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

Nature volume 523, pages 106–110 (2015)Cite this article

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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 filaments1,2,3,4,5,6. ParR links the filaments with centromeric parC plasmid DNA, while facilitating the addition of subunits to ParM filaments3,7,8,9. Growing ParMRC spindles push sister plasmids to the cell poles9,10. 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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Figure 1: Cryo‐EM reconstruction at 4.3 A˚ of ParM+AMPPNP filaments.
Figure 2: ParM filaments are made up of two protofilaments held together by salt bridges, which are perturbed when ParM is bound to ADP.
Figure 3: ParM doublets formed in vitro.
Figure 4: ParM doublets in E. coli cells, imaged by cryo‐ET.

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Primary accessions

Electron Microscopy Data Bank

Protein Data Bank

Data deposits

Cryo-EM and cryo-ET data have been deposited in the Electron Microscopy Data Bank under accession codes EMD-2848, EMD-2849 and EMD-2850. Atomic coordinates of the ParM+AMPPNP filament structure and the ParM antiparallel doublet model have been deposited in the Protein Data Bank under accession codes 5AEY and 5AI7.

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Acknowledgements

We thank F. van den Ent, K. Gerdes and P. Gayathri for help with sample preparation, and C. Johnson, C. Savva and F. de Haas for help with data collection. This work was supported by the Medical Research Council (U105184326) and the Wellcome Trust (095514/Z/11/Z). T.A.M.B. is the recipient of Federation of European Biochemical Societies (FEBS) and European Molecular Biology Organization (EMBO) (ALTF 3-2013) long-term fellowships. G.N.M. was funded by Medical Research Council grant MC-UP-A025-1012.

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Authors and Affiliations

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

    Tanmay A. M. Bharat, Garib N. Murshudov & Jan Löwe

  2. Structural and Computational Biology Unit, European Molecular Biology Laboratory, Meyerhofstrasse 1, Heidelberg, 69117, Germany

    Carsten Sachse

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  1. Tanmay A. M. Bharat
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Contributions

T.A.M.B. and J.L. designed experiments; T.A.M.B. performed experiments; T.A.M.B., G.N.M., C.S. and J.L. analysed data; T.A.M.B. and J.L. wrote the paper.

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Correspondence to Jan Löwe.

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Extended data figures and tables

Extended Data Figure 1 Resolution estimate of the ParM+AMPPNP reconstruction.

a, Resolution of the ParM+AMPPNP reconstruction was estimated using ResMap and this estimate was plotted back onto the cryo‐EM density. Blue indicates high resolution; red indicates lower resolution. b, The power spectrum of the aligned segments (left) compared with the power spectrum of the re‐projection of the cryo‐EM reconstruction (right). A reflection is observed in both cases at 4.8 A˚−1, indicating that the resolution extends beyond this shell. See Fig. 2e for Fourier shell correlation curves.

Extended Data Figure 2 Intra‐ and inter‐protofilament interactions in ParM filaments.

a, Atomic model of one protofilament (strand) of ParM is shown with the residues at the protein–protein interface highlighted in red. See Extended Data Table 2 for a detailed list. b, A magnified view of the interface. Three residues at the interface have been labelled. c, The complete ParM filament (that is, both protofilaments/strands) shown end‐on. d, Atomic model of the ParM filament with the inter‐protofilament residues at the protein–protein interface highlighted in orange. e, A magnified view of d. Salt bridging residues are labelled. f, An orthogonal view of d. See Extended Data Table 2 for a detailed list of interacting residues.

Extended Data Figure 3 The ParM inter‐protofilament interface is small but important.

a, Cryo‐EM density for the ParM+AMPPNP filament is shown at an isosurface contour level of 2.0σ from the mean value. Overlaid on the density, refined atomic coordinates from REFMAC are additionally displayed as grey ribbons. Residues forming salt bridges at the inter‐protofilament interface are highlighted. b, The same figure as a, except the cryo‐EM density shown at an isosurface contour level of 1.5σ from the mean; c, 1.0σ from the mean. d–f, A magnified view of the primary salt‐bridged interface consisting of charged residues that form the ParM inter‐protofilament interface. The cryo‐EM density is shown as a mesh at three different contour levels to demonstrate resolved side‐chain densities. Positively charged residues are highlighted in red; negatively charged residues are highlighted in orange. g, Two residues (K258 and R262) that were the best resolved (marked with an asterisk in d), were mutated to aspartic acid to test the importance of this inter‐protofilament interface. A cryo‐EM image of this mutant protein assembled with AMPPNP is shown. A much higher concentration of the protein was required to obtain filaments on cryo‐EM grids (Methods). This experiment was repeated four times. h, Randomly selected cryo‐EM images of ParM+AMPPNP and ParM(K258D, R262D)+AMPPNP were used to count occurrences of straight and bent filaments by visual inspection. The results of this quantification are shown as a percentage bar diagram. For the ParM protein, 82% of all filaments were classified as straight, while 18% were bent (n = 345). Using exactly the same classification criteria, only 15% of the filaments were found to be straight and 85% of the filaments were bent (n = 45) for the ParM(K258D, R262D) mutant protein. i, Reference‐free class averages show that most of the ParM(K258D, R262D) filaments are made up of double protofilaments like wild‐type ParM. Some class averages show evidence of bending. A few class averages show that single protofilaments were present in the sample (lower panels). However, the double mutation destabilizes the entire ParM filament, making filament formation an unfavourable reaction, illustrating that even though the inter‐protofilament interface is small, it is critical for ParM filament formation.

Extended Data Figure 4 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 min. Many filaments were observed on the grid. This experiment was repeated ten times. b, After 2 h, 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 2 h. 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 Extended Data Table 1 for image‐processing statistics.

Extended Data Figure 5 Model of the ParM doublet.

a, A cryo‐EM image of ParM+AMPPNP + 2% PEG 6000. Instances of doublets are marked with yellow arrowheads. This experiment was repeated 15 times. b, More examples of ParM doublets observed in cryo‐EM. c, Class averages of the doublets. d, Directionality assignment of the filaments in the doublet. Individual sub‐segments and their assigned directionality are indicated by triangles, coloured on the basis of the cross‐correlation score in the alignment procedure: red indicates a poor cross correlation score; green indicates a good score. e, A schematic model of the anti‐parallel ParM doublet. Directionality is indicated with a yellow arrow. f, The thickest parts of ParM filaments of the doublet (as they appear in projection) are marked with black arrowheads.

Extended Data Figure 6 Validation of the doublet model.

a, Two ParM filaments arranged in an anti‐parallel orientation, as obtained from the ParM cryo‐EM doublet model. b, Two ParM filaments arranged in an anti‐parallel orientation, obtained from crystal packing of a monomeric ParM X-ray structure (PDB 4A62)3. c, Two residues at the interface of the doublet (see Extended Data Table 2), S19 and G21, were mutated to arginine to improve affinity of ParM filaments to each other. Cryo‐EM images of the mutant protein with AMPPNP show spontaneous doublet formation and filament bundling without crowding agent, validating the doublet model. This experiment was repeated six times.

Extended Data Figure 7 ParM bundles and doublets observed in vivo.

a–k, E. coli B/-R266 cells were transformed with a high‐copy (pDD19) or medium‐copy (pKG321) plasmid containing the ParMRC locus. Transformed cells were grown to log phase and then prepared for cryo‐EM. This figure shows a gallery of ParM bundles (blue arrows) and doublets (yellow arrows) observed in these cells. a, c, e, h, Cells transformed with the high‐copy‐number plasmid; b, d, f, g, i, j, k, cells transformed with the medium‐copy‐number plasmid. Each experiment with different copy‐number plasmids was performed only once owing to the low‐throughput nature of cryo‐ET.

Extended Data Table 1 Image processing statistics for cryo-EM reconstructions of ParM filaments
Full size table
Extended Data Table 2 Interfaces forming residues of ParM
Full size table
Extended Data Table 3 Instances of single, double and bundled ParM filaments seen in bacterial cells with different copy-number plasmids
Full size table

Supplementary information

4.3 Å cryo-EM reconstruction of ParM+AMPPNP filaments

The cryo-EM structure of the ParM+AMPPNP filaments is shown as an isosurface contoured 2 σ away from the mean. A small interface holds the two protofilaments together. Five ParM monomers that were refined into the density are shown. (MOV 12530 kb)

Morph between the ParM+AMPPNP filament and the ParM+ADP filament structures

A morph was generated between the PDB coordinates of the ParM+AMPPNP and the ParM+ADP filament structure at the filament level (top) and at the monomer level(bottom). Only main chain atoms are shown in the morph as ribbon diagrams. ParM+AMPPNP is shown in green while ParM+ADP is shown in blue. All intermediate morph states are shown at a lower brightness level because they do not represent observed data, and are merely shown for illustrative purposes. (MOV 7632 kb)

ParM doublets do not show any super-helical twist

Sequential z-slices of a reconstructed electron cryotomogram of ParM doublets in vitro. These tomography data show clearly the absence of any super-helical twist in the filaments. (MOV 4069 kb)

ParM doublet model

Model of the ParM antiparallel doublet is shown. The volumes have been filtered down to 40 Å to better illustrate how the two filaments in the doublet are out of phase with each other. (MOV 12048 kb)

ParM doublets are observed in E. coli cells containing plasmids with the ParMRC locus

A video containing sequential z-slices of a reconstructed electron cryotomogram of an E. coli cell transformed with the low-copy number plasmid (pKG491) ('mini-R1' replicon). A ParM doublet is highlighted. (MOV 3931 kb)

ParM bundles and doublets observed in E. coli cells containing plasmids with the ParMRC locus

Video containing sequential z-slices of a reconstructed electron cryotomogram of an E. coli cell transformed with the medium-copy number plasmid (pKG321). Both ParM doublets and bundles were observed in this cell. (MOV 12780 kb)

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Bharat, T., Murshudov, G., Sachse, C. et al. Structures of actin-like ParM filaments show architecture of plasmid-segregating spindles. Nature 523, 106–110 (2015). https://doi.org/10.1038/nature14356

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