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- EMDB-2754: Electron cryo-tomography of Campylobacter jejuni -

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Basic information

Entry
Database: EMDB / ID: EMD-2754
TitleElectron cryo-tomography of Campylobacter jejuni
Map dataElectron cryo-tomogram of two Campylobacter jejuni cells
Sample
  • Sample: Campylobacter jejuni subsp. jejuni ATCC 29428 (whole bacterial cells)
  • Organelle or cellular component: Campylobacter jejuni
Keywordsfood poisoning / cryo electron tomography / cryo-EM / chemoreceptors / acidocalcisomes
Biological speciesCampylobacter jejuni (Campylobacter)
Methodelectron tomography / cryo EM
AuthorsMuller A / Beeby M / McDowall AW / Chow J / Jensen GJ / Clemons WM
CitationJournal: Microbiologyopen / Year: 2014
Title: Ultrastructure and complex polar architecture of the human pathogen Campylobacter jejuni.
Authors: Axel Müller / Morgan Beeby / Alasdair W McDowall / Janet Chow / Grant J Jensen / William M Clemons /
Abstract: Campylobacter jejuni is one of the most successful food-borne human pathogens. Here we use electron cryotomography to explore the ultrastructure of C. jejuni cells in logarithmically growing cultures. ...Campylobacter jejuni is one of the most successful food-borne human pathogens. Here we use electron cryotomography to explore the ultrastructure of C. jejuni cells in logarithmically growing cultures. This provides the first look at this pathogen in a near-native state at macromolecular resolution (~5 nm). We find a surprisingly complex polar architecture that includes ribosome exclusion zones, polyphosphate storage granules, extensive collar-shaped chemoreceptor arrays, and elaborate flagellar motors.
History
DepositionAug 12, 2014-
Header (metadata) releaseSep 10, 2014-
Map releaseSep 10, 2014-
UpdateOct 22, 2014-
Current statusOct 22, 2014Processing 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

EMD-2754.png

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Map

FileDownload / File: emd_2754.map.gz / Format: CCP4 / Size: 390.6 MB / Type: IMAGE STORED AS SIGNED BYTE
AnnotationElectron cryo-tomogram of two Campylobacter jejuni cells
Voxel sizeX=Y=Z: 19.24 Å
Density
Minimum - Maximum-128.0 - 127.0
Average (Standard dev.)7.5194006 (±3.19345403)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin00400
Dimensions10241024400
Spacing10241024400
CellA: 19701.76 Å / B: 19701.76 Å / C: 7696.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeenvelope stored as signed bytes (from -128 lowest to 127 highest)
Å/pix. X/Y/Z19.2419.2419.24
M x/y/z10241024400
origin x/y/z0.0000.0000.000
length x/y/z19701.76019701.7607696.000
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ442626
MAP C/R/S123
start NC/NR/NS00400
NC/NR/NS10241024400
D min/max/mean-128.000127.0007.519

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Supplemental data

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Sample components

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Entire : Campylobacter jejuni subsp. jejuni ATCC 29428 (whole bacterial cells)

EntireName: Campylobacter jejuni subsp. jejuni ATCC 29428 (whole bacterial cells)
Components
  • Sample: Campylobacter jejuni subsp. jejuni ATCC 29428 (whole bacterial cells)
  • Organelle or cellular component: Campylobacter jejuni

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Supramolecule #1000: Campylobacter jejuni subsp. jejuni ATCC 29428 (whole bacterial cells)

SupramoleculeName: Campylobacter jejuni subsp. jejuni ATCC 29428 (whole bacterial cells)
type: sample / ID: 1000 / Number unique components: 1

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Supramolecule #1: Campylobacter jejuni

SupramoleculeName: Campylobacter jejuni / type: organelle_or_cellular_component / ID: 1 / Number of copies: 2 / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Campylobacter jejuni (Campylobacter) / Strain: ATCC 29428 / synonym: Campylobacter jejuni

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Experimental details

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Structure determination

Methodcryo EM
Processingelectron tomography
Aggregation statecell

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Sample preparation

GridDetails: Quantifoil
VitrificationCryogen name: ETHANE-PROPANE MIXTURE / Chamber humidity: 100 % / Chamber temperature: 77 K / Instrument: FEI VITROBOT MARK III
Method: Sample preparation closely followed established procedures (Iancu et al., 2007). In brief: 4 microlitres of a 10 nm colloidal gold (Sigma, USA) in 5% BSA was added to 16 microlitres of a C. ...Method: Sample preparation closely followed established procedures (Iancu et al., 2007). In brief: 4 microlitres of a 10 nm colloidal gold (Sigma, USA) in 5% BSA was added to 16 microlitres of a C. jejuni culture that had been allowed to grow to an optical density of 0.5. 3 microlitres of this mix were then placed onto a glow discharged carbon-coated R 2/2 Quantifoil grid in a Vitrobot (FEI Company, Hillsboro, OR, USA). Prior to this a 10 nm colloidal gold suspension in 5% BSA solution was added to the Quantifoil grid and allowed to dry. The temperature in the Vitrobot chamber was kept at 22 degrees Celsius with 100% humidity. Placing the sample onto the grid was followed by a 1 second blot with an offset of -1.5 degrees, a drain time of 1 second, and plunge-frozen in a mixture of liquid ethane (63%) and propane (37%). The frozen grids were than stored in liquid nitrogen until further use.

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Electron microscopy

MicroscopeFEI POLARA 300
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.2 mm / Nominal defocus max: -12.0 µm / Nominal magnification: 22500
Specialist opticsEnergy filter - Name: GATAN GIF / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 20.0 eV
Sample stageSpecimen holder: Liquid nitrogen cooled / Specimen holder model: GATAN HELIUM / Tilt series - Axis1 - Min angle: -60 ° / Tilt series - Axis1 - Max angle: 60 ° / Tilt series - Axis1 - Angle increment: 1 °
TemperatureMin: 76.9 K / Max: 77.1 K / Average: 77 K
DetailsCamera post-energy filter
DateJan 1, 2014
Image recordingNumber real images: 121 / Average electron dose: 200 e/Å2 / Bits/pixel: 16
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

Final reconstructionAlgorithm: OTHER / Software - Name: IMOD, tomo3d
Details: SIRT reconstruction of a fine-aligned stack low-pass filtered to first zero at approximately 5.5 nanometres.
Number images used: 121
DetailsSingle-axis tilt series from -60 degrees to 60 degrees with images were collected in 1 degree increments and an under-focus of 12 microns using a 300 keV FEI Polara FEG TEM controlled by Leginon software (Suloway et al., 2009) and the cumulative dose was not allowed to exceed 200 e A2. The images were recorded on a 4096 x 4096 pixel Ultracam (Gatan, Pleasanton, CA, USA) at a magnification of 22500 (0.98 nm/pixel). The IMOD software package (Kremer et al., 1996) was used to calculate 3D reconstructions.

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