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- EMDB-13867: Structure of the bacterial type VI secretion system effector RhsA. -

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Basic information

Entry
Database: EMDB / ID: EMD-13867
TitleStructure of the bacterial type VI secretion system effector RhsA.
Map data
Sample
  • Organelle or cellular component: Structure of RhsA.
    • Protein or peptide: Rhs family protein
Keywordsbacterial type VI secretion system / effector / Rhs repeats / TOXIN
Function / homology
Function and homology information


membrane => GO:0016020
Similarity search - Function
RHS protein / RHS protein / RHS repeat / RHS Repeat / PAAR motif / PAAR motif / YD repeat / Rhs repeat-associated core
Similarity search - Domain/homology
Biological speciesPseudomonas protegens Pf-5 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsGuenther P / Quentin D
Funding support3 items
OrganizationGrant numberCountry
Max Planck Society
Natural Sciences and Engineering Research Council (NSERC, Canada)RGPIN 2017 05350
Other private
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 12, 2021-
Header (metadata) releaseDec 22, 2021-
Map releaseDec 22, 2021-
UpdateJul 17, 2024-
Current statusJul 17, 2024Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0331
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.0331
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7q97
  • Surface level: 0.025
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7q97
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_13867.png

Downloads & links

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Map

FileDownload / File: emd_13867.map.gz / Format: CCP4 / Size: 91.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.91 Å
Density
Contour LevelBy AUTHOR: 0.0331 / Movie #1: 0.0331
Minimum - Maximum-0.09305289 - 0.16611847
Average (Standard dev.)0.00004750165 (±0.0051871827)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions288288288
Spacing288288288
CellA=B=C: 262.08002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.910.910.91
M x/y/z288288288
origin x/y/z0.0000.0000.000
length x/y/z262.080262.080262.080
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS288288288
D min/max/mean-0.0930.1660.000

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Supplemental data

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Mask #1

Fileemd_13867_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_13867_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_13867_half_map_2.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Sample components

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Entire : Structure of RhsA.

EntireName: Structure of RhsA.
Components
  • Organelle or cellular component: Structure of RhsA.
    • Protein or peptide: Rhs family protein

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Supramolecule #1: Structure of RhsA.

SupramoleculeName: Structure of RhsA. / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Pseudomonas protegens Pf-5 (bacteria)
Molecular weightTheoretical: 164.91297 KDa

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Macromolecule #1: Rhs family protein

MacromoleculeName: Rhs family protein / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Pseudomonas protegens Pf-5 (bacteria) / Strain: ATCC BAA-477 / NRRL B-23932 / Pf-5
Molecular weightTheoretical: 159.719453 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MGSSHHHHHH SQDPSKTGAD KGLSNLCEGI ANALFPPSIQ ATIASGSKKV LTNNKPAARA AGIAPPTQIS GDGAEEGEAT DTPSATPEE GPGFLDMAKE FFSQMWRPTV ASPAPGSQTK ELDLVTCTKH PPMPVQYLAE GSEKVSIDGQ PAVRSGDRST C DAKVVSAG ...String:
MGSSHHHHHH SQDPSKTGAD KGLSNLCEGI ANALFPPSIQ ATIASGSKKV LTNNKPAARA AGIAPPTQIS GDGAEEGEAT DTPSATPEE GPGFLDMAKE FFSQMWRPTV ASPAPGSQTK ELDLVTCTKH PPMPVQYLAE GSEKVSIDGQ PAVRSGDRST C DAKVVSAG PISPNVTIGG GSVVVREIRS GKTPGVGLAV TVLMMLKGGK GKFLSNLPCM LMAGVNSFVV SQATNALTQA VV ASTHPVH AATGAKVLGA DDDLDFTLPG LMPIEWQRFY NSRDERRDGL LGAGWSLPYE ISVQIEPHPE GGERLLYIDE QGR RIDMGS IALGAGAFSP GEGLSVRRNE NGQLLIESSD GIYRLFEPLP GDSSRLRLSL LGDRNDNRLH LEYDSSGRLW LLRD TFEQA QLELSYSTLW PGRIAQVERL YPDQQREVLV SYDYDAAGDL AQVRDSEGQV QRRFAYDAGH RMIEHQLATG LRCFY QWTQ FADQQWRVTR HWTDEGDSYQ YDYDLEAGIT QVTDSLQRLS RRRWNSQLQI VEYTDNLNQT WAFEWNDERQ LLSATD PLG GRYAYSYDET GNLIAETDPL GHSHSTLWLE HWSLPQTLTD PAGESWHLRY DPRGNCIAEV DPLGQVTQYR HDAHGQV VE IIDAAGRSRK MRWNSSGQLL EHLDCSGYPT RFEYDRRGNV KAIIDALGEH TRFHYDCQGR LLSSQLADGR AEHFQRSS N GQLQGYTDPS GYTTLYQYNR RGQIRQRIDA HGRQVRFDYD AYGRLLALTN ENGESYRFAW DAGNRLSEQQ DLDGSAKRY GYDLLNNVVE LQSHPAPYGT GLSAVPAQPI APIIHRFERD AVGRLLAKIT DDGRSQYTYD PLNQLTAASF TDNLGNQQAL SFSYDALGQ LLEEHTVAGS LIHRYDELGN LTQTQLPDKR WLNRLYYGSG HLHQINLDGQ VVSDFQRDRL HREVLRTQGQ I STRSEYDR CGRLRARLQH SSLLANTLPT APERHLEYDD SDNLVGLAER HATGQHRQLF HYDATGRIIA SQDGLQGQKE TF AYDAAAN LLDGPKAGAG LVVHNKLLTY QDKRYRYDAF GRMIEKRSGR RGVQRFAYDA EHRLIEVRNQ TSSGETLVRM RYD VLGRRI SKSEYDSQGN LLGETRFVWD GLRLLQEQRN QQTSLYVYED LGHAPLARVD GLGAQQKIRY YHNDLNGLPQ QLCE PDGHS VWQARYQVWG NTVEEIREPY YIEEQNLRFQ GQYLDRETGL HFNTFRFYDP DIGRFTTPDP IGLAGGLNLY QYAPN PIGW IDPLGWICKS AYSGRRGTTK AKADLERNGF TVVGEELTMK VKHPVTQKSY RIRADIIAKD KAGNYHVFEV KNGSGK LTK NQQGSGMFDM SNPANTNGGL GGGLIKPSGA TQGQFEVATK TARGTQFGGN GAQHSATFWL LRY

UniProtKB: Rhs family protein

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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

Concentration4 mg/mL
BufferpH: 8
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / 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
TemperatureMin: 70.0 K / Max: 70.0 K
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K2 (4k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number grids imaged: 1 / Number real images: 13090 / Average exposure time: 2.0 sec. / Average electron dose: 61.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal magnification: 105000
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: INSILICO MODEL
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C2 (2 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: 454740
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: SPHIRE (ver. 1.5)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: SPHIRE (ver. 1.5)
Final 3D classificationNumber classes: 4 / Software - Name: RELION (ver. 3.1)
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: AB INITIO MODEL / Overall B value: 50
Output model

PDB-7q97:
Structure of the bacterial type VI secretion system effector RhsA.

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