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- EMDB-3792: Electron cryotomogram of Vibrio cholerae O395 N1 -

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Basic information

Entry
Database: EMDB / ID: EMD-3792
TitleElectron cryotomogram of Vibrio cholerae O395 N1
Map data
Sample
  • Cell: Vibrio Cholerae
    • Cell: Vibrio Cholerae
    • Organelle or cellular component: Toxin-coregulated pilus machine
Biological speciesVibrio cholerae (bacteria)
Methodelectron tomography / cryo EM
AuthorsChang YW / Kjaer A / Ortega DR / Kovacikova G / Sutherland JA / Rettberg LA / Taylor RK / Jensen GJ
CitationJournal: Nat Microbiol / Year: 2017
Title: Architecture of the Vibrio cholerae toxin-coregulated pilus machine revealed by electron cryotomography.
Authors: Yi-Wei Chang / Andreas Kjær / Davi R Ortega / Gabriela Kovacikova / John A Sutherland / Lee A Rettberg / Ronald K Taylor / Grant J Jensen /
Abstract: Type IV pili (T4P) are filamentous appendages found on many Bacteria and Archaea. They are helical fibres of pilin proteins assembled by a multi-component macromolecular machine we call the basal ...Type IV pili (T4P) are filamentous appendages found on many Bacteria and Archaea. They are helical fibres of pilin proteins assembled by a multi-component macromolecular machine we call the basal body. Based on pilin features, T4P are classified into type IVa pili (T4aP) and type IVb pili (T4bP). T4aP are more widespread and are involved in cell motility, DNA transfer, host predation and electron transfer. T4bP are less prevalent and are mainly found in enteropathogenic bacteria, where they play key roles in host colonization. Following similar work on T4aP machines, here we use electron cryotomography to reveal the three-dimensional in situ structure of a T4bP machine in its piliated and non-piliated states. The specific machine we analyse is the Vibrio cholerae toxin-coregulated pilus machine (TCPM). Although only about half of the components of the TCPM show sequence homology to components of the previously analysed Myxococcus xanthus T4aP machine (T4aPM), we find that their structures are nevertheless remarkably similar. Based on homologies with components of the M. xanthus T4aPM and additional reconstructions of TCPM mutants in which the non-homologous proteins are individually deleted, we propose locations for all eight TCPM components within the complex. Non-homologous proteins in the T4aPM and TCPM are found to form similar structures, suggesting new hypotheses for their functions and evolutionary histories.
History
DepositionJul 10, 2017-
Header (metadata) releaseAug 16, 2017-
Map releaseAug 16, 2017-
UpdateAug 16, 2017-
Current statusAug 16, 2017Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Solid view (volume rendering)
  • Imaged by UCSF Chimera
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  • Solid view (volume rendering)
  • Imaged by UCSF Chimera
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  • Simplified surface model
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Structure viewerEM map:
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Supplemental images

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Map

FileDownload / File: emd_3792.map.gz / Format: CCP4 / Size: 1.3 GB / Type: IMAGE STORED AS SIGNED INTEGER (2 BYTES)
Voxel sizeX=Y=Z: 7.8 Å
Density
Minimum - Maximum0. - 32767.
Average (Standard dev.)22828.34400000000096 (±596.131960000000049)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin00800
Dimensions19181856800
Spacing18561918800
CellA: 14476.801 Å / B: 14960.4 Å / C: 6240.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Integer*27
Å/pix. X/Y/Z7.80000053879317.87.8
M x/y/z18561918800
origin x/y/z0.0000.0000.000
length x/y/z14476.80114960.4006240.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS00800
NC/NR/NS18561918800
D min/max/mean0.00032767.00022828.344

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Supplemental data

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Additional map: #1

Fileemd_3792_additional.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Sample components

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Entire : Vibrio Cholerae

EntireName: Vibrio Cholerae (bacteria)
Components
  • Cell: Vibrio Cholerae
    • Cell: Vibrio Cholerae
    • Organelle or cellular component: Toxin-coregulated pilus machine

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Supramolecule #1: Vibrio Cholerae

SupramoleculeName: Vibrio Cholerae / type: cell / ID: 1 / Parent: 0
Details: Bacteria which is the causative agent of the diarrhoeal disease cholera.

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Supramolecule #3: Vibrio Cholerae

SupramoleculeName: Vibrio Cholerae / type: cell / ID: 3 / Parent: 1
Details: Bacteria which is the causative agent of the diarrhoeal disease cholera.
Source (natural)Organism: Vibrio cholerae (bacteria) / Strain: Wild-type

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Supramolecule #2: Toxin-coregulated pilus machine

SupramoleculeName: Toxin-coregulated pilus machine / type: organelle_or_cellular_component / ID: 2 / Parent: 1
Details: Pilus - Bacteria which is the causative agent of the diarrhoeal disease cholera.
Source (natural)Organism: Vibrio cholerae (bacteria) / Strain: Wild-type
Recombinant expressionOrganism: Vibrio cholerae (bacteria) / Recombinant strain: Wild-type / Recombinant plasmid: pMT5-ToxT

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Experimental details

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Structure determination

Methodcryo EM
Processingelectron tomography
Aggregation statecell

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Sample preparation

BufferpH: 7
Component:
ConcentrationFormulaName
100.0 ug/ml^-lC16H19N3O4SAmpcillin
1.0 mMC9H18O5Sisopropyl-B-D-thiogalactoside

Details: V. cholerae O395 N1 strains (wild-type or TCPM component knockout mutants) harbouring the pMT5-ToxT plasmid were grown in 5 ml lysogeny broth (LB) medium.
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE-PROPANE
DetailsAfter 24 h of growth, the tube of V. cholerae culture was placed on a bench at room temperature for 1 min to sediment visible microcolonies. After the pellet was formed, the supernatant was gently removed by pipetting and the pellet was resuspended in fresh LB. The cells were incubated for 15 min prior to mixing with 10 nm colloidal gold.
SectioningOther: NO SECTIONING
Fiducial markerManufacturer: Sigma-Aldrich / Diameter: 10 nm

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Electron microscopy

MicroscopeFEI POLARA 300
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 6.0 µm / Nominal defocus min: 6.0 µm / Nominal magnification: 27500
Specialist opticsEnergy filter - Name: GIF
Sample stageSpecimen holder model: OTHER
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Digitization - Dimensions - Width: 1856 pixel / Digitization - Dimensions - Height: 1918 pixel / Number grids imaged: 1 / Average electron dose: 1.5 e/Å2
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: IMOD
Final reconstructionSoftware - Name: TOMO3D / Software - details: SIRT reconstructions were then produced / Number images used: 121

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