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- PDB-6h3l: Structure of VgrG1 in the Type VI secretion "pre-firing" VgrG1-Ts... -

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Basic information

Entry
Database: PDB / ID: 6h3l
TitleStructure of VgrG1 in the Type VI secretion "pre-firing" VgrG1-Tse6-EagT6-EF-Tu-Tsi6 complex
ComponentsVgrG1
KeywordsTOXIN / Bacterial Type VI effector complex / T6SS chaperone-effector complex / Tse6-loaded VgrG1 complex / NAD(P)+ Glycohydrolase
Function / homology
Function and homology information


type VI protein secretion system complex / protein secretion by the type VI secretion system / extracellular region
Similarity search - Function
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
Type VI secretion system spike protein VgrG1a
Similarity search - Component
Biological speciesPseudomonas aeruginosa (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.2 Å
AuthorsQuentin, D. / Raunser, S.
Funding support Germany, 1items
OrganizationGrant numberCountry
European Research Council615984 Germany
CitationJournal: Nat Microbiol / Year: 2018
Title: Mechanism of loading and translocation of type VI secretion system effector Tse6.
Authors: Dennis Quentin / Shehryar Ahmad / Premy Shanthamoorthy / Joseph D Mougous / John C Whitney / Stefan Raunser /
Abstract: The type VI secretion system (T6SS) primarily functions to mediate antagonistic interactions between contacting bacterial cells, but also mediates interactions with eukaryotic hosts. This molecular ...The type VI secretion system (T6SS) primarily functions to mediate antagonistic interactions between contacting bacterial cells, but also mediates interactions with eukaryotic hosts. This molecular machine secretes antibacterial effector proteins by undergoing cycles of extension and contraction; however, how effectors are loaded into the T6SS and subsequently delivered to target bacteria remains poorly understood. Here, using electron cryomicroscopy, we analysed the structures of the Pseudomonas aeruginosa effector Tse6 loaded onto the T6SS spike protein VgrG1 in solution and embedded in lipid nanodiscs. In the absence of membranes, Tse6 stability requires the chaperone EagT6, two dimers of which interact with the hydrophobic transmembrane domains of Tse6. EagT6 is not directly involved in Tse6 delivery but is crucial for its loading onto VgrG1. VgrG1-loaded Tse6 spontaneously enters membranes and its toxin domain translocates across a lipid bilayer, indicating that effector delivery by the T6SS does not require puncturing of the target cell inner membrane by VgrG1. Eag chaperone family members from diverse Proteobacteria are often encoded adjacent to putative toxins with predicted transmembrane domains and we therefore anticipate that our findings will be generalizable to numerous T6SS-exported membrane-associated effectors.
History
DepositionJul 19, 2018Deposition site: PDBE / Processing site: PDBE
Revision 1.0Sep 12, 2018Provider: repository / Type: Initial release
Revision 1.1Oct 3, 2018Group: Data collection / Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.2May 15, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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Assembly

Deposited unit
A: VgrG1
B: VgrG1
C: VgrG1


Theoretical massNumber of molelcules
Total (without water)216,2953
Polymers216,2953
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: microscopy, electron cryo microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area46980 Å2
ΔGint-178 kcal/mol
Surface area64150 Å2
MethodPISA

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Components

#1: Protein VgrG1


Mass: 72098.375 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas aeruginosa (strain ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1) (bacteria)
Gene: vgrG1, PA0091 / Production host: Escherichia coli BL21 (bacteria) / References: UniProt: Q9I741

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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
IDNameTypeDetailsEntity IDParent-IDSource
1Type VI secretion "pre-firing" VgrG1-Tse6-EagT6-EF-Tu-Tsi6 complexCOMPLEXThe type VI "pre-firing" complex consists of a single copy of Tse6, EF-Tu, Tsi6 and trimeric VgrG1 as well as two copies of dimeric EagT6.all0RECOMBINANT
2Valine-glycine repeat protein 1COMPLEXVgrG1 forms a trimer - UniProt Identifier: Q9I741all1RECOMBINANT
3Type VI secretion exported 6COMPLEXTse6 - UniProt identifier: Q9I7391RECOMBINANT
4Type VI secretion immunity 6COMPLEXTsi6 - UniProt identifier: Q9I7401RECOMBINANT
5Effector associated gene with tse6COMPLEXEagT6 forms dimers - UniProt identifier: Q9I738. Two dimers are present in the EM map.1RECOMBINANT
6Elongation factor Tu 1COMPLEXEF-Tu - UniProt identifier: P0CE471NATURAL
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.35 MDaNO
210.072 MDaNO
310.045 MDaNO
410.011 MDaNO
510.016 MDaNO
610.043 MDaNO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
32Pseudomonas aeruginosa PAO1 (bacteria)208964
43Pseudomonas aeruginosa PAO1 (bacteria)208964
54Pseudomonas aeruginosa PAO1 (bacteria)208964
65Pseudomonas aeruginosa PAO1 (bacteria)208964
76Escherichia coli BL21 (bacteria)511693
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
32Escherichia coli BL21 (bacteria)511693
43Escherichia coli BL21 (bacteria)511693
54Escherichia coli BL21 (bacteria)511693
65Escherichia coli BL21 (bacteria)511693
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTrisTris-HCl1
2300 mMsodium chlorideNaCl1
SpecimenConc.: 0.015 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: GATAN CRYOPLUNGE 3 / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 298 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: OTHER
Electron lensMode: BRIGHT FIELD / Nominal magnification: 59000 X / Nominal defocus max: 4200 nm / Nominal defocus min: 1700 nm / Cs: 0.01 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 1.5 sec. / Electron dose: 60 e/Å2 / Detector mode: INTEGRATING / Film or detector model: FEI FALCON II (4k x 4k) / Num. of real images: 11642
EM imaging opticsSpherical aberration corrector: Cs corrected microscope
Image scansMovie frames/image: 24

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Processing

EM software
IDNameVersionCategoryDetails
2EPU1.8image acquisition
12SPHIREclassificationsort3D
13SPHIRE3D reconstructionmeridien
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 55000 / Symmetry type: POINT

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