Figure 1: The structure of the SB100X transposase catalytic domain.

(a) The SB100X catalytic domain (blue) assumes an RNaseH-fold with all catalytic residues (red) assembled in the active site. Conserved α-helices (α) and β-strands (β) are indicated. Insert: superposition of active-site residues in SB100X and Mos1 (grey, PDB 3HOS)⁶. (b) The SB100X dimer (dark and light blue) observed in our crystal structure (molecules I and II) superposed onto the Mos1 PEC structure (grey). Arrow illustrates the rearrangement (∼50° swing to left with ∼20° rotation backwards) that can bring Molecule I into the PEC conformation. (c) Top view of the SB100X catalytic domain dimer highlights the role of the clamp loops in the interaction. (d) Residues N280 and K339 of the RNaseH core form sequence-specific interactions (hydrogen bonds indicated with dashed lines) with the clamp loops of the partner molecules, anchoring them to cover the active sites. Insert: close-up of the dimer contacts mediated by N280 and K339, respectively. (e) β-stranded interactions between the backbones of the clamp loop and the inter-domain linker. (f) Transposition assay for N280A and K339A mutants in HeLa cells. D3 indicates the negative control using a catalytically inactive transposase mutant, E279D. Error bars represent the means±s.e.m. of three independent experiments. Statistical analysis was performed by a one-sample t-test and P values are as follows: N280A, 0.061; K339A, 0.34; and N280A/K339A, 1.7 × 10⁻⁴.