EPR-aided approach for solution structure determination of large RNAs or protein–RNA complexes

High-resolution structural information on RNA and its functionally important complexes with proteins is dramatically underrepresented compared with proteins but is urgently needed for understanding cellular processes at the molecular and atomic level. Here we present an EPR-based protocol to help solving large RNA and protein–RNA complex structures in solution by providing long-range distance constraints between rigid fragments. Using enzymatic ligation of smaller RNA fragments, large doubly spin-labelled RNAs can be obtained permitting the acquisition of long distance distributions (>80 Å) within a large protein–RNA complex. Using a simple and fast calculation in torsion angle space of the spin-label distributions with the program CYANA, we can derive simple distance constraints between the spin labels and use them together with short-range distance restraints derived from NMR to determine the structure of a 70 kDa protein–RNA complex composed of three subcomplexes.