Reconciling Cryo-EM and Crystallographic Views of RNA Folding through Atomistic Structure-Based Simulations: Roles of Ionic Conditions and Truncation of Flexible Elements.

Discrepancies between biomolecular structures resolved by cryo-electron microscopy (cryo-EM) and X-ray crystallography (XRD) arise from differences in ionic conditions and construct design, yet how these shape RNA folding remains unresolved. The SARS-CoV-2 frameshifting stimulatory element provides a representative case: cryo-EM captures a conformation with the slippery segment, whereas XRD reveals a higher-resolution, coaxially stacked structure lacking this segment but displaying base-triple interactions absent in cryo-EM. To reconcile these condition-dependent views, we integrate explicit-solvent molecular dynamics simulations with a structure-based electrostatic model (STEM) and show that Mg2+ ions drive transitions between these states by stabilizing long-range tertiary interactions involving the slippery site and stem3. Energy landscape analysis further reveals distinct folding pathways, while truncation of the slippery segment reshapes intermediates and yields pathways inconsistent with single-molecule optical tweezers experiments. The STEM framework demonstrates how information from condition-dependent experiments can be integrated to yield a coherent mechanistic picture of RNA folding.