Protein-Cofactor Docking

Hello Everyone,
I was trying to perform docking of a NAD cofactor to my protein of interest. But whenever i run the HADDOCK server, i get an error message saying :

“There was an inconsistency in your data
Error message
Second pdb file contains multiple forms of the same residue. This is not supported in the current form. If you would like to supply multiple conformations, please create an ensemble
**ATOM 2177 PA ANAD A 1 7.894 -8.750 -4.846 0.80 9.95 P”
OR
“There was an inconsistency in your data
Error message
Second pdb file is incorrectly formatted. Please align the columns of your PDB properly”.

My co-factor pdb file looks like:
HETATM 2177 PA ANAD A 1 7.894 -8.750 -4.846 0.80 9.95 P
HETATM 2178 O1AANAD A 1 6.602 -8.421 -4.177 0.80 10.48 O
HETATM 2179 O2AANAD A 1 9.095 -7.874 -5.020 0.80 9.13 O
HETATM 2180 O5BANAD A 1 7.599 -9.298 -6.354 0.80 11.17 O
HETATM 2181 C5BANAD A 1 6.411 -10.021 -6.665 0.80 11.21 C
HETATM 2182 C4BANAD A 1 6.289 -10.222 -8.167 0.80 11.06 C
HETATM 2183 O4BANAD A 1 5.134 -11.007 -8.428 0.80 11.79 O
HETATM 2184 C3BANAD A 1 6.072 -8.916 -8.851 0.80 11.59 C
HETATM 2185 O3BANAD A 1 6.853 -8.905 -10.030 0.80 11.19 O
HETATM 2186 C2BANAD A 1 4.586 -8.877 -9.086 0.80 12.25 C
HETATM 2187 O2BANAD A 1 4.350 -8.085 -10.213 0.80 13.50 O
HETATM 2188 C1BANAD A 1 4.253 -10.322 -9.334 0.80 12.03 C
HETATM 2189 N9AANAD A 1 2.942 -10.854 -8.942 0.80 12.31 N
HETATM 2190 C8AANAD A 1 2.313 -10.700 -7.756 0.80 12.31 C
HETATM 2191 N7AANAD A 1 1.180 -11.455 -7.746 0.80 13.76 N
HETATM 2192 C5AANAD A 1 1.067 -12.072 -8.943 0.80 12.69 C
HETATM 2193 C6AANAD A 1 0.122 -12.940 -9.661 0.80 13.17 C
HETATM 2194 N6AANAD A 1 -0.979 -13.399 -9.070 0.80 12.23 N
HETATM 2195 N1AANAD A 1 0.418 -13.340 -10.930 0.80 13.06 N
HETATM 2196 C2AANAD A 1 1.513 -12.912 -11.553 0.80 13.33 C
HETATM 2197 N3AANAD A 1 2.396 -12.108 -10.996 0.80 12.93 N
HETATM 2198 C4AANAD A 1 2.239 -11.675 -9.717 0.80 12.46 C
HETATM 2199 O3 ANAD A 1 8.314 -10.172 -4.133 0.80 9.86 O
HETATM 2200 PN ANAD A 1 9.706 -11.006 -4.202 0.80 11.10 P
HETATM 2201 O1NANAD A 1 10.318 -10.953 -5.573 0.80 11.19 O
HETATM 2202 O2NANAD A 1 9.381 -12.346 -3.568 0.80 10.61 O
HETATM 2203 O5DANAD A 1 10.626 -10.380 -3.082 0.80 13.23 O
HETATM 2204 C5DANAD A 1 11.670 -9.440 -3.106 0.80 20.65 C
HETATM 2205 C4DANAD A 1 11.416 -8.566 -1.892 0.80 23.06 C
HETATM 2206 O4DANAD A 1 11.760 -9.260 -0.716 0.80 23.57 O
HETATM 2207 C3DANAD A 1 12.166 -7.244 -1.830 0.80 27.59 C
HETATM 2208 O3DANAD A 1 11.456 -6.273 -2.603 0.80 30.98 O
HETATM 2209 C2DANAD A 1 12.190 -6.967 -0.327 0.80 26.74 C
HETATM 2210 O2DANAD A 1 11.443 -5.800 0.085 0.80 26.31 O
HETATM 2211 C1DANAD A 1 11.608 -8.259 0.268 0.80 24.01 C
HETATM 2212 N1NANAD A 1 12.330 -8.608 1.479 0.80 23.61 N
HETATM 2213 C2NANAD A 1 13.472 -9.249 1.333 0.80 23.62 C
HETATM 2214 C3NANAD A 1 14.215 -9.578 2.454 0.80 22.51 C
HETATM 2215 C7NANAD A 1 15.479 -10.272 2.125 0.80 23.68 C
HETATM 2216 O7NANAD A 1 16.019 -10.027 1.033 0.80 21.89 O
HETATM 2217 N7NANAD A 1 15.953 -11.127 3.014 0.80 21.48 N
HETATM 2218 C4NANAD A 1 13.762 -9.299 3.752 0.80 21.12 C
HETATM 2219 C5NANAD A 1 12.554 -8.623 3.868 0.80 20.47 C
HETATM 2220 C6NANAD A 1 11.850 -8.292 2.721 0.80 22.27 C

I tried to change the name from HETATM to ATOM but it also shows the above mentioned error report. I have tried all possible ways to rename and also tried the ways suggested in the forum. But no one is working for me. Please let me know the exact solution which would make the cofactor dockable to my protein of interest. I am still not sure if there is anything wrong with the co-factor pdb file naming or numbering. Thanks for your valuable time and advice.

Check carefully your columns in the PDB file… Something looks wrong.
Did you manually edit the file?

Compare with a file freshly downloaded from the PDB.

And/or try to read it in Pymol and write it out again as PBD file

Dear Prof. Bonvin,

The NAD co-factor is a part of the crystal structure which was in complex with the protein. I have deleted the protein from the complex and saved this as NAD.pdb file in order to dock with my protein of interest. I have opened the file in chimera and then i re-saved as pdb and then tried to dock again. The re-formatted file with Chimera looks like this:
HETATM 1 PA ANAD 1392 7.291 -8.893 -5.018 0.80 9.95 P
HETATM 2 O1AANAD 1392 6.264 -8.692 -3.955 0.80 10.48 O
HETATM 3 O2AANAD 1392 8.113 -7.857 -5.718 0.80 9.13 O
HETATM 4 O5BANAD 1392 6.643 -9.756 -6.242 0.80 11.17 O
HETATM 5 C5BANAD 1392 5.630 -10.732 -6.011 0.80 11.21 C
HETATM 6 C4BANAD 1392 5.058 -11.227 -7.329 0.80 11.06 C
HETATM 7 O4BANAD 1392 4.108 -12.247 -7.058 0.80 11.79 O
HETATM 8 C3BANAD 1392 4.315 -10.134 -8.017 0.80 11.59 C
HETATM 9 O3BANAD 1392 4.622 -10.196 -9.396 0.80 11.19 O
HETATM 10 C2BANAD 1392 2.874 -10.416 -7.685 0.80 12.25 C
HETATM 11 O2BANAD 1392 2.087 -9.904 -8.720 0.80 13.50 O
HETATM 12 C1BANAD 1392 2.835 -11.918 -7.638 0.80 12.03 C
HETATM 13 N9AANAD 1392 1.903 -12.603 -6.733 0.80 12.31 N
HETATM 14 C8AANAD 1392 1.697 -12.351 -5.421 0.80 12.31 C
HETATM 15 N7AANAD 1392 0.851 -13.289 -4.911 0.80 13.76 N
HETATM 16 C5AANAD 1392 0.490 -14.127 -5.908 0.80 12.69 C
HETATM 17 C6AANAD 1392 -0.404 -15.273 -6.127 0.80 13.17 C
HETATM 18 N6AANAD 1392 -1.094 -15.811 -5.124 0.80 12.23 N
HETATM 19 N1AANAD 1392 -0.469 -15.839 -7.366 0.80 13.06 N
HETATM 20 C2AANAD 1392 0.212 -15.338 -8.392 0.80 13.33 C
HETATM 21 N3AANAD 1392 1.011 -14.295 -8.292 0.80 12.93 N
HETATM 22 C4AANAD 1392 1.198 -13.669 -7.099 0.80 12.46 C
HETATM 23 O3 ANAD 1392 8.257 -10.055 -4.367 0.80 9.86 O
HETATM 24 PN ANAD 1392 9.700 -10.614 -4.861 0.80 11.10 P
HETATM 25 O1NANAD 1392 9.778 -10.703 -6.358 0.80 11.19 O
HETATM 26 O2NANAD 1392 9.941 -11.850 -4.014 0.80 10.61 O
HETATM 27 O5DANAD 1392 10.766 -9.631 -4.236 0.80 13.23 O
HETATM 28 C5DANAD 1392 11.481 -8.534 -4.745 0.80 20.65 C
HETATM 29 C4DANAD 1392 11.452 -7.514 -3.623 0.80 23.06 C
HETATM 30 O4DANAD 1392 12.331 -7.902 -2.593 0.80 23.57 O
HETATM 31 C3DANAD 1392 11.837 -6.087 -3.984 0.80 27.59 C
HETATM 32 O3DANAD 1392 10.696 -5.425 -4.535 0.80 30.98 O
HETATM 33 C2DANAD 1392 12.303 -5.537 -2.636 0.80 26.74 C
HETATM 34 O2DANAD 1392 11.482 -4.473 -2.101 0.80 26.31 O
HETATM 35 C1DANAD 1392 12.286 -6.782 -1.735 0.80 24.01 C
HETATM 36 N1NANAD 1392 13.437 -6.760 -0.850 0.80 23.61 N
HETATM 37 C2NANAD 1392 14.580 -7.193 -1.342 0.80 23.62 C
HETATM 38 C3NANAD 1392 15.715 -7.165 -0.549 0.80 22.51 C
HETATM 39 C7NANAD 1392 16.920 -7.660 -1.250 0.80 23.68 C
HETATM 40 O7NANAD 1392 16.982 -7.526 -2.484 0.80 21.89 O
HETATM 41 N7NANAD 1392 17.858 -8.233 -0.516 0.80 21.48 N
HETATM 42 C4NANAD 1392 15.677 -6.740 0.787 0.80 21.12 C
HETATM 43 C5NANAD 1392 14.455 -6.290 1.272 0.80 20.47 C
HETATM 44 C6NANAD 1392 13.346 -6.314 0.441 0.80 22.27 C
CONECT 12 7 10 13
CONECT 35 30 33 36
CONECT 20 19 21
CONECT 10 8 11 12
CONECT 33 34 35 31
CONECT 37 36 38
CONECT 8 6 9 10
CONECT 31 29 32 33
CONECT 38 37 39 42
CONECT 22 13 16 21
CONECT 6 5 7 8
CONECT 29 28 30 31
CONECT 42 38 43
CONECT 16 15 17 22
CONECT 5 4 6
CONECT 28 27 29
CONECT 43 42 44
CONECT 17 18 16 19
CONECT 44 36 43
CONECT 39 38 40 41
CONECT 14 13 15
CONECT 19 17 20
CONECT 36 35 37 44
CONECT 21 20 22
CONECT 18 17

However, when i am trying to use this chimera generated pdb file, i am getting the error saying:
There was an inconsistency in your data
Error message
Second pdb file contains multiple forms of the same residue. This is not supported in the current form. If you would like to supply multiple conformations, please create an ensemble
ATOM 1 PA ANAD 1392 7.291 -8.893 -5.018 0.80 9.95 P

I am not sure what is wrong with the format and how can i create an ensemble? Should i change the name of all the residue numbering from 1392, 1393, 1394,… and so on? Thanks for your time.

The PBD format is very strict and follows a column formatting - removing or adding space will mess up the file.
Which is what you have now…

Take a PDB file freshly downloaded from the PDB database and compare/match the columns!

Hello Everyone,

After banging my head for three days i was able to solve all the errors of docking Protein-Zn-Co-factor together. Here’s how i did it.
The protein of interest and co-factor should be in pdb format. There’s nothing to change with the protein. however, if you have a metal ion in your protein then you have to add in the format HADDOCK recognises it. In my case i had a Zinc ion in my protein. Thus the protein with Zinc ion looks in this format:
ATOM 4391 HG CYS A 314 39.125 78.402 67.775 1.00 1.00 H
ATOM 4392 C CYS A 314 36.273 77.320 68.053 1.00 1.00 C
ATOM 4393 O CYS A 314 36.387 77.018 66.865 1.00 1.00 O
TER 4394 CYS A 314
HETATM 4395 ZN+2 ZN2 A 315 84.481 65.009 76.144 1.00 19.29 ZN
END

In terms of co-factor, it should be renamed to different chain identifier or HADDOCK will show error such as:
Second pdb file contains multiple forms of the same residue. This is not supported in the current form. If you would like to supply multiple conformations, please create an ensemble. **
** OR

Second pdb file is incorrectly formatted. Please align the columns of your PDB properly

Thus in order to avoid this, change the co-factor pdb format. In my case it was NAD and I formatted the pdb like this:
HETATM 40 O7N NAD B 400 11.246 46.818 16.230 1.00 72.85 O
HETATM 41 N7N NAD B 400 9.340 47.583 15.291 1.00 72.17 N
HETATM 42 C4N NAD B 400 10.140 46.365 12.831 1.00 69.14 C
HETATM 43 C5N NAD B 400 10.566 46.086 11.537 1.00 67.89 C
HETATM 44 C6N NAD B 400 11.923 46.126 11.235 1.00 66.35 C
END

And finally I was able to run the docking successfully with these small changes in the pdb files. Hope this helps everyone !!

Looking at those two lines you still have problems… The column are not aligned!
There is a fixed number of characters for each column. This is not the case now.

Most likely you NAD must look pretty weird.