Fig. 1: Overall architecture of E5 in the apo state and the conformational rearrangement of the primase domains after ATP incorporation.

a DNA polymerization assays catalyzed by wild-type (WT) E5 or E5 mutant (DDD/A). 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 DNA substrate used in the assay is shown diagrammatically at the left side, with the end-labeled FAM is shown as a red star. The experiments were repeated three times independently with similar results, and the representative image is provided. b The schematic representation of E5. The domain organization of the helicase domain is highlighted. c Overall structure of the hexameric E5, with protomers A–F shown in different colors and distances between protomers marked. d Close-up view of the collar domain in E5. Amino acids involved in hydrophobic interactions (upper box) and polar interactions (lower box) are shown as sticks and labeled. e The overall architecture of DNA-bound E5 complex (structure 0). f Close-up view of the primase domain. g Local structure of the primase domain after 3D focused refinement. The metal ion is depicted as a sphere, and coordinating amino acids in ZBD-F are shown as sticks. The EM density of the metal binding site is highlighted in the inset. h Representative 2D class averages of wild-type (WT) E5 or E5 mutants after addition of different substrates. The pink arrow indicates the conformation of the inter-primase assembly and the green arrow points to the bound DNA.