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- EMDB-5739: Electron microscopy map of the T6SS TssK component -

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Basic information

Entry
Database: EMDB / ID: EMD-5739
TitleElectron microscopy map of the T6SS TssK component
Map dataEM Reconstruction of TssK
Sample
  • Sample: Purified TssK sample
  • Protein or peptide: Type VI secretion system protein TssK
KeywordsTssK / Type VI secretion / Hcp / sheath assembly / EM / single-particle / three-fold
Function / homologycellular_component / biological_process / molecular_function / Bacterial Type VI secretion, VC_A0110, EvfL, ImpJ, VasE / Type VI secretion system TssK / Type VI secretion system TssK / Type VI secretion system protein TssK
Function and homology information
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / negative staining / Resolution: 26.0 Å
AuthorsZoued A / Durand E / Bebeacua C / Brunet YR / Douzi B / Cambillau C / Cascales E / Journet L
CitationJournal: J Biol Chem / Year: 2013
Title: TssK is a trimeric cytoplasmic protein interacting with components of both phage-like and membrane anchoring complexes of the type VI secretion system.
Authors: Abdelrahim Zoued / Eric Durand / Cecilia Bebeacua / Yannick R Brunet / Badreddine Douzi / Christian Cambillau / Eric Cascales / Laure Journet /
Abstract: The Type VI secretion system (T6SS) is a macromolecular machine that mediates bacteria-host or bacteria-bacteria interactions. The T6SS core apparatus assembles from 13 proteins that form two sub- ...The Type VI secretion system (T6SS) is a macromolecular machine that mediates bacteria-host or bacteria-bacteria interactions. The T6SS core apparatus assembles from 13 proteins that form two sub-assemblies: a phage-like complex and a trans-envelope complex. The Hcp, VgrG, TssE, and TssB/C subunits are structurally and functionally related to components of the tail of contractile bacteriophages. This phage-like structure is thought to be anchored to the membrane by a trans-envelope complex composed of the TssJ, TssL, and TssM proteins. However, how the two sub-complexes are connected remains unknown. Here we identify TssK, a protein that establishes contacts with the two T6SS sub-complexes through direct interactions with TssL, Hcp, and TssC. TssK is a cytoplasmic protein assembling trimers that display a three-armed shape, as revealed by TEM and SAXS analyses. Fluorescence microscopy experiments further demonstrate the requirement of TssK for sheath assembly. Our results suggest a central role for TssK by linking both complexes during T6SS assembly.
History
DepositionAug 9, 2013-
Header (metadata) releaseSep 11, 2013-
Map releaseSep 11, 2013-
UpdateOct 9, 2013-
Current statusOct 9, 2013Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.04
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.04
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5739.png

Downloads & links

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Map

FileDownload / File: emd_5739.map.gz / Format: CCP4 / Size: 1.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationEM Reconstruction of TssK
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
3.5 Å/pix.
x 72 pix.
= 252. Å		3.5 Å/pix.
x 72 pix.
= 252. Å		3.5 Å/pix.
x 72 pix.
= 252. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 3.5 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.04 / Movie #1: 0.04
Minimum - Maximum0.0 - 0.13164011
Average (Standard dev.)0.00123267 (±0.00891095)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-36-36-36
Dimensions727272
Spacing727272
CellA=B=C: 252.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.53.53.5
M x/y/z727272
origin x/y/z0.0000.0000.000
length x/y/z252.000252.000252.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-132-122-147
NX/NY/NZ250274261
MAP C/R/S123
start NC/NR/NS-36-36-36
NC/NR/NS727272
D min/max/mean-0.0000.1320.001

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Supplemental data

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Sample components

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Entire : Purified TssK sample

EntireName: Purified TssK sample
Components
  • Sample: Purified TssK sample
  • Protein or peptide: Type VI secretion system protein TssK

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Supramolecule #1000: Purified TssK sample

SupramoleculeName: Purified TssK sample / type: sample / ID: 1000 / Details: The sample was monodisperse. / Oligomeric state: 3 / Number unique components: 1
Molecular weightExperimental: 160 KDa / Method: Analytical size exclusion chromatography analysis

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Macromolecule #1: Type VI secretion system protein TssK

MacromoleculeName: Type VI secretion system protein TssK / type: protein_or_peptide / ID: 1 / Name.synonym: TssK / Number of copies: 1 / Oligomeric state: Trimer / Recombinant expression: Yes
Source (natural)Organism: Escherichia coli (E. coli) / Strain: 42
Molecular weightExperimental: 160 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: T7 Iq pLys / Recombinant plasmid: pETG20-TssK1
SequenceUniProtKB: Type VI secretion system protein TssK
GO: biological_process, molecular_function, cellular_component
InterPro: Type VI secretion system TssK

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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

Concentration0.05 mg/mL
BufferpH: 8
Details: HBS-EP buffer (10mM HEPES, 150mM NaCl, 3mM EDTA, 0.005% Polysorbate 20)
StainingType: NEGATIVE
Details: Purified protein was immobilized on a glow- discharged carbon grid by incubation for 1 minute, and then stained with 2% uranyl acetate for 10 seconds.
GridDetails: 300 mesh copper grid with thin carbon support, glow discharged for 20 seconds
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI SPIRIT
DateNov 1, 2012
Image recordingCategory: CCD / Film or detector model: GENERIC CCD / Number real images: 10 / Average electron dose: 10 e/Å2 / Details: CCD images
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 2.0 µm / Nominal magnification: 60000
Sample stageSpecimen holder: Room temperature holder / Specimen holder model: SIDE ENTRY, EUCENTRIC
Experimental equipment
Model: Tecnai Spirit / Image courtesy: FEI Company

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Image processing

DetailsThe particles were submitted to maximum likelihood classification and alignment.
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 26.0 Å / Resolution method: OTHER / Software - Name: EMAN, SPIDER, XMIPP / Number images used: 5000
Final angle assignmentDetails: ML
Final two d classificationNumber classes: 500

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