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Structures of actin-like ParM filaments show architecture of plasmid-segregating spindles.

Bharat TA, Murshudov GN, Sachse C, Löwe J - Nature (2015)

Bottom Line: Growing ParMRC spindles push sister plasmids to the cell poles.The ParM filament structure shows strong longitudinal interfaces and weaker lateral interactions.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.

View Article: PubMed Central - PubMed

Affiliation: Structural Studies Division, MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK.

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 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.

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4.3 Å cryo-EM reconstruction of ParM+AMPPNP filaments(a) Cryo-EM image of ParM+AMPPNP filaments. Inset: class average. This experiment was repeated nine times. (b) A 4.3 Å reconstruction of the filaments, isosurface contoured at 2 σ away from the mean (see ED Fig. 1 and Video 1). (c) The same reconstruction as b), overlaid with the refined atomic model with individual ParM subunits coloured differently. (d-g) Enlarged regions of the cryo-EM map showing resolved secondary structure elements and side chain densities, contoured at 1 σ. (h) Density for the nucleotide is stronger than that of the protein (contoured at 3 σ).
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Figure 1: 4.3 Å cryo-EM reconstruction of ParM+AMPPNP filaments(a) Cryo-EM image of ParM+AMPPNP filaments. Inset: class average. This experiment was repeated nine times. (b) A 4.3 Å reconstruction of the filaments, isosurface contoured at 2 σ away from the mean (see ED Fig. 1 and Video 1). (c) The same reconstruction as b), overlaid with the refined atomic model with individual ParM subunits coloured differently. (d-g) Enlarged regions of the cryo-EM map showing resolved secondary structure elements and side chain densities, contoured at 1 σ. (h) Density for the nucleotide is stronger than that of the protein (contoured at 3 σ).

Mentions: Using electron cryomicroscopic (cryo-EM) images collected on a direct-electron detector, we performed real-space helical reconstruction to elucidate a 4.3 Å structure of ParM filaments assembled with the adenosine triphosphate (ATP) analogue AMPPNP (Fig. 1a-c, ED Fig. 1, ED Table 1, Video 1). Densities corresponding to alpha helices, beta strands and many side-chains were clearly observed (Fig. 1d-g). The nucleotide AMPPNP was also observed in our map as strong density, especially on the phosphates (Fig. 1h). No significant resolution anisotropy was detected in the reconstruction (ED Fig. 1), indicating that the entire ParM protein is rigidly held in the filament. To derive an atomic model of the ParM filament, a previous, monomeric crystal structure of ParM and AMPPNP bound to the tail of ParR (PDB 4A62) was fitted into the map and the filament model iteratively rebuilt and all-atom refined using stereochemical restraints with REFMAC.


Structures of actin-like ParM filaments show architecture of plasmid-segregating spindles.

Bharat TA, Murshudov GN, Sachse C, Löwe J - Nature (2015)

4.3 Å cryo-EM reconstruction of ParM+AMPPNP filaments(a) Cryo-EM image of ParM+AMPPNP filaments. Inset: class average. This experiment was repeated nine times. (b) A 4.3 Å reconstruction of the filaments, isosurface contoured at 2 σ away from the mean (see ED Fig. 1 and Video 1). (c) The same reconstruction as b), overlaid with the refined atomic model with individual ParM subunits coloured differently. (d-g) Enlarged regions of the cryo-EM map showing resolved secondary structure elements and side chain densities, contoured at 1 σ. (h) Density for the nucleotide is stronger than that of the protein (contoured at 3 σ).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4493928&req=5

Figure 1: 4.3 Å cryo-EM reconstruction of ParM+AMPPNP filaments(a) Cryo-EM image of ParM+AMPPNP filaments. Inset: class average. This experiment was repeated nine times. (b) A 4.3 Å reconstruction of the filaments, isosurface contoured at 2 σ away from the mean (see ED Fig. 1 and Video 1). (c) The same reconstruction as b), overlaid with the refined atomic model with individual ParM subunits coloured differently. (d-g) Enlarged regions of the cryo-EM map showing resolved secondary structure elements and side chain densities, contoured at 1 σ. (h) Density for the nucleotide is stronger than that of the protein (contoured at 3 σ).
Mentions: Using electron cryomicroscopic (cryo-EM) images collected on a direct-electron detector, we performed real-space helical reconstruction to elucidate a 4.3 Å structure of ParM filaments assembled with the adenosine triphosphate (ATP) analogue AMPPNP (Fig. 1a-c, ED Fig. 1, ED Table 1, Video 1). Densities corresponding to alpha helices, beta strands and many side-chains were clearly observed (Fig. 1d-g). The nucleotide AMPPNP was also observed in our map as strong density, especially on the phosphates (Fig. 1h). No significant resolution anisotropy was detected in the reconstruction (ED Fig. 1), indicating that the entire ParM protein is rigidly held in the filament. To derive an atomic model of the ParM filament, a previous, monomeric crystal structure of ParM and AMPPNP bound to the tail of ParR (PDB 4A62) was fitted into the map and the filament model iteratively rebuilt and all-atom refined using stereochemical restraints with REFMAC.

Bottom Line: Growing ParMRC spindles push sister plasmids to the cell poles.The ParM filament structure shows strong longitudinal interfaces and weaker lateral interactions.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.

View Article: PubMed Central - PubMed

Affiliation: Structural Studies Division, MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK.

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 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.

Show MeSH
Related in: MedlinePlus