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ACCURACY.

The accuracy of H++ was evaluated for a set of 23 high quality structures (x-ray structures with resolution < 2.50 and no missing residues in the middle of the protein sequence), see table 1. The experimental values were obtained mainly from: A summary of the measured pK values of the ionizable groups in folded proteins. by G.R. Grimsley, J.M Scholtz, and C.N. Pace; Protein Science, 18(1) 247--251, 2009.

Table 2 shows the results of the membrane-embedded protein bacteriorhodopsin, a system with functionally relevant extreme pK shifts ( see e.g. Proton Affinity Changes Driving Unidirectional Proton Transport in the Bacteriorhodopsin Photocycle. by Alexey Onufriev, A. Smondyrev, and D. Bashford; J. Mol. Biol. 232 , 1183-1193 (2003). ).

Finally table 3 shows the experimental and computed pK values for the internal LYS residues of 12 Variants of staphylococcal nuclease (SNase) at two different values of internal dielectric constants. The PDB codes and experimental pK values of the LYS residues were obtained from Structural Reorganization Triggered by Charging of Lys Residues in the Hydrophobic Interior of a Protein, by M.S Chiment, V.S. Khangulov, A.C Robinson, A. Heroux, A. Majumdar, J.L. Schlessman, and B. Garcia-Moreno; Structure 20 1071-1085, 2012. .

The dataset of 23 protein structures, the bacteriorhodopsin, and the 12 variants of SNase can be downloaded here.


Table 1: RMS error in pK values computed by H++ relative to experiment, based on 23 protein structures with a total of 201 titratable groups. Results are broken down by groups with large ( >1 ) pK shifts (66 groups) and small (≤1 ) pK shifts (135 groups). Table shows the RMS error, accuracy of predicted protonation at pH 7.0, and accuracy of predicted direction of pK shift, compared to experimentally determined values. The Null model assumes a pK shift of 0 from the model compound pK for the purpose of computing RMS error and predicted protonation state. For direction of the predicted pK shift the Null model assumes equal probabilities of +/- shift.
ε_in = 10.0
ΔpK RMS error Prot. accuracy (%) at pH 7 ΔpK direction accuracy (%)
  H++ Null model H++ Null model H++ Null model
≥1 0.98 2.05 97.0 88 95.5 50
<1 1.04 0.51 97.0 95 75.6 50
ε_in = 6.0
ΔpK RMS error Prot. accuracy (%) at pH 7 ΔpK direction accuracy (%)
  H++ Null model H++ Null model H++ Null model
≥1 1.28 2.05 98.5 88 93.9 50
<1 1.39 0.51 97.0 95 74.8 50


Table 2: Experimental and computed pK values of the membrane-embedded Bacteriorhodopsin. We used three values of protein dielectric (ε_in) constant. The structure corresponds to the BR resting state of the photocycle, and is constructed from PDB ID 1QHJ.
    H++
Site Experiment ε_in = 10 ε_in = 6 ε_in = 4
    (default)    
ARG 82 ≥ 13.8 ≥ 12 ≥ 12 ≥ 12
ASP 85 2.6 3.0 3.1 3.2
ASP 96 ≥ 12 6.0 8.9 ≥ 12
ASP 115 ≥ 9.5 6.5 8.4 11.0
ASP 212 ≤ 2.5 ≤ 0 ≤ 0 ≤0
Schiff Base (216) ≥ 12 ≥ 12 ≥ 12 ≥ 12

Table 3: Experimental and computed pK values of internal LYS residues of 12 variants of SNase.
PDBIDSite exp pK H++
ε_in=10ε_in=6
3QOJLYS-23 7.3 8.3 2.3
3ERQLYS-25 6.3 7.5 1.3
3ITPLYS-34 7.1 6.8 3.2
3EJILYS-36 7.2 9.2 6.3
2RKSLYS-38 10.411.4 11.1
3DMULYS-62 8.1 8.3 4.4
3HZXLYS-66 5.6 7.3 1.9
2RBMLYS-72 8.6>12.0>12.0
1TT2LYS-92 5.36.8 0.7
3E5SLYS-1038.2>12.0>12.0
3C1FLYS-1047.7 8.8 7.1
3C1ELYS-1256.2 7.4 <0.0
RMSD relative to experiment 1.86 3.85