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- EMDB-3282: Negative stain electron microscopy structure of TssA from EAEC ty... -

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Basic information

Entry
Database: EMDB / ID: EMD-3282
TitleNegative stain electron microscopy structure of TssA from EAEC type VI secretion system
Map data3D Reconstruction of TssA dodecamer
Sample
  • Sample: TssA dodecamer
  • Protein or peptide: TssA
KeywordsType VI protein secretion system / bacterial contractile tail structure
Biological speciesEscherichia coli 042 (bacteria)
Methodsingle particle reconstruction / negative staining / Resolution: 19.0 Å
AuthorsDurand E / Fronzes R / Cambillau C / Cascales E
CitationJournal: Nature / Year: 2016
Title: Priming and polymerization of a bacterial contractile tail structure.
Authors: Abdelrahim Zoued / Eric Durand / Yannick R Brunet / Silvia Spinelli / Badreddine Douzi / Mathilde Guzzo / Nicolas Flaugnatti / Pierre Legrand / Laure Journet / Rémi Fronzes / Tâm Mignot / ...Authors: Abdelrahim Zoued / Eric Durand / Yannick R Brunet / Silvia Spinelli / Badreddine Douzi / Mathilde Guzzo / Nicolas Flaugnatti / Pierre Legrand / Laure Journet / Rémi Fronzes / Tâm Mignot / Christian Cambillau / Eric Cascales /
Abstract: Contractile tails are composed of an inner tube wrapped by an outer sheath assembled in an extended, metastable conformation that stores mechanical energy necessary for its contraction. Contraction ...Contractile tails are composed of an inner tube wrapped by an outer sheath assembled in an extended, metastable conformation that stores mechanical energy necessary for its contraction. Contraction is used to propel the rigid inner tube towards target cells for DNA or toxin delivery. Although recent studies have revealed the structure of the contractile sheath of the type VI secretion system, the mechanisms by which its polymerization is controlled and coordinated with the assembly of the inner tube remain unknown. Here we show that the starfish-like TssA dodecameric complex interacts with tube and sheath components. Fluorescence microscopy experiments in enteroaggregative Escherichia coli reveal that TssA binds first to the type VI secretion system membrane core complex and then initiates tail polymerization. TssA remains at the tip of the growing structure and incorporates new tube and sheath blocks. On the basis of these results, we propose that TssA primes and coordinates tail tube and sheath biogenesis.
History
DepositionDec 15, 2015-
Header (metadata) releaseDec 23, 2015-
Map releaseFeb 24, 2016-
UpdateMar 16, 2016-
Current statusMar 16, 2016Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0175
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.0175
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-3282_Tss...

Downloads & links

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Map

FileDownload / File: emd_3282.map.gz / Format: CCP4 / Size: 29.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation3D Reconstruction of TssA dodecamer
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.9 Å/pix.
x 200 pix.
= 380. Å		1.9 Å/pix.
x 200 pix.
= 380. Å		1.9 Å/pix.
x 200 pix.
= 380. 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: 0.0175 / Movie #1: 0.0175
Minimum - Maximum-0.35052326 - 0.19176249
Average (Standard dev.)0.00028877 (±0.00857623)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 380.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.91.91.9
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z380.000380.000380.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-300-64
NX/NY/NZ6161129
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS200200200
D min/max/mean-0.3510.1920.000

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Supplemental data

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Sample components

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Entire : TssA dodecamer

EntireName: TssA dodecamer
Components
  • Sample: TssA dodecamer
  • Protein or peptide: TssA

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Supramolecule #1000: TssA dodecamer

SupramoleculeName: TssA dodecamer / type: sample / ID: 1000 / Details: The sample was mono disperse / Oligomeric state: 12 / Number unique components: 1
Molecular weightExperimental: 952 MDa / Theoretical: 891 MDa
Method: on-line multi-angle laser light scattering/quasi-elastic light scattering/absorbance/refractive index (MALS/QELS/UV/RI)

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Macromolecule #1: TssA

MacromoleculeName: TssA / type: protein_or_peptide / ID: 1 / Name.synonym: TssA / Number of copies: 12 / Oligomeric state: Dodecamer / Recombinant expression: Yes
Source (natural)Organism: Escherichia coli 042 (bacteria) / Strain: strain 042 / synonym: Enteroaggregative E. coli / Location in cell: Cytoplasm
Molecular weightExperimental: 74 KDa / Theoretical: 74 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: Escherichia coli BL21 / Recombinant plasmid: pIBA3C

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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: 20mM Tris-HCL, 150mM NaCl
StainingType: NEGATIVE
Details: TssA 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 ...Details: TssA 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 glow-discharged carbon-coated copper grids (Agar Scientific)
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI F20
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 100,000 times magnification
Specialist opticsEnergy filter - Name: FEI
DateJun 10, 2015
Image recordingCategory: CCD / Film or detector model: FEI FALCON II (4k x 4k) / Number real images: 500 / Average electron dose: 14 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Cs: 2.10 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 100000 particles were automatically selected from 500 independent images and extracted within boxes of 180 pixels by 180 pixels using EMAN2/BOXER. The CTF was estimated and corrected by phase flipping using EMAN2. All two- and three-dimensional 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 20000 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 were used for refinement. Relion auto-refine procedure was used to obtain a final reconstruction at 19A resolution after masking and with D6 symmetry imposed. Reported resolutions are based on the gold-standard Fourier shell correlation 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: D6 (2x6 fold dihedral) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 19.0 Å / Resolution method: OTHER / Software - Name: EMAN2, RELION / Number images used: 18000
Final two d classificationNumber classes: 10

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