An evaluation review of the prediction of protonation states in proteins versus crystallographic experiment

The known protonation states of protein crystal structures obtained using X-ray and neutron crystallographic data, and including relevant NMR derived experimental information, have been predicted using three pK(a) calculation tools, namely PROPKA, H++ and MCCE. Comparisons between the experimental and predicted protonation states have been carried out in order to assess whether the results are of sufficient quality to validate their use in predicting the protonation states of two key histidine residues in the lobster carapace colouration protein beta-crustacyanin as an example. Significant interest has been shown in the protonation states of these residues, which have been out of reach of experiment thus far and are likely to remain so.