The three-dimensional conformations of non-coding RNAs underpin their biochemical functions but have largely eluded experimental characterization. Here, we report that integrating a classic mutation/rescue strategy with high-throughput chemical mapping enables rapid RNA structure inference with unusually strong validation. We revisit a paradigmatic 16S rRNA domain for which SHAPE (selective 2´-hydroxyl acylation with primer extension) suggested a conformational change between apo- and holo-ribosome conformations. Computational support estimates, data from alternative chemical probes, and mutate-and-map (M2) experiments expose limitations of prior methodology and instead give a near-crystallographic secondary structure. Systematic interrogation of single base pairs via a high-throughput mutation/rescue approach then permits incisive validation and refinement of the M2-based secondary structure and further uncovers the functional conformation as an excited state (25±5% population) accessible via a single-nucleotide register shift. These results correct an erroneous SHAPE inference of a ribosomal conformational change and suggest a general mutate-map-rescue approach for dissecting RNA dynamic structure landscapes.