a, Superimposition of 5-bp TSD region with the cryo-EM map contoured at 5.5σ. b, Front and top views of Transib STC structure superimposed on the Transib HFC structure. c, Comparison of target DNA from H. zea Transib, retrovirus integrases, Mos1 transposase and Mu transposase STC structures. Target site DNAs are shown as green and red. The approximate degree of bending in each target DNA is indicated. H. zea Transib is the only DDE/D-family transposase–integrase for which a STC structure has been reported that lacks a bend or base-unpairing at the centre of the target site DNA. Instead, Transib strongly bends target DNA near both edges of the target site DNA (between position −2 and −1 and positions 1 and 2), leading to a total 150° directional change of target DNA. Target DNAs in retroviral integrase STC structures exhibit relatively mild bends with one backbone kink at the centre of target site DNA, regardless of its length (ranging from 4 bp in PFV integrase to 6 bp in RSV integrase). The sharp bending (about 150°) at the centre of the Mos1 target DNA is achieved by flipping of the adenines in the TA target site. The target DNA in Mu STC exhibits a more continuous bending pattern through the 5-bp target site DNA, with one bend before the target site (between position −3 and −2), one at the centre and one immediately after the target site DNA (between position 2 and 3). The central bend is facilitated by the T–T mismatch in the target site. d, Transposon end–target DNA junction region of the Transib STC model superimposed on the cryo-EM map contoured at 5.5σ. Nucleotide residues in target DNA are labelled with a subscript T. e, Difference density between the Transib STC cryo-EM map and the model, showing the uncleaved target DNA phosphodiester bond in a portion of the particles used for cryo-EM map reconstruction. The difference map was contoured at 6σ. f, Superimposition of Transib TCC (protein in orange and metal ions in green) active site with Transib HFC active site (protein in purple and metal ions in grey). Distances are expressed in Å. Attacking oxygen atoms in HFC and TCC are highlighted in black and red circles, respectively. In TCC, the phosphorus is 2.4 Å from the attacking oxygen and the two metal ions are 3.2 Å apart. These distances are 3.6 Å and 4.2 Å in HFC. g, Sequence alignment of Transib transposases, vertebrate RAG1 and deuterostome invertebrate RAG1L proteins, showing the regions corresponding to three RAG2-binding interfaces in RAG1. Residue numbers are for H. zea Transib. Hs, Homo sapiens (human); Mm, Mus musculus (mouse); Dr, Danio rerio (zebrafish); Gg, Gallus gallus (chicken); Bb, Branchiostoma belcheri (amphioxus); Sp, Strongylocentrotus purpuratus (purple sea urchin); Pf, Ptychodera flava (acorn worm); Pm, Petromyzon marinus (sea lamprey) and Af, Asterias forbesi (sea star).