What can we learn by computing ¹³C^α 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 Å 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 ¹³C^α 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 R_free factors similar to those of the deposited X-ray structure, the ¹³C^α 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 ¹³C^α 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 ¹³C^α 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 ¹³C^α 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 ¹³C^α 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 ¹³C^α chemical shifts in solution.