Extended Data Fig. 2: Cryo-EM analysis of the strand-exchange reaction with non-homologous 67-bp dsDNA.

a, Sequences of the 27-nt ssDNA (left, brown) and the 67-bp non-homologous dsDNA (right, black) used in the strand exchange reaction. b, Micrograph from the reaction containing 9-RecA, (dT)₂₇, ATPγS, and non-homologous 67-bp dsDNA. The micrograph is similar to the rest of the 14,762 micrographs except for variations in particle numbers, ice thickness and other parameters across the grid. c, Representative 2D classes of the particles after polishing. Box size is 279 Å. 2D classifications, performed two to three successive times before polishing resulted in similar classes, except for classes with low-quality 2D projections that were discarded. d, Left, chart shows the gold-standard FSC plot of the consensus reconstruction. Dashed line marks the FSC cutoff of 0.143. Second from the left is the consensus reconstruction map coloured by local resolution estimated with the RELION3 post-processing program. The resolution range is mapped to the colours in the inset below the map; the terminal RecA proteins are less ordered than the rest. Third, cartoon representation of the refined model of the consensus refinement. As in Fig. 1a, primary ssDNA is coloured in brown, S2 ssDNA is in red. The 9-RecA protein is coloured uniformly khaki for simplicity. Fourth and fifth, cartoon representations of duplexes A to I in the 5′- and 3′-tilt conformations, respectively coloured cyan and purple, in the same relative orientation as the refined model. Lastly, duplexes with both tilts are superimposed on the protein to highlight the difference in the 5′ and 3′ tilts. The 5′- and 3′-tilted duplexes were combined to generate the masks for the 3D classifications as described in methods. e, The masks used for 3D classification with partial signal subtraction at each duplex are at the top, and the maps of the 3D classes at the bottom. For each RecA position, the classes with duplexes are labelled with percentage and particle numbers (in parentheses). Because of the poor order of the terminal RecAs, and in particular at the 3′ end of the filament where the CTDs extend the farthest away from the filament, we could not reliably classify particles at CTD^A, and for the same reason the penultimate CTD^B was an outlier with a low 4% duplex occupancy (hereinafter we will be referring to individual RecA protomers with letters, starting with A from the 3′ end of the primary ssDNA). At the 5′ end, even though RecA^I was overall poorly ordered, duplex^I-containing particles could readily be identified, as CTD^I points towards the mid-portion of the filament, and its density is considerably better defined than that of CTD^A and CTD^B that point away from the 3′ end of the filament. Masks and maps of the 3D classification for all 28 combinations of duplex pairs are shown in Supplementary Fig. 2 and their details are listed in Supplementary Data 2. f, Histogram of the number of duplexes per particle. The chart shows the percentage of particles that have the indicated number of duplexes for this dataset. The dataset was collected once. Also see Supplementary Data 1 for details. g, The accessibility of the CTDs to dsDNA is highest at the 3′ end of the filament, where the DNA-binding tips of CTD^A to CTD^C point into empty solvent and the dsDNA can approach from a roughly half-spherical volume (left). CTD^A is even more accessible as it has no neighbouring RecA 3′ to it. Moving towards the 5′ end, the CTDs become increasingly encumbered by the presence of RecA protomers 3′ to them. Thus, CTD^D becomes slightly hindered by the RecA^A L2 loop that is 47 Å away (right; C_α–C_α distance from CTD Gly288 to L2 loop Gly200 in a direction that would approximately bisect the axis of dsDNA). CTD^E is more encumbered because, in addition to the RecA^B L2 loop, it is within 35 Å of the RecA^A L1 loop (right panel; C_α-C_α distance from CTD Gly288 to L1 loop Glu158). Additionally, CTD^F is obstructed not only by the RecA^C L2 and RecA^B L1 loops, but also by the helicase domain of RecA^A, which is within 35 Å (right; C_α–C_α distance from CTD Gly288 to helicase Ala131). CTD^G and CTD^H are encumbered the most, by the full turn of filament 3′ to them (left). Their DNA-binding tip is 28 Å away from the N-terminal helices of RecA^A and RecA^B, respectively (C_α–C_α distance from CTD Gly288 to Lys19 of αN), a distance that is only fractionally larger than the approximately 20 Å width of a DNA duplex. The terminal CTD^I is similarly close to the N-terminal helix of RecA^C, although the absence of a 5-neighbouring RecA would substantially increase its accessibility to dsDNA compared to those of CTD^G and CTG^H. The figure shows the molecular surface of the 9-RecA filament with the aforementioned structural elements coloured for each RecA as in Fig. 1a and labelled. Black dotted lines indicate the shortest RecA–RecA distances (marked) that would approximately bisect the axis of dsDNA bound at each CTD. Primary ssDNA is coloured brown. The homologous ssDNA is not shown for clarity. The view in the right panel is rotated by 180°, roughly half a turn of the filament, about the vertical axis to show the environment of CTD^D, CTD^E and CTD^F that are obscured in the left view.