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- PDB-6sk0: The VgrG spike from the Type 6 secretion system -

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Basic information

Entry
Database: PDB / ID: 6sk0
TitleThe VgrG spike from the Type 6 secretion system
ComponentsPutative type VI secretion proteinType VI secretion system
KeywordsCHAPERONE / Puncture / spike / tip / carrier / inhibitor
Function / homology
Function and homology information


Type VI secretion system spike protein VgrG2, C-terminal domain of unknown function DUF2345 / Putative type VI secretion system, Rhs element associated Vgr domain / Uncharacterized protein conserved in bacteria (DUF2345) / Putative type VI secretion system Rhs element Vgr / Type VI secretion system, RhsGE-associated Vgr family subset / Phage tail baseplate hub (GPD) / Type VI secretion system, RhsGE-associated Vgr protein / Gp5/Type VI secretion system Vgr protein, OB-fold domain / Type VI secretion system/phage-baseplate injector OB domain / Vgr protein, OB-fold domain superfamily
Similarity search - Domain/homology
Putative type VI secretion protein
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.3 Å
AuthorsRapisarda, C. / Fronzes, R.
Funding support France, Germany, 2items
OrganizationGrant numberCountry
French National Research Agency France
European Communitys Seventh Framework Programme653706 Germany
CitationJournal: EMBO J / Year: 2020
Title: Structural basis for loading and inhibition of a bacterial T6SS phospholipase effector by the VgrG spike.
Authors: Nicolas Flaugnatti / Chiara Rapisarda / Martial Rey / Solène G Beauvois / Viet Anh Nguyen / Stéphane Canaan / Eric Durand / Julia Chamot-Rooke / Eric Cascales / Rémi Fronzes / Laure Journet /
Abstract: The bacterial type VI secretion system (T6SS) is a macromolecular machine that injects effectors into prokaryotic and eukaryotic cells. The mode of action of the T6SS is similar to contractile phages: ...The bacterial type VI secretion system (T6SS) is a macromolecular machine that injects effectors into prokaryotic and eukaryotic cells. The mode of action of the T6SS is similar to contractile phages: the contraction of a sheath structure pushes a tube topped by a spike into target cells. Effectors are loaded onto the spike or confined into the tube. In enteroaggregative Escherichia coli, the Tle1 phospholipase binds the C-terminal extension of the VgrG trimeric spike. Here, we purify the VgrG-Tle1 complex and show that a VgrG trimer binds three Tle1 monomers and inhibits their activity. Using covalent cross-linking coupled to high-resolution mass spectrometry, we provide information on the sites of contact and further identify the requirement for a Tle1 N-terminal secretion sequence in complex formation. Finally, we report the 2.6-Å-resolution cryo-electron microscopy tri-dimensional structure of the (VgrG) -(Tle1) complex revealing how the effector binds its cargo, and how VgrG inhibits Tle1 phospholipase activity. The inhibition of Tle1 phospholipase activity once bound to VgrG suggests that Tle1 dissociation from VgrG is required upon delivery.
History
DepositionAug 14, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 29, 2020Provider: repository / Type: Initial release
Revision 1.1Nov 11, 2020Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _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.identifier_ORCID / _citation_author.name

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Structure visualization

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Assembly

Deposited unit
A: Putative type VI secretion protein
B: Putative type VI secretion protein
C: Putative type VI secretion protein


Theoretical massNumber of molelcules
Total (without water)277,9723
Polymers277,9723
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area42710 Å2
ΔGint-202 kcal/mol
Surface area23140 Å2
MethodPISA

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Components

#1: Protein Putative type VI secretion protein / Type VI secretion system / Type VI secretion system tip protein VgrG


Mass: 92657.219 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli)
Gene: vgrG, DL654_23170, DM280_18435, EF082_25505, EHD42_22735, EIT12_22235, EIT27_22025, NCTC9044_02519
Production host: Escherichia coli (E. coli) / References: UniProt: A0A3W2RZ19

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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: Tle1 effector bound to the VgrG spike of the type 6 secretion system
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.956 MDa / Experimental value: NO
Source (natural)Organism: Escherichia coli (E. coli)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenConc.: 0.6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 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 FIELDBright-field microscopy
Image recordingElectron dose: 1.28 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

EM software
IDNameCategory
2SerialEMimage acquisition
4GctfCTF correction
9cryoSPARCinitial Euler assignment
10cryoSPARCfinal Euler assignment
11cryoSPARCclassification
12RELION3D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 468438 / Symmetry type: POINT
Atomic model buildingProtocol: BACKBONE TRACE

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