Three Key Files
After a successful four-step run of MCCE, there are three key files used to interpret MCCE’s output: pK.out, sum_crg.out, and fort.38. In this guide we will look at these files and discuss how they should be interpreted.
fort.38
“fort.38” is a table of conformer occupancies at various pH points. By default, these titrations occur from 0 to 14 along whole numbers, but this can be changed. The pKa saved in pK.out is extrapolated from where the occupancy is .5.
Let’s look at some sample output of fort.38:
ph 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0
NTR01A0001_001 0.000 0.000 0.000 0.000 0.000 0.026 0.183 0.666 0.951 0.993 1.000 1.000 1.000 1.000 1.000
NTR+1A0001_002 1.000 1.000 1.000 1.000 1.000 0.974 0.817 0.334 0.049 0.007 0.000 0.000 0.000 0.000 0.000
Because the occupancies for NTR+A0001 cross .5 between pHs 6 and 7, we can expect the pKa reported in “pK.out” for this residue to fall between 6 and 7.
pK.out
pK.out returns a table of information relating to titrated residues of the original PDB file.
Let’s look at some sample output of pK.out:
pH pKa/Em n(slope) 1000*chi2 vdw0 vdw1 tors ebkb dsol offset pHpK0 EhEm0 -TS residues total
NTR+A0001_ 6.682 0.973 0.029 -0.00 -0.05 -0.26 0.64 4.31 -0.95 -1.32 0.00 0.00 -1.30 1.08
LYS+A0001_ 9.503 1.006 0.030 -0.03 -0.00 0.00 0.07 0.59 0.29 -0.90 0.00 0.00 -0.02 -0.00
ARG+A0005_ 12.841 0.858 0.061 -0.05 -0.01 0.00 -0.74 0.79 0.00 0.34 0.00 0.23 -0.34 0.23
TYR-A0053_ >14.0 0.00 0.00 0.00 -1.69 4.65 -0.37 -3.80 0.00 0.00 7.32 6.12
Column Descriptions
-
pH
Name of the titrated residue. -
pKa/Em
pH of the pKa. -
n (slope)
Slope of titration curve (extrapolated fromfort.38). -
1000×chi2
1000 times the chi-squared value. Higher the number, the less accurate the result. -
vdw0
Van der Waals interaction within the residue. Usually contributes minimally to pKa. -
vdw1
Van der Waals interaction to the protein backbone. Usually contributes minimally to pKa. -
tors
Side chain torsion energy. Usually contributes minimally to pKa. -
ebkb
Protein backbone electrostatic interaction.
The protein secondary structure—especially helices—has a dipole that may affect the ionized form more than the neutral form, therefore it could be a factor in pKa. -
dsolv
The desolvation energy.
The ionized residue is less stabilized in the protein than in solution. This makes ionization inside the protein harder and links to a positive free energy for the reaction from the neutral residue to the ionized residue. -
offset
The impact of any extra term fromextra.tplon the residue. Can be changed by the user to control for bias. - pHpK0
Solution pH effect on ionization. It is the environmental pressure on residue ionization.- For an acid, low solution pH makes ionization (releasing a proton) easy, so it contributes favorable energy.
- For a base, low pH makes ionization harder.
- When pH equals the residue’s solution pKa, the environment pH is at a balance point, where the contribution is 0.
-
EhEm0
Environment Eh effect on redox reaction. This works similarly to pHpK0. - TS
Entropy term.
The number of rotamers of neutral and ionized residues generated by MCCE may differ. The effect of different rotamer counts on the two ionization states acts like entropy.- Since this may be undesirable, entropy correction is enabled by default in step 4’s Monte Carlo sampling.
- When entropy correction is enabled in MC, the entropy effect has been eliminated and entropy should be set to 0 in MFE analysis.
- The tool
mfe.pycan detect how MC was done and handle this accordingly, or one can turn it on/off in MFE manually.
-
residues
Total pairwise interaction from other residues.
Other residues may shift the ionization free energy depending on their dipole orientation and charge. - Total
Total free energy of the ionization reaction. It is the sum of all terms above.
sum_crg.out
sum_crg.out simply sums up the charges at each pH of each residue. Again, this information is derived from fort.38.
Let’s look at some sample output of sum_crg.out:
pH 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
NTR+A0001_ 1.00 1.00 1.00 1.00 1.00 1.00 0.97 0.74 0.23 0.04 0.01 0.00 0.00 0.00 0.00
LYS+A0001_ 1.00 1.00 1.00 1.00 1.00 1.00 1.00 0.99 0.95 0.72 0.23 0.03 0.00 0.00 0.00
ARG+A0005_ 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 0.99 0.90 0.47 0.08
GLU-A0007_ -0.00 -0.00 -0.00 -0.06 -0.43 -0.88 -0.99 -1.00 -1.00 -1.00 -1.00 -1.00 -1.00 -1.00 -1.00
LYS+A0013_ 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 0.97 0.76 0.23 0.03 0.00
ARG+A0014_ 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 0.99 0.89 0.49 0.09
HIS+A0015_ 1.00 1.00 1.00 1.00 1.00 0.97 0.76 0.25 0.03 0.00 0.00 0.00 0.00 0.00 0.00
ASP-A0018_ -0.00 -0.00 -0.03 -0.20 -0.68 -0.95 -0.99 -1.00 -1.00 -1.00 -1.00 -1.00 -1.00 -1.00 -1.00
TYR-A0020_ -0.00 -0.00 -0.00 -0.00 -0.00 -0.00 -0.00 -0.00 -0.01 -0.06 -0.35 -0.72 -0.92 -0.99 -1.00
ARG+A0021_ 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 0.99 0.96 0.70 0.18
TYR-A0023_ -0.00 -0.00 -0.00 -0.00 -0.00 -0.00 -0.00 -0.00 -0.00 -0.05 -0.30 -0.78 -0.97 -1.00 -1.00
LYS+A0033_ 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 0.96 0.72 0.22 0.03 0.00 0.00
GLU-A0035_ -0.00 -0.00 -0.00 -0.03 -0.19 -0.63 -0.94 -0.99 -1.00 -1.00 -1.00 -1.00 -1.00 -1.00 -1.00
ARG+A0045_ 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 0.98 0.86 0.48 0.12
ASP-A0048_ -0.00 -0.01 -0.11 -0.52 -0.91 -0.99 -1.00 -1.00 -1.00 -1.00 -1.00 -1.00 -1.00 -1.00 -1.00
ASP-A0052_ -0.00 -0.00 -0.01 -0.08 -0.45 -0.85 -0.98 -1.00 -1.00 -1.00 -1.00 -1.00 -1.00 -1.00 -1.00
TYR-A0053_ -0.00 -0.00 -0.00 -0.00 -0.00 -0.00 -0.00 -0.00 -0.00 -0.00 -0.00 -0.00 -0.15 -0.49 -0.76
ARG+A0061_ 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 0.99 0.95 0.71 0.22
ASP-A0066_ -0.00 -0.05 -0.27 -0.77 -0.96 -0.99 -1.00 -1.00 -1.00 -1.00 -1.00 -1.00 -1.00 -1.00 -1.00
ARG+A0068_ 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 0.96 0.81 0.46
ARG+A0073_ 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 0.99 0.89 0.47 0.10
ASP-A0087_ -0.00 -0.00 -0.02 -0.15 -0.65 -0.95 -0.99 -1.00 -1.00 -1.00 -1.00 -1.00 -1.00 -1.00 -1.00
LYS+A0096_ 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 0.98 0.90 0.64 0.21 0.03 0.00
LYS+A0097_ 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 0.99 0.89 0.50 0.11 0.01 0.00
ASP-A0101_ -0.00 -0.00 -0.00 -0.04 -0.29 -0.80 -0.97 -1.00 -1.00 -1.00 -1.00 -1.00 -1.00 -1.00 -1.00
ARG+A0112_ 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 0.98 0.85 0.33 0.04
ARG+A0114_ 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 0.99 0.90 0.46 0.08
LYS+A0116_ 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 0.95 0.67 0.17 0.02 0.00 0.00
ASP-A0119_ -0.00 -0.00 -0.03 -0.21 -0.73 -0.96 -0.99 -1.00 -1.00 -1.00 -1.00 -1.00 -1.00 -1.00 -1.00
ARG+A0125_ 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 0.99 0.90 0.52 0.11
ARG+A0128_ 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 0.97 0.80 0.31 0.05
CTR-A0129_ -0.00 -0.03 -0.23 -0.74 -0.96 -1.00 -1.00 -1.00 -1.00 -1.00 -1.00 -1.00 -1.00 -1.00 -1.00
----------
Net_Charge 18.99 18.91 18.30 16.19 12.75 9.97 8.87 7.99 7.19 6.54 4.73 1.68 -1.57 -6.66 -11.22
Protons 18.99 18.91 18.30 16.19 12.75 9.97 8.87 7.99 7.19 6.54 4.73 1.68 -1.57 -6.66 -11.22
Electrons 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00