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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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ParM adopts a compact conformation until ATP is hydrolysed to ADP or until phosphate is released(a) ParM protein (10 μM) was incubated with ATP (2 mM) and cryo-EM samples were prepared after 5 minutes. Many filaments were observed on the grid. This experiment was repeated ten times. (b) After two hours, no filaments were seen in the same reaction. Presumably, ATP had been hydrolysed and ParM had returned to monomeric form. This experiment was repeated three times. (c) When sodium orthovanadate (4 mM) was included in the reaction, filaments could be observed even after two hours. This experiment was repeated three times. (d) The same reaction as a), except ATP was replaced by ADP. No filaments were observed in this reaction. This experiment was repeated four times. (e, f) We performed real-space helical reconstruction of the ParM+ATP filaments (red) and ParM+ATP+vanadate filaments (yellow), and compared them with the ParM+AMPPNP filament structure (green). Comparison shows that ParM is held in a very similar conformation until hydrolysis of ATP is complete or until phosphate is released since we currently cannot distinguish these two possible effects of vanadate. See Fig. 2e for resolution estimates and ED Table 1 for image processing statistics.
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Figure 8: ParM adopts a compact conformation until ATP is hydrolysed to ADP or until phosphate is released(a) ParM protein (10 μM) was incubated with ATP (2 mM) and cryo-EM samples were prepared after 5 minutes. Many filaments were observed on the grid. This experiment was repeated ten times. (b) After two hours, no filaments were seen in the same reaction. Presumably, ATP had been hydrolysed and ParM had returned to monomeric form. This experiment was repeated three times. (c) When sodium orthovanadate (4 mM) was included in the reaction, filaments could be observed even after two hours. This experiment was repeated three times. (d) The same reaction as a), except ATP was replaced by ADP. No filaments were observed in this reaction. This experiment was repeated four times. (e, f) We performed real-space helical reconstruction of the ParM+ATP filaments (red) and ParM+ATP+vanadate filaments (yellow), and compared them with the ParM+AMPPNP filament structure (green). Comparison shows that ParM is held in a very similar conformation until hydrolysis of ATP is complete or until phosphate is released since we currently cannot distinguish these two possible effects of vanadate. See Fig. 2e for resolution estimates and ED Table 1 for image processing statistics.

Mentions: ParM’s dynamic instability is caused by intrinsic ATP hydrolysis in the filament and the resulting adenosine diphosphate (ADP)-bound filament being less stable 18, while being temporally protected by an ATP cap. We therefore assembled ParM+ATP filaments and obtained a 7.5 Å cryo-EM structure of these filaments (ED Fig. 4). Since the nucleotide state of this structure may be mixed, we devised a way to inhibit ParM’s ATPase with vanadate. Addition of sodium orthovanadate to the ParM+ATP solution retarded filament disassembly and we captured these ParM+ATP+vanadate filaments before complete disassembly and obtained a 6.4 Å structure (ED Fig. 4). Comparison of the three cryo-EM structures (+AMPPNP, +ATP, +ATP+vanadate) indicates that ParM is held in the same rigid, compact conformation, either until ATP is hydrolysed to ADP or until phosphate is released (ED Fig. 4e-f).


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

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

ParM adopts a compact conformation until ATP is hydrolysed to ADP or until phosphate is released(a) ParM protein (10 μM) was incubated with ATP (2 mM) and cryo-EM samples were prepared after 5 minutes. Many filaments were observed on the grid. This experiment was repeated ten times. (b) After two hours, no filaments were seen in the same reaction. Presumably, ATP had been hydrolysed and ParM had returned to monomeric form. This experiment was repeated three times. (c) When sodium orthovanadate (4 mM) was included in the reaction, filaments could be observed even after two hours. This experiment was repeated three times. (d) The same reaction as a), except ATP was replaced by ADP. No filaments were observed in this reaction. This experiment was repeated four times. (e, f) We performed real-space helical reconstruction of the ParM+ATP filaments (red) and ParM+ATP+vanadate filaments (yellow), and compared them with the ParM+AMPPNP filament structure (green). Comparison shows that ParM is held in a very similar conformation until hydrolysis of ATP is complete or until phosphate is released since we currently cannot distinguish these two possible effects of vanadate. See Fig. 2e for resolution estimates and ED Table 1 for image processing statistics.
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Related In: Results  -  Collection

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Figure 8: ParM adopts a compact conformation until ATP is hydrolysed to ADP or until phosphate is released(a) ParM protein (10 μM) was incubated with ATP (2 mM) and cryo-EM samples were prepared after 5 minutes. Many filaments were observed on the grid. This experiment was repeated ten times. (b) After two hours, no filaments were seen in the same reaction. Presumably, ATP had been hydrolysed and ParM had returned to monomeric form. This experiment was repeated three times. (c) When sodium orthovanadate (4 mM) was included in the reaction, filaments could be observed even after two hours. This experiment was repeated three times. (d) The same reaction as a), except ATP was replaced by ADP. No filaments were observed in this reaction. This experiment was repeated four times. (e, f) We performed real-space helical reconstruction of the ParM+ATP filaments (red) and ParM+ATP+vanadate filaments (yellow), and compared them with the ParM+AMPPNP filament structure (green). Comparison shows that ParM is held in a very similar conformation until hydrolysis of ATP is complete or until phosphate is released since we currently cannot distinguish these two possible effects of vanadate. See Fig. 2e for resolution estimates and ED Table 1 for image processing statistics.
Mentions: ParM’s dynamic instability is caused by intrinsic ATP hydrolysis in the filament and the resulting adenosine diphosphate (ADP)-bound filament being less stable 18, while being temporally protected by an ATP cap. We therefore assembled ParM+ATP filaments and obtained a 7.5 Å cryo-EM structure of these filaments (ED Fig. 4). Since the nucleotide state of this structure may be mixed, we devised a way to inhibit ParM’s ATPase with vanadate. Addition of sodium orthovanadate to the ParM+ATP solution retarded filament disassembly and we captured these ParM+ATP+vanadate filaments before complete disassembly and obtained a 6.4 Å structure (ED Fig. 4). Comparison of the three cryo-EM structures (+AMPPNP, +ATP, +ATP+vanadate) indicates that ParM is held in the same rigid, compact conformation, either until ATP is hydrolysed to ADP or until phosphate is released (ED Fig. 4e-f).

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