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- EMDB-3812: Tomogram of e. coli carrying the ple7 plasmid carrying YFP-MreB h... -

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Basic information

Entry
Database: EMDB / ID: EMD-3812
TitleTomogram of e. coli carrying the ple7 plasmid carrying YFP-MreB hyper-overexpressed by induction with 1uM IPTG
Map data
Sample
  • Cell: Escherichia Coli
    • Organelle or cellular component: Helical MreB Cytoskeleton
Biological speciesEscherichia coli (E. coli)
Methodelectron tomography
AuthorsSwulius MT / Jensen GJ
CitationJournal: J Bacteriol / Year: 2012
Title: The helical MreB cytoskeleton in Escherichia coli MC1000/pLE7 is an artifact of the N-Terminal yellow fluorescent protein tag.
Authors: Matthew T Swulius / Grant J Jensen /
Abstract: Based on fluorescence microscopy, the actin homolog MreB has been thought to form extended helices surrounding the cytoplasm of rod-shaped bacterial cells. The presence of these and other putative ...Based on fluorescence microscopy, the actin homolog MreB has been thought to form extended helices surrounding the cytoplasm of rod-shaped bacterial cells. The presence of these and other putative helices has come to dominate models of bacterial cell shape regulation, chromosome segregation, polarity, and motility. Here we use electron cryotomography to show that MreB does in fact form extended helices and filaments in Escherichia coli when yellow fluorescent protein (YFP) is fused to its N terminus but native (untagged) MreB expressed to the same levels does not. In contrast, mCherry fused to an internal loop (MreB-RFP(SW)) does not induce helices. The helices are therefore an artifact of the placement of the fluorescent protein tag. YFP-MreB helices were also clearly distinguishable from the punctate, "patchy" localization patterns of MreB-RFP(SW), even by standard light microscopy. The many interpretations in the literature of such punctate patterns as helices should therefore be reconsidered.
History
DepositionJul 14, 2017-
Header (metadata) releaseAug 30, 2017-
Map releaseAug 30, 2017-
UpdateAug 30, 2017-
Current statusAug 30, 2017Processing site: PDBe / Status: Released

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Structure visualization

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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_3812.png

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Map

FileDownload / File: emd_3812.map.gz / Format: CCP4 / Size: 984.4 MB / Type: IMAGE STORED AS SIGNED BYTE
Voxel sizeX=Y=Z: 9.46 Å
Density
Minimum - Maximum-128. - 127.
Average (Standard dev.)29.99577 (±2.205534)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions101610161000
Spacing101610161000
CellA: 9611.36 Å / B: 9611.36 Å / C: 9460.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeenvelope stored as signed bytes (from -128 lowest to 127 highest)
Å/pix. X/Y/Z9.469.469.46
M x/y/z101610161000
origin x/y/z0.0000.0000.000
length x/y/z9611.3609611.3609460.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS101610161000
D min/max/mean-128.000127.00029.996

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Supplemental data

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Additional map: #1

Fileemd_3812_additional_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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Additional map: #2

Fileemd_3812_additional_2.map
Projections & Slices
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Additional map: #3

Fileemd_3812_additional_3.map
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Additional map: #4

Fileemd_3812_additional_4.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Sample components

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Entire : Escherichia Coli

EntireName: Escherichia Coli (E. coli)
Components
  • Cell: Escherichia Coli
    • Organelle or cellular component: Helical MreB Cytoskeleton

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Supramolecule #1: Escherichia Coli

SupramoleculeName: Escherichia Coli / type: cell / ID: 1 / Parent: 0
Source (natural)Organism: Escherichia coli (E. coli)

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Supramolecule #2: Helical MreB Cytoskeleton

SupramoleculeName: Helical MreB Cytoskeleton / type: organelle_or_cellular_component / ID: 2 / Parent: 1
Source (natural)Organism: Escherichia coli (E. coli)

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Experimental details

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Structure determination

Processingelectron tomography
Aggregation statecell

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Sample preparation

BufferpH: 7 / Component - Concentration: 50.0 ug/ml / Component - Formula: C16H19N3O4S / Component - Name: Ampicillin
Details: MC1000, MC1000/pLE6, and MC1000/pLE7 were grown in LB at 37C with 50ug/ml ampicillin when appropriate.
GridModel: Quantifoil R2/2 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
SectioningOther: NO SECTIONING

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Electron microscopy

MicroscopeFEI POLARA 300
TemperatureMax: 123.15 K
Specialist opticsEnergy filter - Name: FEI
Image recordingFilm or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Digitization - Dimensions - Width: 1016 pixel / Digitization - Dimensions - Height: 1016 pixel / Average electron dose: 0.4 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

Final reconstructionSoftware - Name: IMOD
Software - details: Tomograms were reconstructed and modelled
Number images used: 501

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