF correction
7Coot0.9.2model fitting
9PHENIX1.17.1model refinement
10RELION3.1initial Euler assignment
11RELION3.1final Euler assignment
12RELION3.1classification
13RELION3.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2623687
3D reconstructionResolution: 3.17 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 140577 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 75.76 / Protocol: BACKBONE TRACE / Space: REAL
RefinementHighest resolution: 3.17 Å / Cross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 47.88 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00468745
ELECTRON MICROSCOPYf_angle_d0.830111879
ELECTRON MICROSCOPYf_chiral_restr0.05221224
ELECTRON MICROSCOPYf_plane_restr0.00551583
ELECTRON MICROSCOPYf_dihedral_angle_d19.72581209

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