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- EMDB-13587: Photorhabdus laumondii T6SS-associated Rhs protein carrying the T... -

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Basic information

Entry
Database: EMDB / ID: EMD-13587
TitlePhotorhabdus laumondii T6SS-associated Rhs protein carrying the Tre23 toxin domain
Map data
Sample
  • Complex: Tre23
    • Protein or peptide: Tre23
Function / homologyRHS repeat / RHS Repeat / PAAR motif / PAAR motif / YD repeat / Rhs repeat-associated core / membrane => GO:0016020 / Uncharacterized protein
Function and homology information
Biological speciesPhotorhabdus laumondii (bacteria) / Photorhabdus laumondii subsp. laumondii TTO1 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.17 Å
AuthorsJurenas D / Talachia Rosa L / Rey M / Chamot-Rooke J / Fronzes R / Cascales E
Funding support France, 1 items
OrganizationGrant numberCountry
Centre National de la Recherche Scientifique (CNRS) France
CitationJournal: Nat Commun / Year: 2021
Title: Mounting, structure and autocleavage of a type VI secretion-associated Rhs polymorphic toxin.
Authors: Dukas Jurėnas / Leonardo Talachia Rosa / Martial Rey / Julia Chamot-Rooke / Rémi Fronzes / Eric Cascales /
Abstract: Bacteria have evolved toxins to outcompete other bacteria or to hijack host cell pathways. One broad family of bacterial polymorphic toxins gathers multidomain proteins with a modular organization, ...Bacteria have evolved toxins to outcompete other bacteria or to hijack host cell pathways. One broad family of bacterial polymorphic toxins gathers multidomain proteins with a modular organization, comprising a C-terminal toxin domain fused to a N-terminal domain that adapts to the delivery apparatus. Polymorphic toxins include bacteriocins, contact-dependent growth inhibition systems, and specialized Hcp, VgrG, PAAR or Rhs Type VI secretion (T6SS) components. We recently described and characterized Tre23, a toxin domain fused to a T6SS-associated Rhs protein in Photorhabdus laumondii, Rhs1. Here, we show that Rhs1 forms a complex with the T6SS spike protein VgrG and the EagR chaperone. Using truncation derivatives and cross-linking mass spectrometry, we demonstrate that VgrG-EagR-Rhs1 complex formation requires the VgrG C-terminal β-helix and the Rhs1 N-terminal region. We then report the cryo-electron-microscopy structure of the Rhs1-EagR complex, demonstrating that the Rhs1 central region forms a β-barrel cage-like structure that encapsulates the C-terminal toxin domain, and provide evidence for processing of the Rhs1 protein through aspartyl autoproteolysis. We propose a model for Rhs1 loading on the T6SS, transport and delivery into the target cell.
History
DepositionSep 16, 2021-
Header (metadata) releaseDec 1, 2021-
Map releaseDec 1, 2021-
UpdateMar 2, 2022-
Current statusMar 2, 2022Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.015
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.015
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7pq5
  • Surface level: 0.015
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_13587.png

Downloads & links

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Map

FileDownload / File: emd_13587.map.gz / Format: CCP4 / Size: 83.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.93 Å/pix.
x 280 pix.
= 260.4 Å		0.93 Å/pix.
x 280 pix.
= 260.4 Å		0.93 Å/pix.
x 280 pix.
= 260.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.93 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.015 / Movie #1: 0.015
Minimum - Maximum-0.031466432 - 0.06747829
Average (Standard dev.)2.0773203e-05 (±0.0017983569)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions280280280
Spacing280280280
CellA=B=C: 260.4 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.930.930.93
M x/y/z280280280
origin x/y/z0.0000.0000.000
length x/y/z260.400260.400260.400
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ352352352
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS280280280
D min/max/mean-0.0310.0670.000

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Supplemental data

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Mask #1

Fileemd_13587_msk_1.map
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_13587_half_map_1.map
Projections & Slices
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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_13587_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 : Tre23

EntireName: Tre23
Components
  • Complex: Tre23
    • Protein or peptide: Tre23

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Supramolecule #1: Tre23

SupramoleculeName: Tre23 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Photorhabdus laumondii (bacteria)
Recombinant expressionOrganism: Escherichia coli K-12 (bacteria)

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Macromolecule #1: Tre23

MacromoleculeName: Tre23 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Photorhabdus laumondii subsp. laumondii TTO1 (bacteria)
Molecular weightTheoretical: 165.588828 KDa
Recombinant expressionOrganism: Escherichia coli K-12 (bacteria)
SequenceString: MSLGDEIVTR IARVGARHAV HVSPPQGSPG PAAAREGDPI KHASFLGALA GAITGALIGA AVFAAASALV GLTILTGGLA TVAVIALGT AATFALGDVI SAASSAVTNM VDSIGPPSGT LTSGSPNVFI EGQPAARATT DIAVCSKHPA PPLIAQGSET V LINGSPAA ...String:
MSLGDEIVTR IARVGARHAV HVSPPQGSPG PAAAREGDPI KHASFLGALA GAITGALIGA AVFAAASALV GLTILTGGLA TVAVIALGT AATFALGDVI SAASSAVTNM VDSIGPPSGT LTSGSPNVFI EGQPAARATT DIAVCSKHPA PPLIAQGSET V LINGSPAA RVDDKLACGA TIKSGAKTVF IGSGQGTYLA VADEFSAWER AILIAVEFLV PPSRGALKGL GKLFTKPGQG IQ GVLKGAK AGAKKAKALL SNRISCAKKA FRETEGAKRY REATKKFFTG DPIDVTTGQL FDQRTDITLG QTLPLVFLRS WVP EEQGLL GPGWTDSFSE CALATGDRVE IRTTEGASLY FALPAAYTHS VNPDHPDFTL SRGEQGYILR HRDSPVSKYF TLPH PSSAS KEAPRRWLLT EHRDVYDNRL RFIYNKHCQL TQVLHSDGPE LTLLYNLRGQ LTEIRRTDER LQEVMARYHY HDNGR LAEA DSTQNFHLYY EYNAQGLISR WSDGDQTWVD YRYDKQGRCT DSVGAGGFYP VHLDYAPGIT RSTTPQGHTT TGHYND QQL ITEIHTPCGG VTRYEYDRWG NLVRQILPEG ETLTLTYLAD TGRVTSLTEA TGAVWQYSYE ADSLQLTGMT DPLQRTW LP QYDEQGQPAG FIAPDGRKTT LTRNAFGLVT SETDPDGNSR TQEYDKHQRL VRVLDEENRT VSLGYDSQDR LRSLTAAG A LWRWRYDRHH RVAVSDRPDN QLEHFTHDRH GNLTCWTDAR GVKWQVEYGP FDLPVARRDG EGHRWQYRYD ADTLQLTQV INPQGETYSY TLDADGRVIT EQDYAGTQWH YRYDRSGNCI EKRDGEENVT RYDYDAARRL TTLHTPEGPT RYHYDSVGRL LTVDSPDST LHFEYDGQDR IVREIQPHGE IQRHYPDNRT AERQLLTGET APVTGPARFS GQTHPGRWQS RREVNRVGEL I TLTLAGQA PLTIERDDAG RDTGRYVDGG FILRQQYSLM GQLTAQRAGR NPYFFRPAEP DEIEGPAYAG VARRYEYDTA LN LTAASDD GQQVNYLLNG NGQVISVGEG RTLREHYQYD ETGYPSRRFD GVQEIMGETL YQEGHRLNWV GSHRFVYDRA GRM QEKQFL AEGCRLALTK YRWNSQNQLT GLITPDGIPW EYRYDAFGRR TEKRCIQSGK LTTYLWDGNV PAEIREYQHG RLKM IRHLV FDGWELVAQQ TQAFTLNLDN RVELMAGEVQ TQYAVSAPTG EPLALFDPAG KRVWRRPKQS LYGLRLGGYG ENPQL DPGL RFAGQLFDEE SGLFYNRFRY YLPEATCYLS PDPTGLWGGE NTYRYVQNPT KFINPLGLAG ENVFIHATNK AGFDGI MKS GVLHPNVSGK VAITDVLMSP KDVMRDLLIN NPNHIGRGDY AIIFKIDPGE RGNIRSPSRL EYTHEGKLKL NNILYAG KN PYAILEHLDY DTRLKLTDNQ VKIRKCGGKV DDYKDDDDK

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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

Concentration0.5 mg/mL
BufferpH: 8.5
Details: 50 mM Tris-HCl pH8.5, 250 mM NaCl, 1 mM TCEP, cOmpleteTM protease inhibitor cocktail (Sigma-Aldrich)
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Pressure: 25.0 kPa / Details: 2 mAu current
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK III

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 1-37 / Number grids imaged: 1 / Number real images: 8550 / Average exposure time: 3.7 sec. / Average electron dose: 49.75 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 20.0 µm / Nominal defocus min: 5.0 µm / Nominal magnification: 45000
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 2623687
CTF correctionSoftware - Name: RELION (ver. 3.1)
Final reconstructionNumber classes used: 1 / Resolution.type: BY AUTHOR / Resolution: 3.17 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 140577
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final 3D classificationNumber classes: 8 / Avg.num./class: 140000 / Software - Name: RELION (ver. 3.1)
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: BACKBONE TRACE / Overall B value: 75.76
Output model

PDB-7pq5:
Photorhabdus laumondii T6SS-associated Rhs protein carrying the Tre23 toxin domain

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