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- EMDB-13843: Structure of VgrG1 from Pseudomonas protegens. -

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Basic information

Entry
Database: EMDB / ID: EMD-13843
TitleStructure of VgrG1 from Pseudomonas protegens.
Map data
Sample
  • Organelle or cellular component: Structure of the VgrG1 trimer from Pseudomonas protegens.
    • Protein or peptide: Type VI secretion protein VgrGType VI secretion system
Function / homologyType VI secretion system, RhsGE-associated Vgr family subset / 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 / Type VI secretion protein VgrG
Function and homology information
Biological speciesPseudomonas protegens Pf-5 (bacteria) / Pseudomonas fluorescens (strain ATCC BAA-477 / NRRL B-23932 / Pf-5) (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsGuenther P / Quentin D / Ahmad S / Sachar K / Gatsogiannis C / Whitney JC / Raunser S
Funding support2 items
OrganizationGrant numberCountry
Max Planck Society
Natural Sciences and Engineering Research Council (NSERC, Canada)RGPIN 2017 05350
CitationJournal: PLoS Pathog / Year: 2022
Title: Structure of a bacterial Rhs effector exported by the type VI secretion system.
Authors: Patrick Günther / Dennis Quentin / Shehryar Ahmad / Kartik Sachar / Christos Gatsogiannis / John C Whitney / Stefan Raunser /
Abstract: The type VI secretion system (T6SS) is a widespread protein export apparatus found in Gram-negative bacteria. The majority of T6SSs deliver toxic effector proteins into competitor bacteria. Yet, the ...The type VI secretion system (T6SS) is a widespread protein export apparatus found in Gram-negative bacteria. The majority of T6SSs deliver toxic effector proteins into competitor bacteria. Yet, the structure, function, and activation of many of these effectors remains poorly understood. Here, we present the structures of the T6SS effector RhsA from Pseudomonas protegens and its cognate T6SS spike protein, VgrG1, at 3.3 Å resolution. The structures reveal that the rearrangement hotspot (Rhs) repeats of RhsA assemble into a closed anticlockwise β-barrel spiral similar to that found in bacterial insecticidal Tc toxins and in metazoan teneurin proteins. We find that the C-terminal toxin domain of RhsA is autoproteolytically cleaved but remains inside the Rhs 'cocoon' where, with the exception of three ordered structural elements, most of the toxin is disordered. The N-terminal 'plug' domain is unique to T6SS Rhs proteins and resembles a champagne cork that seals the Rhs cocoon at one end while also mediating interactions with VgrG1. Interestingly, this domain is also autoproteolytically cleaved inside the cocoon but remains associated with it. We propose that mechanical force is required to remove the cleaved part of the plug, resulting in the release of the toxin domain as it is delivered into a susceptible bacterial cell by the T6SS.
History
DepositionNov 4, 2021-
Header (metadata) releaseDec 15, 2021-
Map releaseDec 15, 2021-
UpdateFeb 2, 2022-
Current statusFeb 2, 2022Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.059
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.059
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7q5p
  • Surface level: 0.059
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7q5p
  • Imaged by Jmol
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_13843.png

Downloads & links

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Map

FileDownload / File: emd_13843.map.gz / Format: CCP4 / Size: 259.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.1 Å
Density
Contour LevelBy AUTHOR: 0.059 / Movie #1: 0.059
Minimum - Maximum-0.12288407 - 0.22215113
Average (Standard dev.)0.00011095713 (±0.0030134872)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-204-204-204
Dimensions408408408
Spacing408408408
CellA=B=C: 448.80002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.11.11.1
M x/y/z408408408
origin x/y/z0.0000.0000.000
length x/y/z448.800448.800448.800
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-204-204-204
NC/NR/NS408408408
D min/max/mean-0.1230.2220.000

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Supplemental data

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Mask #1

Fileemd_13843_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_13843_half_map_1.map
Projections & Slices
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Histogram

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Half map: #1

Fileemd_13843_half_map_2.map
Projections & Slices
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Sample components

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Entire : Structure of the VgrG1 trimer from Pseudomonas protegens.

EntireName: Structure of the VgrG1 trimer from Pseudomonas protegens.
Components
  • Organelle or cellular component: Structure of the VgrG1 trimer from Pseudomonas protegens.
    • Protein or peptide: Type VI secretion protein VgrGType VI secretion system

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Supramolecule #1: Structure of the VgrG1 trimer from Pseudomonas protegens.

SupramoleculeName: Structure of the VgrG1 trimer from Pseudomonas protegens.
type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Pseudomonas protegens Pf-5 (bacteria)
Molecular weightTheoretical: 215.994 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)

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Macromolecule #1: Type VI secretion protein VgrG

MacromoleculeName: Type VI secretion protein VgrG / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Pseudomonas fluorescens (strain ATCC BAA-477 / NRRL B-23932 / Pf-5) (bacteria)
Strain: ATCC BAA-477 / NRRL B-23932 / Pf-5
Molecular weightTheoretical: 72.080133 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MLFQQSTRLA QVNCPLGPDV LLLKSLGGGE ELGRLFDYQL QLASSDANID LNQLLGKPMG LSVQLDGGGQ RYFHGIVARC SQNIDTGQF ASYEVTLRPW LWLLSRTSDC RIFQNLSIPQ IIKQVFRDLG FSDFEDALSR PYREWEYCVQ YRETSFDFVS R LMEQEGIY ...String:
MLFQQSTRLA QVNCPLGPDV LLLKSLGGGE ELGRLFDYQL QLASSDANID LNQLLGKPMG LSVQLDGGGQ RYFHGIVARC SQNIDTGQF ASYEVTLRPW LWLLSRTSDC RIFQNLSIPQ IIKQVFRDLG FSDFEDALSR PYREWEYCVQ YRETSFDFVS R LMEQEGIY YFFRHEKDRH VVVLADAYGA HSSVPGYASV PYYPRDEQQR ERDHMFDWHL AQEVQPGSLE LNDYDFQRPS AR IDVRSAM PRPHSAGDYP LYDYPGTYVQ SSDGEHYAQT RIEALQSLHE RIELSGNARG LGVGNLFSLT GFSRQDQNRE YLI VSIRYY LVQESLESGA GGGSAQFESH LTCIDAQQSF RPLATTHKPM VQGPQTARVV GPAGEEIWTD QYGRVKVHFH WDRH DQSNE NSSCWIRVSQ AWAGKNWGSM QIPRIGQEVI VSFLEGDPDR PIITGRVYNA EQTVPYDLPA NATQSGMKSR SSKGG SPAN FNEIRMEDKK GAEQLYIHAE RNQDIVVEVN ESHSVGNNRN KSIGHDEYVT IGNKRTRIVQ HVDELRVGEK KLDSVG QSY VIEVGERLRL VCGASILELN ASGQINLCGV NISVHASADA QINTGGVLHL NNGGGPGTTT EGQGVQGAIS AKAKAPF SA PKG

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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

Concentration1 mg/mL
BufferpH: 8
GridModel: Quantifoil R2/1 / Material: COPPER / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 101.325 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 281 K / Instrument: FEI VITROBOT MARK IV
DetailsThe sample was monodisperse.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 0.01 mm / Nominal magnification: 59000
Specialist opticsSpherical aberration corrector: Cs corrector / Energy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
TemperatureMin: 70.0 K / Max: 70.0 K
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: INTEGRATING / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 1 / Number real images: 1250 / Average exposure time: 1.5 sec. / Average electron dose: 90.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: CTFFIND (ver. 4.1)
Startup modelType of model: EMDB MAP
EMDB ID:

0136

Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: SPHIRE (ver. 1.5)
Final 3D classificationNumber classes: 461 / Avg.num./class: 900 / Software - Name: SPHIRE (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: SPHIRE (ver. 1.5)
Final reconstructionNumber classes used: 396 / Applied symmetry - Point group: C3 (3 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: SPHIRE (ver. 1.5) / Number images used: 423980
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6h3n


Chain - Chain ID: A / Chain - Residue range: 1-643
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-7q5p:
Structure of VgrG1 from Pseudomonas protegens.

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