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- EMDB-0135: Electron cryo-microscopy of the Type VI secretion "pre-firing" Vg... -

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Basic information

Entry
Database: EMDB / ID: EMD-0135
TitleElectron cryo-microscopy of the Type VI secretion "pre-firing" VgrG1-Tse6-EagT6-EF-Tu-Tsi6 complex
Map dataThe "pre-firing" VgrG1-Tse6-EagT6-EF-Tu-Tsi6 complex (PFC)
Sample
  • Complex: Type VI secretion "pre-firing" VgrG1-Tse6-EagT6-EF-Tu-Tsi6 complex
    • Complex: Valine-glycine repeat protein 1
      • Protein or peptide: VgrG1
    • Complex: Type VI secretion exported 6
    • Complex: Type VI secretion immunity 6
    • Complex: Effector associated gene with tse6
    • Complex: Elongation factor Tu 1
KeywordsBacterial Type VI effector complex / T6SS chaperone-effector complex / Tse6-loaded VgrG1 complex / NAD(P)+ Glycohydrolase / TOXIN
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
: / Bacteriophage T4 gp5 C-terminal trimerisation domain / Type VI secretion system, RhsGE-associated Vgr family subset / Type VI secretion system, RhsGE-associated Vgr protein / Phage tail baseplate hub (GPD) / 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) / Escherichia coli BL21 (bacteria) / Pseudomonas aeruginosa (strain ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1) (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.2 Å
AuthorsQuentin D / Raunser S
Funding support Germany, 1 items
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, 2018-
Header (metadata) releaseAug 22, 2018-
Map releaseSep 12, 2018-
UpdateMay 15, 2024-
Current statusMay 15, 2024Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.053
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.053
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-6h3l
  • Surface level: 0.053
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_0135.png

Downloads & links

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Map

FileDownload / File: emd_0135.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThe "pre-firing" VgrG1-Tse6-EagT6-EF-Tu-Tsi6 complex (PFC)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.14 Å/pix.
x 360 pix.
= 410.4 Å		1.14 Å/pix.
x 360 pix.
= 410.4 Å		1.14 Å/pix.
x 360 pix.
= 410.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.14 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.053 / Movie #1: 0.053
Minimum - Maximum-0.04401986 - 0.11461051
Average (Standard dev.)0.0002702165 (±0.0034003535)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 410.4 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.141.141.14
M x/y/z360360360
origin x/y/z0.0000.0000.000
length x/y/z410.400410.400410.400
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS360360360
D min/max/mean-0.0440.1150.000

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Supplemental data

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Sample components

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Entire : Type VI secretion "pre-firing" VgrG1-Tse6-EagT6-EF-Tu-Tsi6 complex

EntireName: Type VI secretion "pre-firing" VgrG1-Tse6-EagT6-EF-Tu-Tsi6 complex
Components
  • Complex: Type VI secretion "pre-firing" VgrG1-Tse6-EagT6-EF-Tu-Tsi6 complex
    • Complex: Valine-glycine repeat protein 1
      • Protein or peptide: VgrG1
    • Complex: Type VI secretion exported 6
    • Complex: Type VI secretion immunity 6
    • Complex: Effector associated gene with tse6
    • Complex: Elongation factor Tu 1

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Supramolecule #1: Type VI secretion "pre-firing" VgrG1-Tse6-EagT6-EF-Tu-Tsi6 complex

SupramoleculeName: Type VI secretion "pre-firing" VgrG1-Tse6-EagT6-EF-Tu-Tsi6 complex
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: The 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.
Molecular weightTheoretical: 43 KDa

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Supramolecule #2: Valine-glycine repeat protein 1

SupramoleculeName: Valine-glycine repeat protein 1 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: all / Details: VgrG1 forms a trimer - UniProt Identifier: Q9I741
Source (natural)Organism: Pseudomonas aeruginosa PAO1 (bacteria)

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Supramolecule #3: Type VI secretion exported 6

SupramoleculeName: Type VI secretion exported 6 / type: complex / ID: 3 / Parent: 1 / Details: Tse6 - UniProt identifier: Q9I739
Source (natural)Organism: Pseudomonas aeruginosa PAO1 (bacteria)

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Supramolecule #4: Type VI secretion immunity 6

SupramoleculeName: Type VI secretion immunity 6 / type: complex / ID: 4 / Parent: 1 / Details: Tsi6 - UniProt identifier: Q9I740
Source (natural)Organism: Pseudomonas aeruginosa PAO1 (bacteria)

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Supramolecule #5: Effector associated gene with tse6

SupramoleculeName: Effector associated gene with tse6 / type: complex / ID: 5 / Parent: 1
Details: EagT6 forms dimers - UniProt identifier: Q9I738. Two dimers are present in the EM map.
Source (natural)Organism: Pseudomonas aeruginosa PAO1 (bacteria)

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Supramolecule #6: Elongation factor Tu 1

SupramoleculeName: Elongation factor Tu 1 / type: complex / ID: 6 / Parent: 1 / Details: EF-Tu - UniProt identifier: P0CE47
Source (natural)Organism: Escherichia coli BL21 (bacteria)

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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 (strain ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / 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

UniProtKB: Type VI secretion system spike protein VgrG1a

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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

Concentration0.015 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
20.0 mMTris-HClTris
300.0 mMNaClsodium chloride
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 2 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 298 K / Instrument: GATAN CRYOPLUNGE 3

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsSpherical aberration corrector: Cs corrected microscope
Image recordingFilm or detector model: FEI FALCON II (4k x 4k) / Detector mode: INTEGRATING / Number real images: 11642 / Average exposure time: 1.5 sec. / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELD / Cs: 0.01 mm / Nominal defocus max: 4.2 µm / Nominal defocus min: 1.7 µm / Nominal magnification: 59000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: EMDB MAP
EMDB ID:

3113

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: SPHIRE / Software - details: meridien / Number images used: 55000
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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