Supplementary Figure 2: Additional data to map the interaction surface of SecA with nascent chains on RNC and with post-translational substrates.

a, FRET experiments to monitor the proximity between Cm (blue star)-labeled RNC_RodZ or RNC_phoA and BDP (green star) labeled at indicated positions on SecA. Cm was incorporated at residue 111 immediately upstream of the RodZ TMD (magenta) or residue 4 upstream of the phoA signal sequence (magenta). Top left panel, scheme of the FRET-based binding assay. Lower left panel, SecA residues for acceptor labeling are mapped onto the structure of SecA from this work. b, Representative equilibrium titrations showing the binding of SecA^BDP to Cm-labeled RNC_RodZ. Reactions used 20 nM RNC^Cm (donor) and indicated concentrations of SecA^BDP (acceptor). All titrations saturated above 20 nM SecA, indicating tight binding of all the fluorescently labeled SecA variants. The data for individual SecA variants are colored as in the lower panel of a. c, Summary of FRET efficiency in the complexes formed between the indicated SecA variants and RNC_RodZ or RNC_PhoA. FRET efficiency was calculated at 500 nM SecA^BDP according to Equation 1 in Supplementary Note 1. The data for individual SecA positions are colored as in the lower panel of a. All values represent mean ± s.d., with n = 2 or 3 independent experiments. d,e, Engineered single cysteines at indicated positions on SecA were tested for crosslinking by BMH to the RodZ TMD (residues 104-133) or the phoA signal sequence (residues 1-21) fused to the C-terminus of SUMO (SMT3 residues 1-101). The cysteines on RodZ and PhoA are at the same locations as in Fig. 1a,b, respectively. Crosslinking reactions used 8.3 μM SUMO fusion proteins and 1 μM SecA. Asterisks denote crosslinked products detected by both the anti-SUMO and anti-T7 antibodies. f,g, Crosslinking efficiency from the data in d and e, respectively, are summarized in the structural model of SecA from this work. Crosslinking efficiencies (normalized) were relative to the crosslinked product formed by SecA (C193), based on western-blots against SUMO and strep-tag. Residues are colored based on crosslinking efficiency as indicated. h, Characterization of samples for cryo-EM. RNCs were tested for crosslinking between the indicated cysteines on the nascent chain and SecA (C12). RNC_{6KR_1A9L} contained a model signal sequence 1A9L in place of the RodZ-TMD preceded by six consecutive basic residues derived from residues 104–109 of RodZ. Single bands were observed for both the tRNA-linked nascent chain and crosslinked products with SecA, probably due to the removal of polysomes during preparation of the samples for cryo-EM. Asterisks denote major crosslinked products detected by anti-strep antibody. Crosslinking efficiency was quantified from the ratio of the intensity of crosslinked nascent chain relative to the total intensity of bands containing the nascent chain.