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- EMDB-2927: Negative stain structure of a type 6 secretion system membrane co... -

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Basic information

Entry
Database: EMDB / ID: EMD-2927
TitleNegative stain structure of a type 6 secretion system membrane core complex
Map dataReconstruction of the whole TssJ-TssM-TssL complex
Sample
  • Sample: Negative stain structure of the TssJ-TssM-TssL type 6 secretion membrane core complex
  • Protein or peptide: TssJ
  • Protein or peptide: TssM
  • Protein or peptide: TssL
KeywordsBacterial secretion / virulence
Function / homology
Function and homology information


IcmF-related / IcmF-related / Intracellular multiplication and human macrophage-killing / : / : / Type VI secretion protein IcmF helical domain / Type VI secretion system IcmF, C-terminal / Type VI secretion system, lipoprotein SciN / Type VI secretion system IcmF, C-terminal / Type VI secretion system, lipoprotein SciN ...IcmF-related / IcmF-related / Intracellular multiplication and human macrophage-killing / : / : / Type VI secretion protein IcmF helical domain / Type VI secretion system IcmF, C-terminal / Type VI secretion system, lipoprotein SciN / Type VI secretion system IcmF, C-terminal / Type VI secretion system, lipoprotein SciN / Type VI secretion protein SciN-like superfamily / Type VI secretion protein IcmF C2-like domain / Type VI secretion lipoprotein, VasD, EvfM, TssJ, VC_A0113 / Type IV / VI secretion system, DotU / Type IV / VI secretion system, DotU / Type IV / VI secretion system, DotU superfamily / Type VI secretion system protein DotU / Prokaryotic membrane lipoprotein lipid attachment site profile.
Similarity search - Domain/homology
Type VI secretion system lipoprotein TssJ / Type VI secretion protein / Type VI secretion protein
Similarity search - Component
Biological speciesEscherichia coli (strain 55989 / EAEC) (bacteria)
Methodsingle particle reconstruction / negative staining / Resolution: 11.5 Å
AuthorsDurand E / Fronzes R
CitationJournal: Nature / Year: 2015
Title: Biogenesis and structure of a type VI secretion membrane core complex.
Authors: Eric Durand / Van Son Nguyen / Abdelrahim Zoued / Laureen Logger / Gérard Péhau-Arnaudet / Marie-Stéphanie Aschtgen / Silvia Spinelli / Aline Desmyter / Benjamin Bardiaux / Annick ...Authors: Eric Durand / Van Son Nguyen / Abdelrahim Zoued / Laureen Logger / Gérard Péhau-Arnaudet / Marie-Stéphanie Aschtgen / Silvia Spinelli / Aline Desmyter / Benjamin Bardiaux / Annick Dujeancourt / Alain Roussel / Christian Cambillau / Eric Cascales / Rémi Fronzes /
Abstract: Bacteria share their ecological niches with other microbes. The bacterial type VI secretion system is one of the key players in microbial competition, as well as being an important virulence ...Bacteria share their ecological niches with other microbes. The bacterial type VI secretion system is one of the key players in microbial competition, as well as being an important virulence determinant during bacterial infections. It assembles a nano-crossbow-like structure in the cytoplasm of the attacker cell that propels an arrow made of a haemolysin co-regulated protein (Hcp) tube and a valine-glycine repeat protein G (VgrG) spike and punctures the prey's cell wall. The nano-crossbow is stably anchored to the cell envelope of the attacker by a membrane core complex. Here we show that this complex is assembled by the sequential addition of three type VI subunits (Tss)-TssJ, TssM and TssL-and present a structure of the fully assembled complex at 11.6 Å resolution, determined by negative-stain electron microscopy. With overall C5 symmetry, this 1.7-megadalton complex comprises a large base in the cytoplasm. It extends in the periplasm via ten arches to form a double-ring structure containing the carboxy-terminal domain of TssM (TssMct) and TssJ that is anchored in the outer membrane. The crystal structure of the TssMct-TssJ complex coupled to whole-cell accessibility studies suggest that large conformational changes induce transient pore formation in the outer membrane, allowing passage of the attacking Hcp tube/VgrG spike.
History
DepositionMar 10, 2015-
Header (metadata) releaseApr 15, 2015-
Map releaseAug 5, 2015-
UpdateAug 5, 2015-
Current statusAug 5, 2015Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.025
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.025
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_2927.png

Downloads & links

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Map

FileDownload / File: emd_2927.map.gz / Format: CCP4 / Size: 81.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of the whole TssJ-TssM-TssL complex
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.9 Å/pix.
x 280 pix.
= 532. Å		1.9 Å/pix.
x 280 pix.
= 532. Å		1.9 Å/pix.
x 280 pix.
= 532. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.9 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.025 / Movie #1: 0.025
Minimum - Maximum-0.11743928 - 0.13615407
Average (Standard dev.)0.00066825 (±0.00788903)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions280280280
Spacing280280280
CellA=B=C: 532.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.91.91.9
M x/y/z280280280
origin x/y/z0.0000.0000.000
length x/y/z532.000532.000532.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS280280280
D min/max/mean-0.1170.1360.001

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Supplemental data

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Sample components

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Entire : Negative stain structure of the TssJ-TssM-TssL type 6 secretion m...

EntireName: Negative stain structure of the TssJ-TssM-TssL type 6 secretion membrane core complex
Components
  • Sample: Negative stain structure of the TssJ-TssM-TssL type 6 secretion membrane core complex
  • Protein or peptide: TssJ
  • Protein or peptide: TssM
  • Protein or peptide: TssL

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Supramolecule #1000: Negative stain structure of the TssJ-TssM-TssL type 6 secretion m...

SupramoleculeName: Negative stain structure of the TssJ-TssM-TssL type 6 secretion membrane core complex
type: sample / ID: 1000 / Oligomeric state: Decamer / Number unique components: 3
Molecular weightExperimental: 1.7 MDa / Theoretical: 1.7 MDa
Method: Relative stoichometry of the individual components is 1:1:1 for TssJ:TssM:TssL. Docking of the crystal structures of some parts of the complex indicates 10 copies of each protein.

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Macromolecule #1: TssJ

MacromoleculeName: TssJ / type: protein_or_peptide / ID: 1 / Name.synonym: T6SS_SciN / Number of copies: 10 / Oligomeric state: 1 / Recombinant expression: Yes
Source (natural)Organism: Escherichia coli (strain 55989 / EAEC) (bacteria) / Strain: strain 55989 / EAEC / synonym: Enteroaggregative Escherichia coli / Location in cell: outer membrane
Molecular weightExperimental: 18.4 KDa / Theoretical: 18.4 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant plasmid: pRSF-Duet
SequenceUniProtKB: Type VI secretion system lipoprotein TssJ / InterPro: Type VI secretion system, lipoprotein SciN

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Macromolecule #2: TssM

MacromoleculeName: TssM / type: protein_or_peptide / ID: 2 / Name.synonym: T6SS_SciS / Number of copies: 10 / Oligomeric state: 1 / Recombinant expression: Yes
Source (natural)Organism: Escherichia coli (strain 55989 / EAEC) (bacteria) / Strain: strain 55989 / EAEC / synonym: Enteroaggregative Escherichia coli / Location in cell: inner membrane
Molecular weightExperimental: 130 KDa / Theoretical: 130 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant plasmid: pRSF-Duet
SequenceUniProtKB: Type VI secretion protein
InterPro: Type VI secretion system IcmF, C-terminal, IcmF-related

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Macromolecule #3: TssL

MacromoleculeName: TssL / type: protein_or_peptide / ID: 3 / Name.synonym: T6SS_SciP / Number of copies: 10 / Oligomeric state: 1 / Recombinant expression: Yes
Source (natural)Organism: Escherichia coli (strain 55989 / EAEC) (bacteria) / Strain: strain 55989 / EAEC / synonym: Enteroaggregative Escherichia coli / Location in cell: inner membrane
Molecular weightExperimental: 24 KDa / Theoretical: 24 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant plasmid: pRSF-Duet
SequenceUniProtKB: Type VI secretion protein / InterPro: Type IV / VI secretion system, DotU

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.01 mg/mL
BufferpH: 8
Details: 50 mM Tris-HCl pH 8.0, 50 mM NaCl, 0.025% w/v Decyl Maltose Neopentyl Glycol (DM-NPG)
StainingType: NEGATIVE
Details: Nine microlitres of TssJLM complex sample was applied to glow-discharged carbon-coated copper grids (Agar Scientific). After 30 sec of absorption, the sample was blotted, washed with three ...Details: Nine microlitres of TssJLM complex sample was applied to glow-discharged carbon-coated copper grids (Agar Scientific). After 30 sec of absorption, the sample was blotted, washed with three drops of water and then stained with 2% uranyl acetate.
GridDetails: 400 mesh carbon-coated copper grids
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI F20
Alignment procedureLegacy - Astigmatism: Astigmatism was checked during imaging and during CTF correction of the micrographs. Micrographs is astigmatism >10% were discarded.
DateNov 20, 2014
Image recordingCategory: CCD / Film or detector model: FEI FALCON II (4k x 4k) / Number real images: 850 / Average electron dose: 20 e/Å2 / Bits/pixel: 16
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 68100 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.1 mm / Nominal defocus max: -2.5 µm / Nominal defocus min: -0.5 µm / Nominal magnification: 50000
Sample stageSpecimen holder model: OTHER
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

DetailsA total of 72,146 particles were automatically selected from 1,200 independent images and extracted within boxes of 280 pixels x 280 pixels using EMAN2/BOXER. The CTF was estimated and corrected by phase flipping using EMAN2 (e2ctf). All two- and three-dimensional (2D and 3D) classifications and refinements were performed using RELION 1.3 . We used three rounds of reference-free 2D class averaging to clean up the automatically selected dataset. Only highly populated classes displaying high-resolution features were conserved during this procedure and a final dataset of 26,544 particles was assembled. An initial 3D-model was generated in EMAN2 using using 30 classes. 3D classification was then performed in Relion with 5 classes. The particles corresponding to most populated class (~ 16,738) were used for refinement. Relion auto-refine procedure was used to obtain a final reconstruction at 11.56-A resolution after masking and with C5 symmetry imposed. Reported resolutions are based on the gold-standard Fourier shell correlation (FSC) 0.143 criterion, and FSC curve were corrected for the effects of a soft mask on the FSC curve using high-resolution noise substitution .
CTF correctionDetails: Each particle
Final reconstructionApplied symmetry - Point group: C5 (5 fold cyclic) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 11.5 Å / Resolution method: OTHER / Software - Name: EMAN2, Relion / Number images used: 16738
FSC plot (resolution estimation)

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