Anisotropic NMR Data Acquisition and Validation

Anisotropic NMR data (RDC measurements) were acquired on Bruker 500 MHz Avance IIIHD instrument equipped with a 5mm CPP TCI cryoprobe. RDC measurements were performed using a stretched gel methodology (detailed in [26 (link), 27 (link)]). ¹H-¹⁵N and ¹H-¹³C RDCs were measured as a difference between respective one-bond ¹H-¹⁵N and ¹H-¹³C coupling constants in the isotropic and anisotropic (stretched gel) environments. ¹H-¹⁵N one-bond coupling constants were measured in the ${}^{15}\text{N}$ dimension of ¹H-¹⁵N HSQC spectra (coupled, 90Hz optimized, 76 scans, 122 indirect increments). ¹H-¹³C one-bond coupling constants were measured in the ${}^{13}\text{C}$ dimension of ¹H-¹³C HSQC data (coupled, 145Hz optimized, 16 scans, 1108 indirect increments). Only backbone (NH and $\text{C}\alpha \text{H}$ ) RDCs were used for validation; this simplified the analysis by eliminating the need to account for RDC averaging in flexible side chains.
RDC data were used for an orthogonal validation of generated conformational ensembles and were not used as restraints. For every structure in the ensemble, a single value decomposition (SVD) analysis was utilized to calculate the alignment tensor and the Q-factor, which measures a quality value if a correlation exists between the experimental and back-calculated RDC values [28 (link)].