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- EMDB-2849: E coli cell with plasmid containing the ParMRC locus -

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Basic information

Entry
Database: EMDB / ID: EMD-2849
TitleE coli cell with plasmid containing the ParMRC locus
Map dataE coli cell with plasmid containing the ParMRC locus
Sample
  • Sample: E coli cell with plasmid containing medium copy number plasmid with ParMRC locus
  • Organelle or cellular component: Escherichia coli cytoskeleton
Keywordsbacterial cytoskeleton / plasmid segregation / actin-like protein
Biological speciesEscherichia coli (E. coli)
Methodelectron tomography / cryo EM
AuthorsBharat TAM / Murshudov GN / Sachse C / Lowe J
CitationJournal: Nature / Year: 2015
Title: Structures of actin-like ParM filaments show architecture of plasmid-segregating spindles.
Authors: Tanmay A M Bharat / Garib N Murshudov / Carsten Sachse / Jan Löwe /
Abstract: Active segregation of Escherichia coli low-copy-number plasmid R1 involves formation of a bipolar spindle made of left-handed double-helical actin-like ParM filaments. ParR links the filaments with ...Active segregation of Escherichia coli low-copy-number plasmid R1 involves formation of a bipolar spindle made of left-handed double-helical actin-like ParM filaments. ParR links the filaments with centromeric parC plasmid DNA, while facilitating the addition of subunits to ParM filaments. Growing ParMRC spindles push sister plasmids to the cell poles. Here, using modern electron cryomicroscopy methods, we investigate the structures and arrangements of ParM filaments in vitro and in cells, revealing at near-atomic resolution how subunits and filaments come together to produce the simplest known mitotic machinery. To understand the mechanism of dynamic instability, we determine structures of ParM filaments in different nucleotide states. The structure of filaments bound to the ATP analogue AMPPNP is determined at 4.3 Å resolution and refined. The ParM filament structure shows strong longitudinal interfaces and weaker lateral interactions. Also using electron cryomicroscopy, we reconstruct ParM doublets forming antiparallel spindles. Finally, with whole-cell electron cryotomography, we show that doublets are abundant in bacterial cells containing low-copy-number plasmids with the ParMRC locus, leading to an asynchronous model of R1 plasmid segregation.
History
DepositionJan 12, 2015-
Header (metadata) releaseFeb 11, 2015-
Map releaseMay 6, 2015-
UpdateJul 1, 2015-
Current statusJul 1, 2015Processing site: PDBe / Status: Released

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Structure visualization

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Structure viewerEM map:
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Supplemental images

emd-2849.png

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Map

FileDownload / File: emd_2849.map.gz / Format: CCP4 / Size: 665.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationE coli cell with plasmid containing the ParMRC locus
Voxel sizeX=Y=Z: 17.8 Å
Density
Contour LevelBy EMDB: 0.422
Minimum - Maximum-15.260960580000001 - 9.1611557
Average (Standard dev.)0.1681698 (±0.30318704)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin00455
Dimensions959927201
Spacing959927201
CellA: 16500.6 Å / B: 17070.2 Å / C: 3577.7998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z17.817.79999895724717.8
M x/y/z927959201
origin x/y/z0.0000.0000.000
length x/y/z16500.60017070.1993577.800
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS00455
NC/NR/NS927959201
D min/max/mean-15.2619.1610.168

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Supplemental data

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Sample components

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Entire : E coli cell with plasmid containing medium copy number plasmid wi...

EntireName: E coli cell with plasmid containing medium copy number plasmid with ParMRC locus
Components
  • Sample: E coli cell with plasmid containing medium copy number plasmid with ParMRC locus
  • Organelle or cellular component: Escherichia coli cytoskeleton

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Supramolecule #1000: E coli cell with plasmid containing medium copy number plasmid wi...

SupramoleculeName: E coli cell with plasmid containing medium copy number plasmid with ParMRC locus
type: sample / ID: 1000 / Number unique components: 1

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Supramolecule #1: Escherichia coli cytoskeleton

SupramoleculeName: Escherichia coli cytoskeleton / type: organelle_or_cellular_component / ID: 1 / Details: 200 mesh copper / rhodium grid with carbon support / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Escherichia coli (E. coli)

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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: 200 mesh copper / rhodium grid with carbon support
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: -8.0 µm / Nominal defocus min: -8.0 µm
Specialist opticsEnergy filter - Name: Gatan Quantum Energy Filter / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 20.0 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Tilt series - Axis1 - Angle increment: 1 °
DateJun 1, 2014
Image recordingCategory: CCD / Film or detector model: GATAN K2 (4k x 4k) / Number real images: 121 / Average electron dose: 120 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionAlgorithm: OTHER / Software - Name: IMOD, Tomo3D / Number images used: 121
DetailsTilt series was aligned using IMOD and reconstructed using Tomo3D

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