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- EMDB-2848: actin-like ParM protein bound to ADP -

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Basic information

Entry
Database: EMDB / ID: EMD-2848
Titleactin-like ParM protein bound to ADP
Map dataactin-like ParM protein bound to ADP
Sample
  • Sample: ParM bound to ADP
  • Protein or peptide: ParM
Keywordsbacterial cytoskeleton / plasmid segregation / actin-like protein
Function / homologyPlasmid segregation protein ParM/StbA / : / Plasmid segregation protein ParM, N-terminal / Plasmid segregation protein ParM, C-terminal / ParM-like / plasmid partitioning / ATPase, nucleotide binding domain / identical protein binding / Plasmid segregation protein ParM
Function and homology information
Biological speciesEscherichia coli (E. coli)
Methodhelical reconstruction / cryo EM / Resolution: 11.0 Å
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-
UpdateApr 27, 2016-
Current statusApr 27, 2016Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.3
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 1.3
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-2848-par...

Downloads & links

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Map

FileDownload / File: emd_2848.map.gz / Format: CCP4 / Size: 733.4 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationactin-like ParM protein bound to ADP
Voxel sizeX=Y=Z: 2.6 Å
Density
Contour LevelBy AUTHOR: 1.3 / Movie #1: 1.3
Minimum - Maximum-2.32683754 - 5.49724817
Average (Standard dev.)0.0 (±0.99999738)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-20-20-60
Dimensions4040120
Spacing4040120
CellA: 104.0 Å / B: 104.0 Å / C: 312.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.62.62.6
M x/y/z4040120
origin x/y/z0.0000.0000.000
length x/y/z104.000104.000312.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-20-20-60
NC/NR/NS4040120
D min/max/mean-2.3275.4970.000

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Supplemental data

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Sample components

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Entire : ParM bound to ADP

EntireName: ParM bound to ADP
Components
  • Sample: ParM bound to ADP
  • Protein or peptide: ParM

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Supramolecule #1000: ParM bound to ADP

SupramoleculeName: ParM bound to ADP / type: sample / ID: 1000 / Details: ParM was incubated with ADP / Oligomeric state: filamentous / Number unique components: 1

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Macromolecule #1: ParM

MacromoleculeName: ParM / type: protein_or_peptide / ID: 1 / Details: ParM was incubated with ADP / Oligomeric state: Filamentous / Recombinant expression: Yes
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightExperimental: 36 KDa / Theoretical: 36 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria) / Recombinant cell: AI
SequenceUniProtKB: Plasmid segregation protein ParM

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

Concentration14.4 mg/mL
BufferpH: 7 / Details: 50 mM Tris-HCl, 100 mM KCl, and 1 mM MgCl2
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: -6.0 µm / Nominal defocus min: -2.0 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
DateJun 1, 2014
Image recordingCategory: CCD / Film or detector model: FEI FALCON II (4k x 4k) / Average electron dose: 25 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Helical parameters - Δz: 23.4 Å
Applied symmetry - Helical parameters - Δ&Phi: 165.1 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 11.0 Å / Resolution method: OTHER / Software - Name: CTFFIND, EMAN2, SPRING
DetailsCTF determination was conducted using CTFFIND. Data was extracted using EMAN2, and processed using SPRING.

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