Combining NMR and EPR to Determine Structures of Large RNAs and Protein-RNA Complexes in Solution.

Abstract

Although functional significance of large noncoding RNAs and their complexes with proteins is well recognized, structural information for this class of systems is very scarce. Their inherent flexibility causes problems in crystallographic approaches, while their typical size is beyond the limits of state-of-the-art purely NMR-based approaches. Here, we review an approach that combines high-resolution NMR restraints with lower resolution long-range constraints based on site-directed spin labeling and measurements of distance distribution restraints in the range between 15 and 80Å by the four-pulse double electron-electron resonance (DEER) EPR technique. We discuss sample preparation, the basic assumptions behind data analysis in the EPR-based distance measurements, treatment of the label-based constraints in generation of the structure, and the back-calculation of distance distributions for structure validation. Step-by-step protocols are provided for DEER distance distribution measurements including data analysis and for CYANA based structure calculation using combined NMR and EPR data.