What can we learn by computing 13Calpha chemical shifts for X-ray protein models?

The room-temperature X-ray structures of ubiquitin (PDB code 1ubq) and of the RNA-binding domain of nonstructural protein 1 of influenza A virus (PDB code 1ail) solved at 1.8 and 1.9 A resolution, respectively, were used to investigate whether a set of conformations rather than a single X-ray structure provides better agreement with both the X-ray data and the observed 13Calpha chemical shifts in solution. For this purpose, a set of new conformations for each of these proteins was generated by fitting them to the experimental X-ray data deposited in the PDB. For each of the generated structures, which show R and Rfree factors similar to those of the deposited X-ray structure, the 13Calpha chemical shifts of all residues in the sequence were computed at the DFT level of theory. The sets of conformations were then evaluated by their ability to reproduce the observed 13Calpha chemical shifts by using the conformational average root-mean-square-deviation (ca-r.m.s.d.). For ubiquitin, the computed set of conformations is a better representation of the observed 13Calpha chemical shifts in terms of the ca-r.m.s.d. than a single X-ray-derived structure. However, for the RNA-binding domain of nonstructural protein 1 of influenza A virus, consideration of an ensemble of conformations does not improve the agreement with the observed 13Calpha chemical shifts. Whether an ensemble of conformations rather than any single structure is a more accurate representation of a protein structure in the crystal as well as of the observed 13Calpha chemical shifts is determined by the dispersion of coordinates, in terms of the all-atom r.m.s.d. among the generated models; these generated models satisfy the experimental X-ray data with accuracy as good as the PDB structure. Therefore, generation of an ensemble is a necessary step to determine whether or not a single structure is sufficient for an accurate representation of both experimental X-ray data and observed 13Calpha chemical shifts in solution.