Hello, My name is Jiajie Xu. I encounter an interesting question when I use Gromacs for MD simulation. 

Currently I am trying on constant pH simulation on Gromacs 5.0.7. The target protein ID is 2LSB. It is under condition of 5.5 pH. I want to modified it into pH 4.5 and 7.4 's  protonation status. So I use H++ online server to achieve this (http://biophysics.cs.vt.edu/). The H++ modified pdb file has more hydrogen (7 more H in total) on 4.5 pH as the I expected. However, when I start running on Gromacs, the force field won't recognise these atoms because they have more number of atoms for HIS ( 12 in normal, modified one has 13).  So I need to use -ignh to ignore hydrogen. 

The error is shown like this:

command line:  

gmx pdb2gmx -f 45.pdb -o 45processed.gro -water spce 

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Fatal error:

Atom HA3 in residue GLY 90 was not found in rtp entry NGLY with 9 atoms

while sorting atoms.

For a hydrogen, this can be a different protonation state, or it

might have had a different number in the PDB file and was rebuilt

(it might for instance have been H3, and we only expected H1 & H2).

Note that hydrogens might have been added to the entry for the N-terminus.

Remove this hydrogen or choose a different protonation state to solve it.

Option -ignh will ignore all hydrogens in the input.

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What really interesting is I actually expect the same results from constant pH 4.5, pH 5.5 and pH 7.4 although results assume to be different based on experiment.  Because after I used this -ignh commend to ignore H I added by H++, the pKa should be the same, right? Then the modified file supposed won't change. However, the results from pH 4.5, 5.5, 7.4 are shown different (I have run twice (4.5 pH, 5.5 pH and 7.4 pH) to confirm this).  

The attached link below are my origional pdb file, modified 4.5 pH pdb file and 45processed.gro after -ignh. You can see the atoms number change back to original one which means Gromacs delete all hydrogen I added. However, the running results are different based on my RMSD analysis and VMD visualisation.

https://drive.google.com/open?id=1enw_6dxAfxPMKYRWl08L9hZ65Rur4Nyf

Do you know why? I am really curious. 

Thanks for your concern. Appreciate your time.

Best Regards,

Jiajie Xu