Extended Data Fig. 2: Cryo-EM analysis of the IS621 synaptic complex.

(a) Preparation of the IS621–bRNA–dDNA–tDNA synaptic complex. The IS621 recombinase, bRNA, and dDNA/tDNA (containing no mismatch (WT) or 6-nt mismatches (MM) at positions 2–7 in dDNA and tDNA) were mixed, and then purified by a Superose 6 Increase 10/300 column. The peak fraction of the synaptic complex with the mismatched DNA (indicated by a red line) was analyzed by SDS-PAGE (10–20%) and TBE–urea PAGE (15%). The proteins and nucleic acids were visualized with CBB and SYBR Gold, respectively. We observed three slower-migrating bands that may correspond to covalent IS621–DNA intermediates during recombination (probably IS621–dDNA, IS621–tDNA, and IS621–dDNA–tDNA). Experiments were repeated at least three times with similar results. (b) In vitro DNA recombination experiments. The 38-bp tDNA and 44-bp dDNA substrates (containing no mismatch (WT) or 6-nt mismatches (MM) at positions 2–7 in their top strands) were incubated with the IS621–bRNA complex at 37 °C for 1 h, and then the reaction was analyzed using an 18% TBE–urea gel. The tDNA was labeled with Cy5 at the 5′ end of the top strand. The band intensities of the product and cleaved DNAs were quantified, and the recombination ratios (product DNA / product DNA + cleaved DNA) were calculated. Experiments were repeated at least three times with similar results. (c) Single-particle cryo-EM image processing workflow. (d) Representative 2D averaged class images. (e) Angular distribution of particles in the final reconstruction. (f) Fourier shell correlation (FSC) curves. The map-to-map FSC curve was calculated between the two independently refined half-maps after masking (blue line), and the overall resolution was determined by the gold standard FSC = 0.143 criterion. The map-to-model FSC curve was calculated between the refined atomic model and the full map (red line). (g, h) Cryo-EM density maps, colored according to the local resolution (g) and the protein domains (h). In (h), the active sites with the ordered and disordered S241 residues are indicated by red solid and dashed circles, respectively. (i) Effects of 5′ SL deletion on bRNA binding to the IS621 recombinase. Binding of the purified IS621 recombinase to the bRNA, its reverse complement (RC), or the bRNA lacking the 5′ SL (nucleotides A1–U33) (Δ5′ SL) was analyzed using microscale thermophoresis. Data are shown as mean ± SEM for three technical replicates. (j) Effects of 5′ SL deletion on IS621-mediated recombination in E. coli. Data are shown as mean ± SD for three biological replicates. (k) Distance between TBL and DBL. Nucleotides U96–A109 are disordered in the present structure. Modeling of nucleotides U96–A109 (colored grey) suggests that C98 and C99 are ~40-Å apart, indicating that the TBL and DBL in the synaptic complex structure are derived from two different bRNA molecules.