Extended Data Fig. 1. Biochemical characterization of HzTransib transposase and single-particle cryo-EM analysis of HzTransib in complex with intact TIR substrates.

a, Size-exclusion chromatography-multiple angle light scattering (SEC-MALS) analysis of purified HzTransib protein, indicating that it forms a dimer in solution. Size-exclusion chromatography was repeated three times and similar profiles were obtained. MALS experiment was not repeated. b, Numbering and sequence of endogenous left end (5’TIR) and right end (3’TIR) of the HzTransib transposon with nucleotide differences in black boxes. The first 16 bp of the TIR sequence are the same as the 16 bp transposon end of the TIR substrates used in structure determination. c, Schematic of the TIR substrate DNA used in the in vitro DNA cleavage assay. 5’TIR and 3’TIR are shown as yellow and purple triangles, respectively. d, Cleavage of DNA substrates bearing one or two TIRs by MBP-tagged wild-type or mutant HzTransib transposases, each with the N-terminal 16 amino acids removed. The experiment was repeated three times and similar results were obtained. For gel source data, see Supplementary Figure 1. e, Cleavage of DNA substrates bearing either full length (lanes 1 and 2) or truncated (lanes 3–8) 5’TIR or 3’TIR, with site of truncation indicated in the substrate name. The experiment was repeated three times and similar results were obtained. Open and closed arrowheads indicate single 5’TIR and single 3’TIR cleavage products, respectively. Red asterisk marks the double cleavage band. The DNA cleavage products were resolved in 5% Tris-borate-EDTA (TBE) polyacrylamide gels and stained with SYBR Gold. f, Flowchart of cryo-EM structure determination of HzTransib in complex with intact TIR substrates. After the first round of 3D classification, 3D auto-refinement using all of the particles in the best class generated a 3.3 Å map. Further 3D classifications focusing on either two ZnB plus flanking DNA regions or on one ZnB domain with symmetry expansion were used to obtain the final 3.4 Å map or a 3.5 Å map with clear ZnB domain density. All three maps were used for cross-references in model building. The final map and accompanying local resolution illustrations are enclosed in the dashed black box.