Extended Data Fig. 6: Cryo-EM analysis of the strand-exchange reaction with partially homologous 67-bp dsDNA.

a, Sequences of the primary 27-nt ssDNA (top, brown) and of the 67-bp donor dsDNA (bottom) containing a 10-nt segment of homology to the ssDNA. The dsDNA region of homology is coloured green and red for the complementary and homologous strands, respectively. The directions of the DNA strands, and every tenth nucleotide are labelled. Dots indicate complementarity. b, Consensus reconstruction as in Extended Data Fig. 2d, except complementary DNA can now be seen and is included in the model (green). c, Masks and maps of the 3D classification for individual duplexes as in Extended Data Fig. 2e. Masks and maps of the 3D classification for all 28 combinations of duplex pairs are shown in Supplementary Fig. 4 and their details listed in Supplementary Data 6. d, Chart of the percentage of particles that have the indicated number of duplexes for this dataset. The dataset was collected once. Also see Supplementary Data 5 for details. e, Charts show the number of particles with the indicated S2-connected duplex pairs for this dataset. Pairs of duplexes starting at RecA^C are shown in Fig. 2c. See Supplementary Data 6. The dataset was collected once. f, Reconstruction after 3D refinement of the minor class from the classification of the 5′/3′ tilted duplex^D–duplex^G particles. This class essentially has no heteroduplex density. g, The same 3D sub-clasification analysis of the class with 5′/5′-tilted duplexes (10,703 particles) identified only 1,297 particles with some heteroduplex density. Their reconstruction, shown in the figure, had overall weak density for both the homologous and complementary strands, and the connections to both duplexes were very weak. We presume that this class was still heterogeneous, but it could not be further sub-classified owing to limited numbers of particles. This suggests that the D-loop forms preferentially in the 5′/3′-tilt conformation. h, Reconstruction after 3D refinement of the major class from the 3D sub-clasification of the 5′/5′ tilted duplex^D–duplex^G particles. The density for the complementary strand of the heteroduplex is even weaker than that of g, and it is discontinuous. We could not further sub-classify this class, presumably owing to extensive heterogeneity and limited numbers of particles.