Extended Data Fig. 1. Optimization of the LR synaptic complex assembly with various DNA substrates.

a, Schematic showing the Y-35 blunt-end DNA substrate. Complex assembly was attempted (supplying DNA-PKcs, Ku, XLF, and LigIV-XRCC4) prior to purification via RNAse-H elution. b, A representative negative staining raw micrograph of the complex assembled as described in a. The raw micrograph is representative of 24 micrographs. c, Representative 2D class-averages of the complex assembled as described in a, showing the appearance of only DNA-PK complex despite the addition of XLF and LigIV-XRCC4. d-f, Same procedure as a-c, showing the complex assembly with the same Y-35 substrate, but adding XLF and LigIV-XRCC4 to purified DNA-PK complex following RNAse-H elution. The raw micrograph is representative of 27 micrographs. In f, 2D class-averages representing the characteristic view of scarce but existing LR complex are obtained. g-i, Same procedure as a-c, showing the complex assembly using Y30-T40-c8 DNA substrate with 40 nt flexible poly-T and 8 bp of complementary ends as 3’ overhang. While the single-stranded poly-T overhang and the 8-bp complementary region contribute to complex stability, they are not observed in any part of the reconstructed density map, presumably because these ssDNA tethers are too flexible to be aligned with the rest of the complex. The raw micrograph is representative of 24 micrographs. The complex was assembled prior to RNAse-H elution as described in a. In i, the majority of the 2D classes correspond to the LR complex. j, A representative cryo-EM raw micrograph (out of 17,114 in total) of the LR complex assembled with the Y30-T40-c8 DNA substrate shown in g. k, Representative 2D class averages of particles (329,784 in total) contributing to the final reconstruction of the LR complex. l, Silver-stained SDS-PAGE (4–12% gradient, biologically replicated three times) showing the input purified subunits (Ku, DNA-PKcs, LigIV-XRCC4, and XLF) and the RNAse-H purified LR and SR complex for cryo-EM data collection. All representative micrographs in b, e, h, j are from at least three biologically replicated experiments. For gel source data, see Supplementary Figure 1. m, Protein-protein interaction network between the components of the LR complex. Major unmodeled regions are shown in gray. Well-documented hetero- or homo-dimers are grouped by red dashed lines. Alternative protein-protein interactions are depicted by black dashed lines. The globular domain within the Ku80 C-terminal region (CTR) is completely flexible in the LR complex, and we do not see evidence of the Ku80 CTR domain swap observed by Chaplin et al.³⁰. The putative distance between one Ku80 CTR globular domain and the other copy of the Ku80 C-terminal helix is too far to be reached by the 18-amino acid linker within Ku80. Abbreviations: N-HEAT: N-terminal HEAT domain; M-HEAT: middle HEAT domain; KD: kinase domain; vWA: von Willebrand A domain; CTD: C-terminal domain; DBD: DNA-binding domain; NTD: N-terminal domain; OBD: OB-fold domain; BRCTs: tandem BRCT domains; HD: head domain; CC: Coiled-coil domain.