Abstract
The stability and function of biomolecules are directly influenced by their myriad interactions with water. In this study, we investigated water through cryogenic electron microscopy (cryo-EM) on a highly solvated molecule, the Tetrahymena ribozyme, determined at 2.2 and 2.3 Å resolutions. By employing segmentation-guided water and ion modeling (SWIM), an approach combining resolvability and chemical parameters, we automatically modeled and cross-validated water molecules and Mg(2+) ions in the ribozyme core, revealing the extensive involvement of water in mediating RNA non-canonical interactions. Unexpectedly, in regions where SWIM does not model ordered water, we observed highly similar densities in both cryo-EM maps. In many of these regions, the cryo-EM densities superimpose with complex water networks predicted by molecular dynamics (MD), supporting their assignment as water and suggesting a biophysical explanation for their elusiveness to conventional atomic coordinate modeling. Our study demonstrates an approach to unveil both rigid and flexible waters that surround biomolecules through cryo-EM map densities, statistical and chemical metrics, and MD simulations.