What are the methodological and theoretical prospects for paramagnetic NMR in structural biology? A glimpse into the crystal ball

• Integrating paramagnetic NMR with other techniques improves structural accuracy. • Paramagnetic NMR data provide information on biomolecular conformational variability. • New quantum chemistry theory is questioning the validity of the traditional theory. NMR spectroscopy is very sensitive to the presence of unpaired electrons, which perturb the NMR chemical shifts, J splittings and nuclear relaxation rates. These paramagnetic effects have attracted increasing attention over the last decades, and their use is expected to increase further in the future because they can provide structural information not easily achievable with other techniques. In fact, paramagnetic data provide long range structural restraints that can be used to assess the accuracy of crystal structures in solution and to improve them by simultaneous refinements with the X-ray data. They are also precious for obtaining information on the conformational variability of biomolecular systems, possibly in conjunction with SAXS and/or DEER data. We foresee that new tools will be developed in the next years for the simultaneous analysis of the paramagnetic data with data obtained from different techniques, in order to take advantage synergistically of the information content of all of them. Of course, the use of the paramagnetic data for structural purposes requires the knowledge of the relationship between these data and the molecular coordinates. Recently, the equations commonly used, dating back to half a century ago, have been questioned by first principle quantum chemistry calculations. Our prediction is that further theoretical/computational improvements will essentially confirm the validity of the old semi-empirical equations for the analysis of the experimental paramagnetic data.