WARNING [file forcefield.itp, line 1]: Too few parameters on line || WARNING: net charge

User discussions
1

GROMACS version:2019.4
Hi,

I would like to simulate a membrane-protein-ligand system. The protein topology (topol.top, topol_Protein.itp) was created by pdb2gmx under amber14SB force fields, while the lipid topology was created by pdb2gmx under lipid17 FFs. The ligand .itp file was generated by acpype.py. Then I combined three .gro files together, and included topologies of the lipid and the ligand into topol .top using #include mechanism.

When I conducted the subsequent equilibration, I kept getting warnings as follows.
//---------------------------------------------------------------------------
WARNING 1 [file forcefield.itp, line 1]:
Too few parameters on line (source file
/THL6/home/bickel/software/gromacs-2019.4/src/gromacs/gmxpreprocess/toppush.cpp, line 290)
….{9 more similar warnings}
NOTE 1 [file topol.top, line 71]:
System has non-zero total charge: -0.000587
Total charge should normally be an integer. See
http://www.gromacs.org/Documentation/Floating_Point_Arithmetic
for discussion on how close it should be to an integer.

WARNING 11 [file topol.top, line 71]:
You are using Ewald electrostatics in a system with net charge. This can
lead to severe artifacts, such as ions moving into regions with low
dielectric, due to the uniform background charge. We suggest to
neutralize your system with counter ions, possibly in combination with a
physiological salt concentration.
//--------------------------------------------------------------------------
Since I have introduced counter-ions to the system, there shouldn’t be net charges. Then I figured out that the total charge of an individual DMPC molecule given by acpype.py is about 3E-6*e instead of absolutely zero, which results in a small non-zero charge of the system. I assume this infinitesimal charge wouldn’t cause problems, so can be ignored?

I am not sure if the first 10 warnings will greatly affect the final results. I am thinking there might be something wrong with the topology but don’t know where. Below is the topol.top file. The [atomtyps] parts of the ligand and the lipid have been placed in the front of the file. The line "#include "ffnonbonded.itp"
" in the lipid17.ff/forcefield.itp file has been commented out. I will attach more details on the topology files if needed. Any help will be much appreciated.
//----------------------------------------------------------------
#define _FF_AMBER
#define _FF_AMBER12SB

[ defaults ]
; nbfunc comb-rule gen-pairs fudgeLJ fudgeQQ
1 2 yes 0.5 0.8333

[ atomtypes ]
;name bond_type mass charge ptype sigma epsilon Amb
oh oh 0.00000 0.00000 A 3.06647e-01 8.80314e-01 ; 1.72 0.2104
n4 n4 0.00000 0.00000 A 3.25000e-01 7.11280e-01 ; 1.82 0.1700
c3 c3 0.00000 0.00000 A 3.39967e-01 4.57730e-01 ; 1.91 0.1094
hc hc 0.00000 0.00000 A 2.64953e-01 6.56888e-02 ; 1.49 0.0157
ca ca 0.00000 0.00000 A 3.39967e-01 3.59824e-01 ; 1.91 0.0860
hx hx 0.00000 0.00000 A 1.95998e-01 6.56888e-02 ; 1.10 0.0157
ha ha 0.00000 0.00000 A 2.59964e-01 6.27600e-02 ; 1.46 0.0150
hn hn 0.00000 0.00000 A 1.06908e-01 6.56888e-02 ; 0.60 0.0157
ho ho 0.00000 0.00000 A 0.00000e+00 0.00000e+00 ; 0.00 0.0000
[ atomtypes ]
; name at.num mass charge ptype sigma epsilon
hL 1 1.008000 0.00000000 A 0.26014243 0.04184 ; LAL_PC_LAL
cD 6 12.010000 0.00000000 A 0.33996695 0.4577296 ; LAL_PC_LAL
cC 6 12.010000 0.00000000 A 0.33996695 0.29288 ; LAL_PC_LAL
oC 8 16.000000 0.00000000 A 0.29399658 0.58576 ; LAL_PC_LAL
oS 8 16.000000 0.00000000 A 0.29399658 0.50208 ; LAL_PC_LAL
cA 6 12.010000 0.00000000 A 0.33996695 0.4577296 ; LAL_PC_LAL
hE 1 1.008000 0.00000000 A 0.2471353 0.0656888 ; LAL_PC_LAL
oT 8 16.000000 0.00000000 A 0.29399658 0.50208 ; LAL_PC_LAL
pA 15 30.970000 0.00000000 A 0.37417746 0.8368 ; LAL_PC_LAL
hX 1 1.008000 0.00000000 A 0.19599772 0.0656888 ; LAL_PC_LAL
nA 7 14.010000 0.00000000 A 0.32499985 0.71128 ; LAL_PC_LAL
oP 8 16.000000 0.00000000 A 0.29399658 0.58576 ; LAL_PC_LAL
cB 6 12.010000 0.00000000 A 0.33996695 0.359824 ; LAL_PC_OL
hB 1 1.008000 0.00000000 A 0.22272468 0.029288 ; LAL_PC_OL
hN 1 1.008000 0.00000000 A 0.10690785 0.0656888 ; LAL_PE_LAL
oO 8 16.000000 0.00000000 A 0.29399658 0.58576 ; LAL_PS_LAL
oH 8 16.000000 0.00000000 A 0.29399658 0.50208 ; LAL_PGR_LAL
hO 1 1.008000 0.00000000 A 0.10690785 0.0656888 ; LAL_PGR_LAL
hA 1 1.008000 0.00000000 A 0.26495328 0.0656888 ; CHL

#include “amber14sb_parmbsc1.ff/forcefield.itp”
#include “lipid17.ff/forcefield.itp”
#include “DOA.itp”
#include “topol_Protein.itp”
#include “topol_DMPC.itp”

; Include water topology
#include “amber14sb_parmbsc1.ff/tip3p.itp”

#ifdef POSRES_WATER
; Position restraint for each water oxygen
[ position_restraints ]
; i funct fcx fcy fcz
1 1 1000 1000 1000
#endif

; Include topology for ions
include “amber14sb_parmbsc1.ff/ions.itp”

[ system ]
system

[ molecules ]
; Compound #mols
Protein 1
DMPC 199
DOA 1
SOL 20434
NA 79
CL 89
//-----------------------------------------------------------------

2

Hi,

Sorry it is not clear to me if you are trying to use different types of force fields. If this is the case, it is not possible. One can include only one forcefield.itp or only once the corresponding directives

Best regards
Alessandra

Best regards

3

Hi,

Yeah, that’s right. I do have seen a similar explanation. But I assume Amber FFs are compatible with lipid17.ff? So it should be OK? Even if only one forcefield.itp is allowed, I have tried to put the content of lipid forcefield.itp into the main Amber forcefield.itp and keep one forcefield.itp, but the warnings still exist.

Or do you have any suggestions to create topologies for the membrane? Super thanks!

4

Hi,

That is correct, since only one of the corresponding directive (e.i [defaults]) is allow. The directive [defaults] include the “basis rules” of the force field see here for more details https://manual.gromacs.org/current/reference-manual/topologies/topology-file-formats.html.

This is not something that one can assume. All the force field parameters have to be parameterized in line with the force field philosophy, otherwise they are not compatible. Moreover you need to define twice [defaults] , that may indicate that are two independent force field. Good to ask to the force field authors.

An option is CHARMM GUI (but I do not know if cover the force field you are interested in), maybe others can have useful suggestion here.

Best regards
Alessandra

5

Thanks Alessandra. I really appreciate!

I have double checked. The directory lipid17.ff is force field parameters in GROMACS format, and shares the same rule (Buckingham potential function) with the AMBER force field. GitHub - xiki-tempula/gmx_lipid17.ff: a gromacs port of the amber lipid force field LIPID17

I did have thought about the Charmm-GUI. But the CGenFF generated charges for the ligand (small molecule) are not as precise as RESP charges and the charge penalty is too high. It seems Amber FFs are my only choice for the membrane-protein-ligand system.

I have also tried another lipid FFs, that is Slipids lipid force fields. Slipids Force Field Downloads. The website indicates clearly that these FFs work with Amber FFs. But I still got the same warning: WARNING 1 [file forcefield.itp, line 1]: Too few parameters on line (source file…
I really have no idea why.

All the best,
Eva

6

Are you still having this issue? You don’t say which ACPYPE version you’re using neither the command you’re issued.

If asking for help with ACPYPE, please, always rerun it with -d and send the detailed output.

Try to use the latest version seen here. ACPYPE does rebalance the charges in order to give integral net charge (never a float), unless you’re obtaining your charges elsewhere.

7

Hi,

Thanks for your concern. The issue has been resolved. The ACPYPE worked well in my case in generating the molecule charges. Sorry I just found out that I described wrong my question. I didn’t create the DMPC charges with ACPYPE, so the integral float charges result from other aspects.

8

Hi, I have meet the same issue as you described. I have creat DOPC by charmm-gui, and the total net-charge of one DOPC is 0.00001. Would you share how you resolve this issue? Thanks

9

Simply take this charge diff and subtract from another atom, preferably the lowest negative one.

10

I disagree.

The charges in the CHARMM residue definition for DOPC have only two decimal places, so any claim of a 0.00001 net charge is a simple rounding error by GROMACS. This magnitude of apparent net charge requires no manual adjustment.