Fig. 2: Time-lapse crystallography and the matched Mg²⁺-reaction state ternary complex.

a Time-lapse x-ray crystallography protocol. Ground state ternary complex crystals (GS, yellow) were grown by mixing pol λ, gapped DNA substrate, nucleotide (dNTP), CaCl₂ and precipitants. Ground state (GS) crystals were then soaked in a cryo-solution containing 50 mM Mg²⁺ (green arrow) or 50 mM Mn²⁺ (purple arrow) to initiate the reaction in the crystal. Crystals were frozen after increasing incubation times (central black arrow) and the structures of the intermediate complexes at increasing degrees of product formation (% incorporation) were determined. b Matched Mg²⁺-reaction state (RS_Mg) structure after a 2 min soak. Partial bond breakage in substrate (P_α–P_β of TTP) and bond formation in product (primer O3´–P_α of TTP) is visible, with inversion about P_α and concurrent PP_i formation. Rotation of Asp427 and presence of Mg²⁺ in the nucleotide metal site (Mg_n) and Mg²⁺/Na⁺ (Mg_c/Na_c) in the catalytic metal site, is shown. Appearance of an alternate water molecule to coordinate the catalytic metal site (Mg_c/Na_c) upon Asp427 rotation is indicated with a red arrow. Active site aspartates are shown in yellow stick representation, the incoming nucleotide in green, DNA in cyan. Mg²⁺ and Na⁺ are shown as green and purple spheres, respectively, water molecules are blue. The simulated annealing omit (F_o-F_c) density (green mesh) shown is contoured at 3σ, carve radius 2.0 Å. c Comparison of the Mg²⁺-reaction (RS_Mg) and ground state (Ca²⁺-GS) ternary complexes. The Ca²⁺-GS complex is shown in yellow, RS_Mg is in magenta and are otherwise displayed as in (b). Ca²⁺ is shown as an orange sphere. The primer terminal (P_n) and preceding (P_n-1) nucleotides are indicated. The overlay was generated by superimposition of C_α atoms of the respective palm domains (residues 386–494).