Abstract
Determination of torsion angles via recoupling of backbone HC and HN dipolar interactions is a well-known method in magic-angle spinning NMR spectroscopy. Torsion angle values can be obtained by comparing simulated and experimental signals, either in the frequency or time domains. Typically, all molecular orientations are assumed to have identical detected amplitudes at zero recoupling time. The changes in these amplitudes during the recoupling period define the dipolar coupling values and the torsion angles. Experimentally, however, orientations may exhibit different detected amplitudes due to additional cross-polarization (CP) blocks that connect different spins in multidimensional experiments. We numerically and experimentally investigate how CP blocks bias backbone φ torsion angle determination and propose CP conditions that minimize this effect, thereby improving accuracy. Applying these conditions in pseudo-4D (H)CANH experiments yields improved agreement of the extracted angles with X-ray crystallographic data for microcrystalline chicken α-spectrin SH3. For the influenza A M2 membrane protein, we identify an unexpected backbone dihedral angle for the I32 residue, which is consistent with TALOS-N predictions but deviates from ideal α-helical transmembrane geometry.