The sequence-energy landscape between naturally occurring TLRs. (A) The sequence-energy landscape for the 11ntR → IC3R conversions (red) and 11ntR → Vc2R conversions (orange) parameterized by the number of mutations from the 11ntR along the x-axis. The y-axis represents ΔΔG relative to the 11ntR (averaged over tectoRNA scaffolds). Each point represents a TLR variant along the conversion landscape, with open circles depicting lower limits for the ΔΔG value. Gray lines connect TLR variants that lie on the same mutational pathway. (B) Distribution of the highest energy intermediate along the mutational pathways from 11ntR to IC3R (left, red) and Vc2R (right, orange). (C,D) The ΔΔG values for TLR sequences along the 11ntR → IC3R pathways (C, red) or the 11ntR → Vc2R pathways (D, orange). ΔΔG values are relative to the 11ntR and averaged over tectoRNA scaffolds; values represent medians of the bootstrapped distributions, and error bars represent 95% confidence interval (CI). The specific mutations in each variant are shown in the tables underneath the bar chart, where blue squares represent the original 11ntR sequence and red/orange squares represent mutated sequences toward the IC3R/Vc2R, respectively. Lighter-colored bars represent lower limits for the ΔΔG measurements since the limit of detection (dashed horizontal line) lies within their CIs. (E,F) The mutational effect of each mutation (ΔΔGmut, measured as ΔΔG from the previous mutational intermediate) along the 11ntR → IC3R conversions (E, red) or 11ntR → Vc2R conversions (F, orange); values represent medians of the bootstrapped distributions, and error bars represent 95% CI (or limits to the CI). The x-axis is grouped by the order in which the mutation is made, e.g., first corresponds to a ΔΔG of a single mutant from the wt 11ntR. Gray bars correspond to upper or lower limits to ΔΔGmut values (negative or positive values, respectively), and each gray “x” refers to mutations for which neither quantitative nor qualitative ΔΔGmut values could be determined.
