(A) Schematic for obtaining mRNA structure and translation efficiency using DMS-seq, mRNA-seq, and ribosome profiling from the same sample. (B) Plot showing the effect of DMS-seq read coverage on the reproducibility of structure determination. X-axis: DMS-seq read depth cutoff (reads/nucleotide); Y-axis: median of Pearson’s R values calculated by comparing two replicates of in vivo DMS-seq signals of the first 200nt of ORFs passing the DMS-seq depth cutoff indicated in X-axis. A read coverage of ~15 reads/nucleotide is sufficient for reproducible structure determination. (C) Receiver operating characteristic (ROC) curve on the in vivo DMS-seq signals for A and C bases in the 16S rRNA using the E.coli ribosome crystal structure (Zhang et al., 2009) as a model. True positives are defined as bases that are both unpaired and solvent-accessible, and true negatives are bases that are paired. The total number of evaluated A/C bases is 438. Signal threshold of 0.2 has 90% agreement with the crystal structure. (D) Structural prediction for rimM. The predicted rimM structure is based on a minimum free-energy prediction constrained by our DMS-seq measurements, using the same 0.2 threshold used for the 16S rRNA in (B), which agrees with the rimM structure proposed and mutationally verified in Wikström et al. (1992). The DMS-seq signal across rimM is shown below the structure. The color bar indicates the intensity of the DMS-seq signal at each position. (E) Calculation of the Gini index from the DMS-seq signal is indicated schematically by comparing highly structured regions to less structured regions. For a region of mRNA, the cumulative fraction of the total DMS-seq signal is plotted against the cumulative fraction of the total number of positions as a Lorenz Curve. The extent to which the curve sags below the diagonal indicates the degree of inequality of distribution, which is quantified by the Gini index defined as the ratio of the area between the diagonal line and the Lorenz Curve (a) to the area below the diagonal line (a + b). A high Gini index indicates high level of mRNA structure, and vice versa. (F) Histogram of Gini indices of E. coli ORFs calculated from in vivo DMS-seq data at 37°C. All ORFs selected have ≥15 DMS-seq reads/nt (N = 1116). The Gini index of 16S rRNA and rimM, and the mean of Gini indices of in vitro heat-denatured mRNAs at 95°C are indicated.