( a ) resolution of the parm+amppnp reconstruction was estimated using resmap and this estimate was plotted back onto the cryo - em density . ( b ) the power spectrum of the aligned segments ( left ) compared to the power spectrum of the re - projection of the cryo - em reconstruction ( right ) . a reflection is observed in both cases at 4.8  indicating that the resolution extends beyond this shell . ( a ) atomic model of one protofilament ( strand ) of parm is shown with the residues at the protein : protein interface highlighted in red . ( c ) the complete parm filament ( i.e. 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 ) . ( f ) an orthogonal view of d ) . see ed table 2 for a detailed list of interacting residues . ( a ) cryo - em density for the parm+amppnp filament is shown at an isosurface contour level of 2.0  from the mean value . overlaid on the density , refined atomic coordinates from refmac are additionally displayed as grey ribbons . ( b ) the same figure as a ) , except the cryo - em density shown at an isosurface contour level of 1.5  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 while negatively charged residues are highlighted in orange . ( g ) two residues ( k258 and r262 ) that were the best resolved ( marked with an * 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 ( supplementary methods ) . ( 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 . 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 . a few class averages show that single protofilaments were present in the sample ( lower panels ) . however , the double mutation destabilises 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 . ( a ) parm protein ( 10 m ) was incubated with atp ( 2 mm ) and cryo - em samples were prepared after 5 minutes . ( b ) after two hours , no filaments were seen in the same reaction . ( c ) when sodium orthovanadate ( 4 mm ) was included in the reaction , filaments could be observed even after two hours . ( d ) the same reaction as a ) , except atp was replaced by adp . ( 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 can not distinguish these two possible effects of vanadate . ( a ) a cryo - em image of parm+amppnp + 2% peg 6000 . instances of doublets are marked with yellow arrowheads . ( b ) more examples of parm doublets observed in cryo - em . ( c ) ( d ) directionality assignment of the filaments in the doublet . individual sub - segments and their assigned directionality are indicated by triangles , coloured based on the cross - correlation score in the alignment procedure : red indicates a poor cross correlation score while green indicates a good score . ( f ) the thickest parts of parm filaments of the doublet ( as they appear in projection ) are marked with black arrowheads . ( a ) two parm monomers arranged in an anti - parallel orientation , as obtained from the parm cryo - em doublet model . ( b ) two parm monomers arranged in an anti - parallel orientation , obtained from crystal packing of a monomeric parm x - ray structure ( pdb 4a62 ) . ( c ) two residues at the interface of the doublet ( see ed 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 . ( a - k ) e. coli b / r266 cells were transformed with a high - copy ( pdd19 ) , or medium - copy ( pkg321 ) plasmid containing the parmrc locus . this figure shows a gallery of parm bundles ( blue arrows ) and doublets ( yellow arrows ) observed in these cells . panels a , c , e and h show cells transformed with the high - copy number plasmid while panels b , d , f , g , i , j and k show cells transformed with the medium copy number plasmid . each experiment with different copy number plasmids was performed only once due to low - throughput nature of cryo - et . image processing statistics for cryo - em reconstructions of parm filaments interfaces forming residues of parm residues of parm that are part of interfaces have been highlighted . inter- and intra - protofilament interface - forming residues have been highlighted in red and green respectively ( web version ) . these residues have been assigned using a 4  distance cut off based on the parm+amppnp structure . residues forming the inter - filament interface in the parm doublet have been highlighted in blue ( web version ) . this assignment was based on a 7  distance cut off for c- atoms in the derived model of the parm doublet because of the lower accuracy of the model . instances of single , double and bundled parm filaments seen in bacterial cells with different copy number plasmids the parmrc locus was inserted into high , medium and low - copy number plasmids ( plasmids pdd19 , pkg321 and pkg491 respectively ) . the ratio of observed doublets per cell ( i.e. the number of doublets observed divided by the number of cells imaged ) was 5.8:1.6:0.3 ( ~19:5:1 ) . these ratios are roughly the same as the expected copy number ratios of the different copy - number plasmids .
active segregation of e. coli low - copy number plasmid r1 involves formation of a bipolar spindle made of left - handed double - helical actin - like parm filaments 1 - 6 . 
 parr links the filaments with centromeric parc plasmid dna , while facilitating the addition of subunits to parm filaments 3,7 - 9 . 
 growing parmrc spindles push sister plasmids to the cell poles 9,10 . here , using modern electron cryomicroscopy methods we have investigated 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 determined structures of parm filaments in different nucleotide states . 
 the structure of filaments bound to amppnp was determined at 4.3  resolution and refined . 
 the parm filament structure shows strong longitudinal interfaces and weaker lateral interactions . also using electron cryomicroscopy , we reconstructed 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 .