RESEARCH ARTICLE Conjoined Use of EM and NMR in RNA Structure Refinement

More than 40 % of the RNA structures have been determined using nuclear magnetic reso-nance (NMR) technique. NMRmainly provides local structural information of protons and works most effectively on relatively small biomacromolecules. Hence structural characteri-zation of large RNAs can be difficult for NMR alone. Electron microscopy (EM) provides global shape information of macromolecules at nanometer resolution, which should be com-plementary to NMR for RNA structure determination. Here we developed a new energy term in Xplor-NIH against the density map obtained by EM. We conjointly used NMR and map restraints for the structure refinement of three RNA systems — U2/U6 small-nuclear RNA, genome-packing motif (ΨCD)2 from Moloney murine leukemia virus, and ribosome-binding element from turnip crinkle virus. In all three systems, we showed that the incorpo-ration of a map restraint, either experimental or generated from known PDB structure, great-ly improves structural precision and accuracy. Importantly, our method does not rely on an initial model assembled from RNA duplexes, and allows full torsional freedom for each nu-cleotide in the torsion angle simulated annealing refinement. As increasing number of mac-romolecules can be characterized by both NMR and EM, the marriage between the two techniques would enable better characterization of RNA three-dimensional structures