Fig. 5: E5 double hexamer formation, overall structure and rupture.

a The representative 2D class averages (left) and schematic models (right) showing structural flexibility and intermolecular arrangements of the E5 double hexamer. b Unsharpened EM density map of E5 double hexamer. The density map displays the local resolution distribution, the diffuse densities of AEPs (black arrows), and the density of the bound dsDNA. The latter is highlighted in the inset, presented in the cut-off view, and indicated with a red dashed box along with a red arrow. c, d Sharpened EM density map of the E5 double hexamer. The ZBDs and the helicase are colored orange and green, respectively. The anti-clockwise arrangement of ZBDs with respect to the corresponding helicase domains is highlighted by black dashed lines. e Cut-off view of the dsDNA binding channel reveals a positively charged inner wall suitable for DNA accommodation. f Model of dsDNA incorporation into the double hexamer. The length and position of the dsDNA is determined from the unsharpened EM density map. B-form DNA was used for the generation of this model. The precise status of the DNA duplex incorporated in the double hexamer remains to be clarified. g Representative 2D class averages of wild-type (WT) E5 and E5 mutant (DDD/A) under varying substrate conditions. “-” indicates substrate absence, and “+” indicates substrate presence. h DNA polymerization assays catalyzed by wild-type E5 (WT) or E5 mutants (DDD/A, KRR/A, RF/A, and ΔHelicase). The “-” is used to represent the mock group, in which an equal volume of protein buffer was added to the reaction to replace the E5 proteins. The experiments were repeated three times independently with similar results, and the representative image is provided.