(a) Crystal structures of the starting primer-template complex (n). Color scheme and PDB codes: new structure (yellow, 6DSW), known structure (grey, 1L3S). (b–f) Post-chemistry structures comparing the active site conformation for n + 1 and n + 2 translocated products generated from solution and in crystallo catalyzed primer-extension reactions. Color scheme and PDB codes: known n + 1 and n + 2 in crystallo catalyzed structures (grey, 1L3T and 1L3U, respectively), new n + 1 and n + 2 solution-catalyzed structures (yellow, 6DSY and 6DSV, respectively), and a new n + 2 in crystallo catalyzed structure obtained from the n + 1 crystal of a solution-catalyzed reaction (blue, 6DSX). (b,c) Overlays comparing the translocated product obtained from solution and in crystallo catalyzed n + 1 and n + 2 primer-extension reactions, respectively. (d) Overlays of the n + 1 and n + 2 structures from solution catalyzed reactions. (e) Overlay of the n + 2 structures for solution and in crystallo catalyzed reactions that initiate from the same polymerase conformation. (f) Overlay of n + 2 in crystallo catalyzed structures that initiate from different polymerase conformations.