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Open data
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Basic information
Entry | Database: EMDB / ID: EMD-2850 | |||||||||
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Title | actin-like ParM protein bound to AMPPNP | |||||||||
![]() | actin-like ParM protein bound to AMPPNP | |||||||||
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![]() | bacterial cytoskeleton / plasmid segregation / actin-like protein | |||||||||
Function / homology | Plasmid 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![]() | |||||||||
Biological species | ![]() ![]() | |||||||||
Method | helical reconstruction / cryo EM / Resolution: 4.3 Å | |||||||||
![]() | Bharat TAM / Murshudov GN / Sachse C / Lowe J | |||||||||
![]() | ![]() 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 |
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Structure visualization
Movie |
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Structure viewer | EM map: ![]() ![]() ![]() |
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 6.1 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 9.9 KB 9.9 KB | Display Display | ![]() |
Images | ![]() | 39.6 KB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 5aeyMC ![]() 2848C ![]() 2849C ![]() 5ai7C M: atomic model generated by this map C: citing same article ( |
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Similar structure data |
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Links
EMDB pages | ![]() ![]() |
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Map
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Annotation | actin-like ParM protein bound to AMPPNP | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.07 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
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Sample components
-Entire : ParM bound to AMPPNP
Entire | Name: ParM bound to AMPPNP |
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Components |
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-Supramolecule #1000: ParM bound to AMPPNP
Supramolecule | Name: ParM bound to AMPPNP / type: sample / ID: 1000 / Details: ParM was incubated with AMPPNP / Oligomeric state: filamentous / Number unique components: 1 |
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-Macromolecule #1: ParM
Macromolecule | Name: ParM / type: protein_or_peptide / ID: 1 / Details: ParM was incubated with AMPPNP / Oligomeric state: Filamentous / Recombinant expression: Yes |
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Source (natural) | Organism: ![]() ![]() |
Molecular weight | Experimental: 36 KDa / Theoretical: 36 KDa |
Recombinant expression | Organism: ![]() ![]() |
Sequence | UniProtKB: Plasmid segregation protein ParM |
-Experimental details
-Structure determination
Method | cryo EM |
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![]() | helical reconstruction |
Aggregation state | filament |
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Sample preparation
Concentration | 0.3 mg/mL |
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Buffer | pH: 7 / Details: 50 mM Tris-HCl, 100 mM KCl, and 1 mM MgCl2 |
Grid | Details: 200 mesh copper / rhodium grid with carbon support |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV |
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Electron microscopy
Microscope | FEI TITAN KRIOS |
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Date | Feb 2, 2014 |
Image recording | Category: CCD / Film or detector model: FEI FALCON II (4k x 4k) / Average electron dose: 28 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: ![]() |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: -4.0 µm / Nominal defocus min: -1.0 µm |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |
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Image processing
Details | CTF determination was conducted using CTFFIND. Data was extracted using EMAN2, and processed using SPRING. |
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Final reconstruction | Applied symmetry - Helical parameters - Δz: 24.7 Å Applied symmetry - Helical parameters - Δ&Phi: 165.1 ° Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric) Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 4.3 Å / Resolution method: OTHER / Software - Name: CTFFIND, EMAN2, SPRING |
-Atomic model buiding 1
Initial model | PDB ID: Chain - Chain ID: A |
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Software | Name: REFMAC, COOT, MAIN |
Details | PDB co-ordinates from PDB ID 4A62 were used as a starting point. Alternate rounds of model building and all-atom refinements with stereochemical restrainsts were carried out in REFMAC. |
Refinement | Space: RECIPROCAL / Protocol: FLEXIBLE FIT / Target criteria: Maximum-likelihood |
Output model | ![]() PDB-5aey: |