(A) Top panel shows a stereo view of the mFo-DFc electron density at the active site, calculated with phases after omitting the two calcium ions (blue spheres), the water molecule (red sphere), a 3-nt segment centered on the scissile phosphate group and the side chain of Lys301, and subjecting the rest of the entire structure to simulated annealing from 3000°K. The dark blue map is contoured at 5 σ, and the light green map at 2.5 σ. The orientation is similar to that of Figure 2B. For comparison, the corresponding residues of the λ nuclease-DNA complex (Zhang et al., 2011) are shown as black sticks (the lysine is mutated to alanine in that structure), and the calcium ions and water molecule as black spheres. The Dna2 Ca-1 ion and water molecule have a coordination shell and position very similar to the first magnesium and associated water in λ nuclease, but the position of Ca-2 is different. The Ca coordination shell and residue labels are shown in bottom panel. For clarity, not shown are the electron density, λ nuclease, and the His164 side chain to which the green-dotted bond from Ca-1 would be connected. (B) Superposition of Dna2 helicase domains 1A (pink) and 2A (cyan) with Ighmbp2 (gold). DNA is colored in red, RNA is colored in green. The only regions of the Dna2 helicase that do not have counterparts in the Upf1 subfamily are the ~40 residue 1B domain, which packs with the nuclease domain, and the ~20 residue 2B domain, which is distal from the rest of the protein and the DNA (Figure 1D). (C) Superposition of individual Dna2 helicase domains 1A (pink) and 2A (cyan) with Upf1 (gold). DNA is colored in red, RNA is colored in green. Motif III residues are shown as sticks. Supplemental discussion of helicase translocation. Based on the proposed SF1B translocation mechanism (Saikrishnan et al., 2009), ATP binding and concomitant closure of the cleft between the 1A and 2A domains would be coupled to domain 2A releasing the DNA and rebinding it at a register shifted by 1 nt in the 5’ to 3’ direction, while domain 1A would retain its DNA. On ATP hydrolysis, domain 1A would be the one releasing its DNA and rebinding it at the +1 nt register, restoring the initial open-cleft conformation. In the Dna2-ssDNA structure, however, the rebinding of 1A to DNA at the shifted register will have to await the transient dissociation of the nuclease domain from DNA. The increased half life of the closed cleft would likely also reduce the rate of exchange of ADP for ATP, as the γ phosphate binding site of the helicase fold is far less solvent accessible in the closed cleft.