NMR structure calculation is inherently integrative, and can incorporate new experimental data as restraints. As RNAs have lower proton densities and are more conformational heterogenous than proteins, the refinement of RNA structures can benefit from additional types of restraints. Paramagnetic relaxation enhancement (PRE) provides distance information between a paramagnetic probe and protein or RNA nuclei. However, covalent conjugation of a paramagnetic probe is difficult for RNAs, thus limiting the use of PRE NMR for RNA structure characterization. Here, we show that the solvent PRE can be accurately measured for RNA labile imino protons, simply with the addition of an inert paramagnetic cosolute. Demonstrated on three RNAs that have increasingly complex topologies, we show that the incorporation of the solvent PRE restraints can significantly improve the precision and accuracy of RNA structures. Importantly, the solvent PRE data can be collected for RNAs without isotope enrichment. Thus, the solvent PRE method can work integratively with other biophysical techniques for better characterization of RNA structures.

Chemical cross-linking coupled with mass spectroscopy (CXMS) is a powerful technique for investigating protein structures. CXMS has been mostly used to characterize the predominant structure for a protein, whereas cross-links incompatible with a unique structure of a protein or a protein complex are often discarded. We have recently shown that the so-called over-length cross-links actually contain protein dynamics information. We have thus established a method called DynaXL, which allow us to extract the information from the over-length cross-links and to visualize protein ensemble structures. In this protocol, we present the detailed procedure for using DynaXL, which comprises five steps. They are identification of highly confident cross-links, delineation of protein domains/subunits, ensemble rigid-body refinement, and final validation/assessment. The DynaXL method is generally applicable for analyzing the ensemble structures of multi-domain proteins and protein–protein complexes, and is freely available atwww.tanglab.org/resources.

Chemical cross-linking coupled with mass spectrometry (CXMS) identifies protein residues that are close in space, and has been increasingly used for modeling the structures of protein complexes. Here we show that a single structure is usually sufficient to account for the intermolecular cross-links identified for a stable complex with sub-µmol/L binding affinity. In contrast, we show that the distance between two cross-linked residues in the different subunits of a transient or fleeting complex may exceed the maximum length of the cross-linker used, and the cross-links cannot be fully accounted for with a unique complex structure. We further show that the seemingly incompatible cross-links identified with high confidence arise from alternative modes of protein-protein interactions. By converting the intermolecular cross-links to ambiguous distance restraints, we established a rigid-body simulated annealing refinement protocol to seek the minimum set of conformers collectively satisfying the CXMS data. Hence we demonstrate that CXMS allows the depiction of the ensemble structures of protein complexes and elucidates the interaction dynamics for transient and fleeting complexes.