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- EMDB-0136: Electron cryo-microscopy of VgrG1 in the Type VI secretion VgrG1-... -

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Basic information

Entry
Database: EMDB / ID: EMD-0136
TitleElectron cryo-microscopy of VgrG1 in the Type VI secretion VgrG1-Tse6-EF-Tu complex embedded in lipid nanodiscs
Map data
Sample
  • Complex: The type VI secretion VgrG1-Tse6-EF-Tu complex embedded in lipid nanodiscsType VI secretion system
    • Protein or peptide: VgrG1
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 PAO1 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.25 Å
AuthorsQuentin D / Raunser S
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, 2018-
Header (metadata) releaseAug 22, 2018-
Map releaseSep 12, 2018-
UpdateOct 3, 2018-
Current statusOct 3, 2018Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.027
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.027
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6h3n
  • Surface level: 0.027
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_0136.png

Downloads & links

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Map

FileDownload / File: emd_0136.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.1 Å
Density
Contour LevelBy AUTHOR: 0.027 / Movie #1: 0.027
Minimum - Maximum-0.06907088 - 0.12727217
Average (Standard dev.)0.000058806607 (±0.0018062005)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 396.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.11.11.1
M x/y/z360360360
origin x/y/z0.0000.0000.000
length x/y/z396.000396.000396.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS360360360
D min/max/mean-0.0690.1270.000

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Supplemental data

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Sample components

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Entire : The type VI secretion VgrG1-Tse6-EF-Tu complex embedded in lipid ...

EntireName: The type VI secretion VgrG1-Tse6-EF-Tu complex embedded in lipid nanodiscsType VI secretion system
Components
  • Complex: The type VI secretion VgrG1-Tse6-EF-Tu complex embedded in lipid nanodiscsType VI secretion system
    • Protein or peptide: VgrG1

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Supramolecule #1: The type VI secretion VgrG1-Tse6-EF-Tu complex embedded in lipid ...

SupramoleculeName: The type VI secretion VgrG1-Tse6-EF-Tu complex embedded in lipid nanodiscs
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Pseudomonas aeruginosa PAO1 (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
Molecular weightTheoretical: 350 KDa

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Macromolecule #1: VgrG1

MacromoleculeName: VgrG1 / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Pseudomonas aeruginosa PAO1 (bacteria)
Molecular weightTheoretical: 72.098375 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString: MQLTRLVQVD CPLGPDVLLL QRMEGREELG RLFAYELHLV SENPNLPLEQ LLGKPMSLSL ELPGGSRRFF HGIVARCSQV AGHGQFAGY QATLRPWPWL LTRTSDCRIF QNQSVPEIIK QVFRNLGFSD FEDALTRPYR EWEYCVQYRE TSFDFISRLM E QEGIYYWF ...String:
MQLTRLVQVD CPLGPDVLLL QRMEGREELG RLFAYELHLV SENPNLPLEQ LLGKPMSLSL ELPGGSRRFF HGIVARCSQV AGHGQFAGY QATLRPWPWL LTRTSDCRIF QNQSVPEIIK QVFRNLGFSD FEDALTRPYR EWEYCVQYRE TSFDFISRLM E QEGIYYWF RHEQKRHILV LSDAYGAHRS PGGYASVPYY PPTLGHRERD HFFDWQMARE VQPGSLTLND YDFQRPGARL EV RSNIARP HAAADYPLYD YPGEYVQSQD GEQYARNRIE AIQAQHERVR LRGVVRGIGA GHLFRLSGYP RDDQNREYLV VGA EYRVVQ ELYETGSGGA GSQFESELDC IDASQSFRLL PQTPVPVVRG PQTAVVVGPK GEEIWTDQYG RVKVHFHWDR HDQS NENSS CWIRVSQAWA GKNWGSMQIP RIGQEVIVSF LEGDPDRPII TGRVYNAEQT VPYELPANAT QSGMKSRSSK GGTPA NFNE IRMEDKKGAE QLYIHAERNQ DNLVENDASL SVGHDRNKSI GHDELARIGN NRTRAVKLND TLLVGGAKSD SVTGTY LIE AGAQIRLVCG KSVVEFNADG TINISGSAFN LYASGNGNID TGGRLDLNSG GASEVDAKGK GVQGTIDGQV QAMFPPP AK G

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.02 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
20.0 mMTris-HClTrisTris
300.0 mMNaClSodium chloridesodium chloride
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 2.0 nm / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK III
Details: 0.01 % Tween-20 was added to improve ice quality..

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 0.01 mm / Nominal defocus max: 3.2 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 59000
Specialist opticsSpherical aberration corrector: Cs corrected microscope
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number real images: 1873 / Average exposure time: 15.0 sec. / Average electron dose: 91.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: EMDB MAP
EMDB ID:

3113

Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionApplied symmetry - Point group: C3 (3 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.25 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: SPHIRE / Software - details: meridien / Number images used: 72000

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