Protein and RNA preparation
The IS621 recombinase gene was cloned into a modified pFastBac1 expression vector (Thermo Fisher Scientific), which encodes an N-terminal His8 tag, a Twin-Strep tag and a human rhinovirus 3C protease cleavage site (Supplementary Table 1). The IS621 protein was expressed in Sf9 cells (Thermo Fisher Scientific), using the Bac-to-Bac baculovirus expression system (Thermo Fisher Scientific). Sf9 cells were cultured in Sf900II medium (Thermo Fisher Scientific), infected with the recombinant baculovirus at a density of approximately 2 × 106 cells per millilitre, and then incubated at 27 °C for 48 h. The cells were collected by centrifugation at 5,000g and stored at −80 °C before use. The Sf9 cells were lysed by sonication in lysis buffer (20 mM Tris-HCl (pH 7.5), 1 M NaCl, 2 mM MgCl2, 2% Triton X-100 and protease inhibitor cocktail), and the lysate was clarified by centrifugation at 40,000g. The supernatant was mixed with Strep-Tactin XT resin (IBA) at 4 °C for 1 h. The resin was washed with wash buffer (20 mM Tris-HCl (pH 7.5), 500 mM NaCl, 2 mM MgCl2, 3 mM 2-mercaptoethanol and 10% glycerol), and the protein was eluted with wash buffer containing 80 mM biotin. The eluted protein was purified by size-exclusion chromatography on a Superdex 200 Increase 10/300 GL column (Cytiva), equilibrated with buffer (20 mM Tris-HCl (pH 7.5), 500 mM NaCl, 1 mM dithiothreitol (DTT), 10% glycerol and 2 mM MgCl2). The peak fractions were collected and stored at −80 °C until use. The S241A and D11A/E60A/D102A/D105A mutants were similarly expressed and purified. The bRNAs were transcribed in vitro with T7 RNA polymerase and purified by 10% denaturing (7 M urea) PAGE (Supplementary Table 1).
Synaptic complex preparation
The IS621–bRNA–dDNA–tDNA synaptic complex was reconstituted by mixing the purified IS621 recombinase, a 177-nt bRNA (177 nt plus 5′-GGG for in vitro transcription), a 44-bp dDNA and a 38-bp tDNA, at a molar ratio of 4:1:1:1. To obtain a synaptic complex with the WT bRNA (pre-strand exchange state), six mismatches were introduced into the top strands of the tDNA and dDNA (Supplementary Table 1). To obtain a synaptic complex with the pre-HSB bRNA (pre-strand exchange locked state) or the post-HSB bRNA (post-strand exchange state), four mismatches were introduced into the top strands of the tDNA and dDNA (Supplementary Table 1). The IS621–bRNA–dDNA–tDNA synaptic complex was purified by size-exclusion chromatography on a Superose 6 Increase 10/300 column (Cytiva), equilibrated with buffer (20 mM Tris-HCl (pH 7.5), 300 mM NaCl, 5 mM MgCl2 and 1 mM DTT). The peak fraction containing the synaptic complex was concentrated to 0.5–1 mg ml−1, using an Amicon Ultra-4 Centrifugal Filter Unit (MWCO 50 kDa; Millipore). Protein concentrations were measured by the Pierce 660 nm Protein Assay Reagent (Thermo Fisher Scientific).
Cryo-EM analysis
The grids were glow-discharged in low-pressure air at a 10-mA current in a PIB-10 ion generator (Vacuum Device). The synaptic complex solution was applied to a freshly glow-discharged Quantifoil Holey Carbon Grid (R1.2/1.3, Au, 300 mesh) (SPT Labtech) using a Vitrobot Mark IV system (Thermo Fisher Scientific) at 4 °C, with a waiting time of 10 s and a blotting time of 6 s under 100% humidity conditions. The grids were plunge-frozen in liquid ethane cooled at liquid nitrogen temperature.
The grids containing the synaptic complex with the WT bRNA or the pre-HSB bRNA were transferred to a Titan Krios G3i electron microscope (Thermo Fisher Scientific) running at 300 kV and equipped with a Gatan Quantum-LS Energy Filter (GIF) and a Gatan K3 Summit direct electron detector. The grid containing the synaptic complex with the post-HSB bRNA was transferred to a Titan Krios G4 electron microscope (Thermo Fisher Scientific) running at 300 kV and equipped with a Gatan Quantum-LS Energy Filter and a Gatan K3 Summit direct electron detector. Imaging was performed at a nominal magnification of ×105,000, corresponding to a calibrated pixel size of 0.83 Å per pixel. For the synaptic complex with the WT bRNA, each movie was dose fractionated to 50 frames and recorded using the correlated double-sampling mode at a dose rate of 7.7 e− px−1 s−1, resulting in a total accumulated exposure of 50 e− Å−2 of the specimen. For the synaptic complex with the pre-HSB bRNA, each movie was dose fractionated to 48 frames and recorded using the normal mode at a dose rate of 14.7 e− px−1 s−1, resulting in a total accumulated exposure of 49 e− Å−2 of the specimen. For the synaptic complex with the post-HSB bRNA, each movie was dose fractionated to 64 frames and recorded using the correlated double-sampling mode at a dose rate of 9.5 e− px−1 s−1, resulting in a total accumulated exposure of 62 e− Å−2 of the specimen. The data were automatically acquired using the image-shift method in the EPU software (Thermo Fisher Scientific), with a defocus range of −0.8 to −2.0 μm.
The data were processed using the cryoSPARC v4.3.0 software package34. The dose-fractionated movies were aligned using Patch motion correction, and the contrast transfer function (CTF) parameters were estimated using patch-based CTF estimation. For the synaptic complex with the WT bRNA, particles were automatically picked using Blob picker and template picker, followed by reference-free 2D classification to curate particle sets. The particles were further curated by heterogeneous refinement, using the map derived from cryoSPARC ab initio reconstruction as the template. The best-class particle set was refined using non-uniform refinement, yielding a map at 2.58 Å resolution. Local motion correction followed by non-uniform refinement with CTF value optimization yielded a map at 2.52 Å resolution, according to the Fourier shell correlation (FSC) = 0.143 criterion35. The local resolution was estimated by BlocRes in cryoSPARC.
For the synaptic complex with the pre-HSB bRNA, particles were automatically picked using template picker, followed by reference-free 2D classification of the WT bRNA sets. The particles were further curated by heterogeneous refinement, using the WT bRNA maps as a template. The best-class particle set was refined using homogeneous refinement and non-uniform refinement, yielding a map at 2.79 Å resolution. Local motion correction followed by non-uniform refinement with CTF value optimization yielded a map at 2.72 Å resolution, according to the FSC = 0.143 criterion. The local resolution was estimated by BlocRes in cryoSPARC.
For the synaptic complex with the post-HSB bRNA, particles were automatically picked using template picker, followed by reference-free 2D classification of the WT bRNA datasets. The particles were further curated by heterogeneous refinement, using the WT bRNA maps as a template. To further distinguish the conformational heterogeneity, the selected particles after homogeneous refinement were divided into four classes using 3D classification. The particle sets in the two selected classes were further refined using homogeneous refinement and heterogeneous refinement. Non-uniform refinement with CTF value optimization yielded maps at 2.88 Å resolution (state 1) and 2.73 Å resolution (state 2), according to the FSC = 0.143 criterion. The local resolution was estimated by BlocRes in cryoSPARC.
Model building and validation
The models of the IS621–bRNA–dDNA–tDNA synaptic complexes were manually built using COOT36, starting from a model predicted by ColabFold37. The models were refined using phenix.real_space_refine38 and Servalcat39 against unsharpened half maps. The models were validated using MolProbity40. The statistics of the 3D reconstruction and model refinement are summarized in Extended Data Table 1. The molecular graphics and cryo-EM density map figures were prepared with CueMol (http://www.cuemol.org) or UCSF ChimeraX41.
In vitro recombination assays
For in vitro recombination assays, linear tDNA and dDNA substrates were synthesized by Eurofins Genomics, and labelled with FAM or Cy5 at the 5′ end of the top or bottom strand (Supplementary Table 1). The tDNA (0.1 μM) and dDNA (0.1 μM) substrates were mixed with the pre-incubated IS621–bRNA complex (1.4 μM) in 100 µl buffer (20 mM Tris-HCl (pH 7.5), 300 mM NaCl, 5 mM MgCl2 and 1 mM DTT), and then the reactions were incubated at 37 °C for 1 h. The reaction mixture was mixed with proteinase K (Nacalai Tesque) and then boiled at 95 °C for 3 min in denaturing buffer (7 M urea). The samples were analysed by 18% urea-PAGE, and fluorescent signals were imaged using FUSION Solo S (Vilber Bio Imaging). For gel source data, see Supplementary Fig. 7.
Microscale thermophoresis
Microscale thermophoresis experiments were performed using a Monolith NT.115pico Series instrument (NanoTemper), as previously described2. The IS621 recombinase was labelled using the RED-MALEIMIDE 2nd generation cysteine reactive kit (NanoTemper). The labelled protein was eluted in buffer (20 mM Tris-HCl (pH 7.5), 500 mM NaCl, 5 mM MgCl2, 1 mM DTT and 0.01% Tween 20). To determine the affinity of the IS621 recombinase for RNA, 20 nM recombinase was incubated with a dilution series (0.076–250 nM) of the bRNA (177 nt), its reverse complement (177 nt) or the bRNA mutant lacking the 5′ stem loop (146 nt). Microscale thermophoresis experiments were performed at 37 °C using premium capillaries (NanoTemper) at medium microscale thermophoresis power with the LED excitation power set to automatic (excitation ranged from 10% to 50%). Data were analysed using the NanoTemper MO.affinity analysis software package, and raw data were plotted using GraphPad Prism 9 (GraphPad).
Bacterial recombination assays
Bacterial recombination assays were performed, as previously described2. In brief, E. coli BL21(DE3) cells (NEB) were co-transformed with a pTarget plasmid encoding a target sequence and a T7-inducible IS621 recombinase, and a pDonor plasmid encoding a bRNA, a donor sequence and GFP, such that, upon recombination into pRecombinant, GFP expression would be activated by the synthetic Bba_R0040 promoter adjacent to the target site (Supplementary Table 1). pDonor encodes the WT IS621 donor sequences and pTarget encodes a DNA sequence not found in the E. coli genome, and the bRNA was programmed to recombine these two DNA sequences. Co-transformed cells were plated on fresh LB agar containing kanamycin, chloramphenicol and 0.07 mM IPTG to induce recombinase expression. Plates were incubated at 37 °C for 16 h and then at room temperature for 8 h. Hundreds of colonies were scraped from the plate, resuspended in terrific broth and diluted to an appropriate concentration for flow cytometry. About 5 × 104 cells were analysed on a NovoCyte Quanteon Flow Cytometer to assess the fluorescence intensity of GFP-expressing cells (Supplementary Fig. 8). The mean fluorescence intensity of the population (including both GFP+ and GFP− cells) was plotted.
Covariation analysis
Covariation analysis to identify base-pairing potential between bRNA and tDNA or dDNA was performed, as previously described2. In brief, IS621 orthologue sequences were searched (blastp) against a curated database of IS110 elements extracted from publicly available genomic sequence archives42. Next, a covariance model (CM) of the bRNA primary and secondary structures was used to identify homologues of the bRNA sequence in the non-coding ends of these orthologous sequences43. Target and donor sequences centred around the predicted core were extracted. Predicted bRNA sequences were aligned using the cmalign tool in the Infernal package. Two paired alignments were then generated that contained concatenated target and bRNA sequences, and concatenated donor and bRNA sequences. These alignments were analysed using CCMpred (‘-n 100’) to identify covarying nucleotides between target–donor and bRNA sequences44. These covariation scores were normalized and multiplied by the sign of a base-pairing concordance score to produce the covariation score scale, which ranged from −1 (top strand base pairing) to +1 (bottom strand base pairing).
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.