Protein NMR Structures Refined with Rosetta Have Higher Accuracy Relative to Corresponding X-ray Crystal Structures

Binchen Mao, Roberto Tejero, David Baker, and Gaetano T. Montelione Journal of the American Chemical Society 2014, 136, 1893

Highlighted by +Jan Jensen 

In a previous study Baker, Mentelione and co-workers showed that refining an NMR structure using Rosetta moved it closer to the x-ray structure, but increased the number of restraint violations (i.e. decreased the apparent agreement with the NMR data). A subsequent study of found the same for two additional proteins and raises intriguing questions: 

Do those violated restraints reflect true structural differences between NMR structures and X-ray crystal structures? If that is the case, then would incorporating those NMR experimental restraints into Rosetta refinement drive the NMR structure away from its X-ray counterpart?

This study sets out to answer these questions in by refining 40 NMR structures using Rosetta or Rosettta + distance and dihedral angle restraints and comparing the resulting ensembles to the corresponding x-ray structure. 

It is found that 

unrestrained Rosetta refinement generally decreases the precision of NMR structures, while restrained Rosetta refinement can increase the precision of the side chain heavy atoms of otherwise well-defined residues. Additionally, restrained Rosetta refined structures fit the unassigned NOESY peak list data significantly better than unrestrained Rosetta refined structures. Rosetta refinement can generally improve the stereochemical quality and geometry of NMR structures. More specifically, the experimental backbone dihedral angle restraints can guide Rosetta to generate models with even better backbone structures than is achieved without restraints.

Thus, it is possible to find structural ensembles that agree better with x-ray structures and the measured NMR data.  The refinement protocol allows for only relative limited movement of the protein compared to that used in protein structure determination, but converges much faster (10-20 minutes for a 100-residue protein).  So this extra step add negligible computational cost to conventional NMR protein structure determination, but probably only applicable to relatively high quality NMR structural ensembles.

Intriguingly, the relaxed X-ray structures have lower energies than the restrained-Rosetta structures for most proteins. This means that the sampling is still incomplete. But does it also point to the different between solution phase and crystal structures?  That may be so if the number of restraint violations in the relaxed X-ray structures are larger than for the restraint-refined structures. 

This work is licensed under a Creative Commons Attribution 4.0 International License.