Extended Data Fig. 7: Optimization of the SR synaptic complex assembly with various DNA substrates.

a, Schematic showing the Y30–c4 DNA substrate with 4-nt 3′ complementary overhang. The complex was assembled before RNase H elution. b, A representative negative-staining raw micrograph of the complex assembled as described in a. The raw micrograph is representative of 23 micrographs. c, Representative two-dimensional class averages of the complex assembled as described in a. d, Cryo-EM map reconstructed from a small dataset using Y30–c4 DNA substrate shown in a. The map is coloured according to the estimation of the local resolution. e–h, Same procedure as a–d, showing the complex assembly with the Y30 blunt-end substrate. Stably assembled SR complexes on DNA substrates with either complementary or blunt ends indicates that these complexes are stable in the absence of any bridging effect from DNA. The raw micrograph is representative of 24 micrographs. i–l, Same procedure as a–d, showing the complex assembly with the Y14–T2–c20–n10–10 substrate, with one central single non-ligatable nick. The raw micrograph is representative of 24 micrographs. Strand e is added last after mixing the two halves together with NHEJ factors. m, A representative cryo-EM raw micrograph (out of 32,723 total images) of the SR complex assembled with the Y14–T2–c20–n10–10 DNA substrate shown in i. n, Representative two-dimensional class averages of particles (175,866 in total) contributing to the final reconstruction of the SR complex. All representative micrographs in b, f, j, m are from at least three biologically replicated experiments. o, Protein–protein interaction network between the components of the SR complex. Major unmodelled regions are shown in grey. Well-documented hetero- or homo-dimers are grouped by red dashed circles. Alternative protein–protein interactions are depicted by black dashed lines.