Supplementary Figure 8: Target DNA binds in a positively charged groove.

a, Plot of target DNA minor groove width. The minor groove width was calculated from the target DNA model using the 3DNA webserver (Li, S., et al., Nucleic Acids Research. 47, W26–W34, 2019), with a 2 bp sliding window, accounting for phosphate van der Waals radii. The target DNA sequence is depicted on the x-axis and colored as in Fig. 5a. Red dots indicate transposition sites, on either the top or bottom strand of the target DNA. b, Electrostatic surface potential of the STC as viewed from below the target DNA binding site. Calculations were performed in UCSF Chimera (Pettersen, E. F., et al., J Comput. Chem. 25, 1605–1612, 2004). Blue denotes a positive charge and red denotes a negative charge. Target DNA is shown as in Fig. 5a. c, Schematic representation of observed base-specific and backbone contacts between transposase and the target DNA. Target DNA (purple border) is numbered as in Fig. 5e (target site duplication, pink fill; AT-rich flanks, green fill). Residue numbers are indicated and outlined in a solid or dashed border to indicate transposase subunit A, or transposase subunit B, respectively. Residues are colored according to domain (RNase H, orange; GBD, blue). Direct contacts are shown as solid lines; aromatic base stacking interactions are shown as dashed lines; major groove, minor groove and main chain contacts are indicated; interacting phosphates are highlighted yellow. d, Unmodeled density at the C-terminus is oriented towards the target DNA. The map is low-pass filtered to 4 Å to more clearly show the presence of additional, poorly-ordered density. While we could not confidently build into the density beyond position 734, the highly basic nature and positioning of the weak density near DNA suggests that this region likely plays a role in target DNA binding. Consistent with this, C-terminal tags on transposase decrease the overall excision and strand transfer activity (unpublished results, D. Rio).