Natural Sciences and Engineering Research Council (NSERC, Canada)
RGPIN 2017 05350
Other private
Citation
Journal: 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.
Mass: 159719.453 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Pseudomonas protegens Pf-5 (bacteria) / Strain: ATCC BAA-477 / NRRL B-23932 / Pf-5 / Gene: PFL_6096 / Plasmid: pETDuet-1 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q4K3M9
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Experimental details
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Experiment
Experiment
Method: ELECTRON MICROSCOPY
EM experiment
Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction
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Sample preparation
Component
Name: Structure of RhsA. / Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: all / Source: RECOMBINANT
Molecular weight
Value: 0.16491297 MDa / Experimental value: NO
Source (natural)
Organism: Pseudomonas protegens Pf-5 (bacteria)
Source (recombinant)
Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solution
pH: 8
Specimen
Conc.: 4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: The sample was monodisperse.
Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 281 K
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Electron microscopy imaging
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
Microscopy
Model: FEI TITAN KRIOS
Electron gun
Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lens
Mode: BRIGHT FIELD / Nominal magnification: 105000 X / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holder
Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 70 K / Temperature (min): 70 K
Image recording
Average exposure time: 2 sec. / Electron dose: 61 e/Å2 / Film or detector model: GATAN K2 (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 13090
EM imaging optics
Energyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 454740 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model building
B value: 50 / Protocol: AB INITIO MODEL / Space: REAL
Refine LS restraints
Refine-ID
Type
Dev ideal
Number
ELECTRONMICROSCOPY
f_bond_d
0.009
17440
ELECTRONMICROSCOPY
f_angle_d
0.648
23610
ELECTRONMICROSCOPY
f_dihedral_angle_d
10.047
6448
ELECTRONMICROSCOPY
f_chiral_restr
0.045
2416
ELECTRONMICROSCOPY
f_plane_restr
0.005
3164
+
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