Mainan, A.; Roy, S.; Kirmizialtin, S.

Discrepancies between biomolecular structures resolved by cryo-electron microscopy (cryo-EM) and X-ray crystallography (XRD) often arise from differences in ionic conditions and construct design, yet their mechanistic impact on RNA folding remains unclear. In the SARS-CoV-2 frameshifting stimulatory element, cryo-EM and XRD structures reveal distinct pseudoknot conformations: a bent and a coaxially stacked state, complicating its structure-function relationship. Here, combining all-atom explicit-solvent simulation results with a structure-based electrostatic model, we show that Mg2+; ions drive transitions between these states by stabilizing long-range tertiary interactions and modulating local dynamical coupling involving the slippery site and stem3. Energy landscape analysis reveals distinct folding pathways, while deletion of the slippery segment in crystallographic constructs alters intermediates and produces pathways inconsistent with single-molecule optical tweezer experiments. This study demonstrates how condition-dependent experiments encode complementary interaction-level information and how physics-based computational approaches integrate these to yield a coherent, mechanistic picture of RNA folding.