An RNA dynamic ensemble at atomic resolution

Biomolecules do not fold into a single 3D structure but rather form dynamic ensembles of many inter-converting conformations1. Knowledge of dynamic ensembles is key for understanding how biomolecules fold and function, and for rationally manipulating their activities in drug discovery and synthetic biology2-4. However, solving dynamic ensembles of biomolecules at atomic resolution is a major challenge in structural biology because the information required to specify the position of all atoms in thousands of conformations in an ensemble far exceeds the information content of experimental measurements. Here we addressed the data gap and dramatically simplified and accelerated RNA ensemble determination by using structure prediction tools that leverage the growing database of RNA structures to generate a conformational library. Library refinement with NMR residual dipolar couplings enabled determination of an atomic-resolution ensemble for HIV-1 TAR as confirmed by quantum-mechanical calculations of NMR chemical shifts, comparison to a crystal structure of a substate, and through the successful redistribution of the ensemble by design using atomic mutagenesis. The ensemble provides an unprecedented view of how bulge residues cooperatively flip out and undergo sugar repuckering to allow the adjoining helices to stack. The generality of this approach will make determination of atomic-resolution RNA ensembles routine.