Extended Data Fig. 3: MsCAPP PP domain structure analyses and comparison.

a. Overall structure of dGTP-complexed MsCAPP PP domain, with N-terminal α/β domain coloured in green and RRM-like domain coloured in yellow (left), and a close-up view showing dGTP (orange), Mn(II) ions (spheres) and residues lining the active site pocket. b. Architecture of MsCAPP PP domain showing the secondary structural elements within the α/β domain (aa 111-164; aa 274–278) in green and the RRM-like domain (aa 169–262; aa 291–328) in yellow. c. Side by side comparison of PP domains from various Prim-Pols with a single nucleotide (ball-and-stick model) bound to the elongation site. N-terminal RRM-like domain (yellow), α/β domain (green) and helical domain (red). d. Simulated annealing Fo-Fc omit map of Co(II) ions in the active site of MsCAPP (contoured at 5 σ-level at 1.90 Å resolution), along with GTP (blue), dATP (orange), and acidic residues D177, D179 and E260 in stick representation. e. Close-up view of Mn(II) bound in the A site of the MsCAPP dGTP complex, showing octahedral coordination to dGTP, ethylene glycol and surrounding acidic residues with distances labelled in Å (left). Close-up view of Mn(II) bound in the B site of MsCAPP dGTP complex, showing octahedral coordination to dGTP, DxD motif and a water molecule, with distances labelled in Å (right). Mn(II) – purple spheres. f. Overlay of Region 1 (aa 130-142) (left), Region 2 (aa 263–274) (middle) and residues around Region 3 (aa 280-289) (right) from the structures of MsCAPP apo (green) and primer initiation complex (grey). Residues 283–286 in Region 3 are flexible and not resolved in the apo structure. g. MsCAPP active site with surface coloured according to hydrophobicity, with regions of high hydrophobicity coloured in red. Three core residues that form part of the active site hydrophobic pocket (L275, I276 and F262) are shown in stick representation (red). I-site GTP (blue) and E-site dATP (orange) are shown in stick representation. Templating DNA (pink) is shown in cartoon representation. h. Comparison of CAPP primer initiation complex active site. Figure on the left – CAPP primer initiation complex shown in the main figures (PDB: 7P6J). Figure on the right shows the structure of an alternative CAPP primer initiation complex (PDB: 7QAZ), where the phosphate tail (red) of I-site GTP adopts a different conformation and coordinate to an extra Co(II) ion. The rest of the GTP molecule is shown in blue and dATP in orange. Templating DNA in pink, surface of α/β domain is coloured in green and RRM-like domain is shown in yellow.