Fig. 1: Overview of the SHAMAN approach.

A Input stage: Selection of the RNA target structure and of the probes to initialize the mother and replica systems, each one with a different probe. B Production stage: the unbiased/unrestrained MD simulation of the mother system communicates the positions of the RNA backbone atoms to the replicas, which are restrained to follow the mother like shadows. The probe exploration of the RNA conformation is accelerated by metadynamics. C Analysis stage (from top to bottom): (i) the sampled RNA ensemble is clustered into a set of representative conformations; (ii) for each cluster and probe, a free-energy map is calculated from the probe occupancy during the course of the simulation; (iii) voxels in the free-energy maps are clustered together into interacting sites; (iv) for each interacting site, free energy and buriedness score are calculated and sites too exposed to solvent are discarded; (v) for each RNA cluster, all interacting sites obtained from all probes are clustered together into SHAMAPs. D Output stage: two RNA representative clusters with population equal to 32% (light brown, left panel) and 28% (pink, right panel) with the corresponding SHAMAPs (green circles). For each SHAMAP, we provide the binding free energy to RNA (\(\varDelta G\)) and the difference with respect to the lowest free energy (top scored).