A hybrid protocol combining Rosetta fullatom refinement and Multi-Conformation Continuum Electrostatics (MCCE) to estimate pKa is applied to the blind prediction of 94 mutated residues in Staphylococcal nuclease (SNase), as part of the pKa-cooperative benchmark test. The standard MCCE method is limited to sidechain conformational changes. The Rosetta refinement protocol is used to add the backbone conformational changes in pKa calculations. The non-electrostatic energy component from Rosetta and the electrostatic energy from MCCE are combined to weight the calculated ionization states. Of 63 measured pKas, the root mean squared deviation (RMSD) between the calculated pKas and the measured values is 4.3, showing an improvement compared to the RMSD of 6.6 in the standard MCCE calculations, using a low protein dielectric constant of 4. The breakdown of pKa shift from the solution values (ΔpKa) shows that the desolvation energy contributes the most in the standard MCCE calculations. Lowering desolvation penalties and optimizing electrostatic interactions with the Rosetta/MCCE protocol reduces the ΔpKa to favor the charged states. Analysis also showed that the Rosetta/MCCE protocol samples conformations with pKas close to the solution values. The question remains whether the correct conformational changes coupled to the ionization changes are found here. Nevertheless, a challenge emerges to accurately estimate the reorganization energy, which is not directly measured from the electrostatic environment of the site of interest. Possible improvements to the protocol are also discussed. Proteins 2011; © 2011 Wiley-Liss, Inc.