Excluded distance larger than cutoff in ligand with virtual site

GROMACS version: 2023
GROMACS modification: Yes, patched with plumed 2.9.0

I have prepared a membrane protein-ligand complex using CHARMM-GUI where parametrization of the ligand includes a virtual site. After minimization, I get the following error during the first step of equilibration:

ERROR 1 [file step6.1_equilibration.mdp]:
The largest distance between excluded atoms is 1.201 nm between atom 5040
and 5126, which is larger than the cut-off distance. This will lead to
missing long-range corrections in the forces and energies.

Atom 5040 is the first atom of the ligand and 5126 is the virtual site. When I check the excluded pairs in the ligand topology, atom 5040 is not included in the list of exclusions.
Any ideas why gromacs is considering atoms 5040 and 5126 to be excluded?

Are these two atoms connected by bonds? If so, by how many? What is the exclusion range that is set for this molecule type?

The two atoms are connected by bonds. You would need to go through 10 bonds to connect atom1 to the virtual site.
nrexcl=3 for this molecule.

Somehow it seems to be excluded. Are there explicit exclusions in the topology for the virtual site?

Yes there are, does nrexcl=3 stack with the explicitly defined exclusions in the topology?

Here are the exclusions:
[ exclusions ]
; ai aj
87 42 1
87 36 1
87 34 1
87 33 1
87 35 1
87 37 1
87 38 1
87 80 1

Atom 87 is the virtual site. The error that I mention in the post is due to atom 87 and atom 1 being excluded for some reason

Update: I tried a simulation where I commented out all the exclusions section while leaving nrexcl=3 and the simulation is running now.

The exclusion section was automatically written during ligand parametrization using CGenFF. I am worried removing exclusions would lead to wrong energy calculations including the virtual site, any insight regarding that point?
Thanks a lot!

Lone pairs used in CHARMM must have the same exclusions as their parent atom, so the explicitly listed exclusions apply to all 1-4 pairs that are also excluded for that parent atom.

Thanks for the clarification, if I understand correctly, this leads to a “transmission” of exclusions eventually leading to the error.

Since the lone pair site will have the same exclusions as its parent atom, then I assume there’s no need for the explicitly defined exclusions including the lone pair?

No, you need to explicitly add the exclusions.

But then I guess the coordinates of the lone pair are wrong in your initial structure. They should be very close to the parent atom, but then you wouldn’t get the error.

Then it seems I am in a predicament, since the exclusion error is raised even if I only have the parent atom (the atom directly connected to the lone pair) explicitly defined in the exclusion list.
I checked the trajectory of the equilibration steps and it seems that the distance between the lone pair and atom1 in the beginning is right under 1.2nm (1.187), but then when the restraints are gradually decreased, the ligand relaxes and the distance increased to 1.205 nm, leading to the error.
Upon visual inspection, the coordinates of the lone pair is correct.
I think the issue is that the lone pair and atom1 are considered excluded when they should not be?

Sure there should be an exclusion between the atom and lone pair. But I would think that the distance between those two should never be more than 0.1 or 0.2 nm.

@mHijazi did you eventually resolve this issue? Using a topology file prepared by CHARMM-GUI, I am also having the same issue: my virtual site (lone pair of a chloride atom) is 12 bonds away from the C1 atom involved in the error and I was also using nrexcl=3. The parent atom closest to the C1 atom is still 9 bonds away from the C1 atom, so I don’t understand why GROMACS considered the C1 atom and my virtual site to be excluded. If they are more than 3 bonds away, why should there be an exclusion as @hess mentioned?

Thanks a lot in advance!

For your information, here is a screenshot of my molecule. The two atoms involved in the error are coloured in black. The left one is the C1 atom and the right one is the virtual site.

The parent atoms of the virtual site are the three carbon atoms vertically closest to the virtual site in the figure. The type of the virtual site is 3fad.

To me, the issue does not seem to be that C1 is too far away from the virtual site but that C1 and the virtual site should not be excluded.

Could it be that you are doing free-energy (de)coupling calculations? Then all pair interactions in the ligand are excluded.

@hess thank so much for the quick reply. No, I did not have any free energy settings in my MDP file. Here I am attaching the input MDP if you are interested in taking a look at. The version of GROMACS I used was 2023.1 and the error I had is as follows:

  The largest distance between excluded atoms is 1.328 nm between atom 8722
  and 8755, which is larger than the cut-off distance. This will lead to
  missing long-range corrections in the forces and energies. If you expect
  that minimization will bring such distances within the cut-off, you can
  ignore this warning.

I have been having a hard time telling why GROMACS see the involved atoms as excluded, so any insights would be incredibly helpful. I have also pasted the involved itp file below. (Atoms 8722 and 8755 correspond to indices 1 and 34 in the atoms directive, respectively.) Thanks so much!

step6.0_minimization.mdp (581 Bytes)

;;
;; Generated by CHARMM-GUI FF-Converter
;;
;; Correspondance:
;; jul316@lehigh.edu or wonpil@lehigh.edu
;;
;; GROMACS topology file for FEN
;;


[ moleculetype ]
; name	nrexcl
FEN	     3

[ atoms ]
; nr	type	resnr	residu	atom	cgnr	charge	mass
     1     CG2R61    701      FEN     C1      1    -0.115000    12.0110   ; qtot -0.115
     2     CG2R51    701      FEN     C2      2     0.155000    12.0110   ; qtot  0.040
     3      CG321    701      FEN     C3      3    -0.274000    12.0110   ; qtot -0.234
     4      CG2O3    701      FEN     C4      4     0.628000    12.0110   ; qtot  0.394
     5     CG2R61    701      FEN     C5      5     0.017000    12.0110   ; qtot  0.411
     6     CG2R61    701      FEN     C6      6    -0.111000    12.0110   ; qtot  0.300
     7     CG2R61    701      FEN     C7      7    -0.105000    12.0110   ; qtot  0.195
     8     CG2R61    701      FEN     C8      8     0.055000    12.0110   ; qtot  0.250
     9     CG2R61    701      FEN     C9      9    -0.117000    12.0110   ; qtot  0.133
    10     CG2R61    701      FEN    C10     10    -0.105000    12.0110   ; qtot  0.028
    11     CG2R61    701      FEN    C11     11    -0.111000    12.0110   ; qtot -0.083
    12     CG2R61    701      FEN    C12     12    -0.119000    12.0110   ; qtot -0.202
    13     CG2R61    701      FEN    C13     13     0.066000    12.0110   ; qtot -0.136
    14     CG2R61    701      FEN    C14     14    -0.119000    12.0110   ; qtot -0.255
    15     CG2R61    701      FEN    C15     15    -0.117000    12.0110   ; qtot -0.372
    16     CG2R53    701      FEN    C16     16     0.435000    12.0110   ; qtot  0.063
    17     CG2R51    701      FEN    C17     17     0.260000    12.0110   ; qtot  0.323
    18     NG2R50    701      FEN      N     18    -0.792000    14.0070   ; qtot -0.469
    19      OG2D2    701      FEN     O1     19    -0.760000    15.9994   ; qtot -1.229
    20      OG2D2    701      FEN     O2     20    -0.760000    15.9994   ; qtot -1.989
    21     SG2R50    701      FEN      S     21    -0.145000    32.0600   ; qtot -2.134
    22      CLGR1    701      FEN     CL     22    -0.201000    35.4530   ; qtot -2.335
    23      HGR61    701      FEN     H1     23     0.115000     1.0080   ; qtot -2.220
    24       HGA2    701      FEN     H2     24     0.090000     1.0080   ; qtot -2.130
    25       HGA2    701      FEN     H3     25     0.090000     1.0080   ; qtot -2.040
    26      HGR61    701      FEN     H4     26     0.115000     1.0080   ; qtot -1.925
    27      HGR62    701      FEN     H5     27     0.150000     1.0080   ; qtot -1.775
    28      HGR61    701      FEN     H6     28     0.115000     1.0080   ; qtot -1.660
    29      HGR62    701      FEN     H7     29     0.150000     1.0080   ; qtot -1.510
    30      HGR61    701      FEN     H8     30     0.115000     1.0080   ; qtot -1.395
    31      HGR61    701      FEN     H9     31     0.115000     1.0080   ; qtot -1.280
    32      HGR61    701      FEN    H10     32     0.115000     1.0080   ; qtot -1.165
    33      HGR61    701      FEN    H11     33     0.115000     1.0080   ; qtot -1.050
    34        LPH    701      FEN    LP1     34     0.050000     0.0000   ; qtot -1.000

[ bonds ]
; ai	aj	funct	b0	Kb
    1     9     1
   15     1     1
    1    23     1
    3     2     1
    2    17     1
   21     2     1
    4     3     1
    3    24     1
    3    25     1
   19     4     1
   20     4     1
    5     6     1
   11     5     1
   17     5     1
    6     7     1
    6    26     1
    8     7     1
    7    27     1
   10     8     1
    8    22     1
   12     9     1
    9    28     1
   11    10     1
   10    29     1
   11    30     1
   13    12     1
   12    31     1
   14    13     1
   16    13     1
   14    15     1
   14    32     1
   15    33     1
   16    18     1
   21    16     1
   17    18     1

[ pairs ]
; ai	aj	funct	c6	c12 or
; ai	aj	funct	fudgeQQ	q1	q2	c6	c12
    1    13     1
    1    31     1
    1    32     1
    2     6     1
    2    11     1
    2    13     1
    2    19     1
    2    20     1
    3     5     1
    3    16     1
    3    18     1
    4    17     1
    4    21     1
    5     8     1
    5    16     1
    5    21     1
    5    27     1
    5    29     1
    6    10     1
    6    18     1
    6    22     1
    6    30     1
    6    34     1
    7    11     1
    7    17     1
    7    29     1
    8    26     1
    8    30     1
    9    14     1
    9    16     1
    9    33     1
   10    17     1
   10    27     1
   11    18     1
   11    22     1
   11    26     1
   11    34     1
   12    15     1
   12    18     1
   12    21     1
   12    23     1
   12    32     1
   13    17     1
   13    28     1
   13    33     1
   14    18     1
   14    21     1
   14    23     1
   14    31     1
   15    16     1
   15    28     1
   16    31     1
   16    32     1
   17    24     1
   17    25     1
   17    26     1
   17    30     1
   19    24     1
   19    25     1
   20    24     1
   20    25     1
   21    24     1
   21    25     1
   22    27     1
   22    29     1
   23    28     1
   23    33     1
   26    27     1
   27    34     1
   28    31     1
   29    30     1
   29    34     1
   32    33     1

[ angles ] 
; ai	aj	ak	funct	th0	cth	S0	Kub
    9     1    15     5
    9     1    23     5
   15     1    23     5
    3     2    17     5
    3     2    21     5
   17     2    21     5
    2     3     4     5
    2     3    24     5
    2     3    25     5
    4     3    24     5
    4     3    25     5
   24     3    25     5
    3     4    19     5
    3     4    20     5
   19     4    20     5
    6     5    11     5
    6     5    17     5
   11     5    17     5
    5     6     7     5
    5     6    26     5
    7     6    26     5
    6     7     8     5
    6     7    27     5
    8     7    27     5
    7     8    10     5
    7     8    22     5
   10     8    22     5
    1     9    12     5
    1     9    28     5
   12     9    28     5
    8    10    11     5
    8    10    29     5
   11    10    29     5
    5    11    10     5
    5    11    30     5
   10    11    30     5
    9    12    13     5
    9    12    31     5
   13    12    31     5
   12    13    14     5
   12    13    16     5
   14    13    16     5
   13    14    15     5
   13    14    32     5
   15    14    32     5
    1    15    14     5
    1    15    33     5
   14    15    33     5
   13    16    18     5
   13    16    21     5
   18    16    21     5
    2    17     5     5
    2    17    18     5
    5    17    18     5
   16    18    17     5
    2    21    16     5

[ dihedrals ]
; ai	aj	ak	al	funct	phi0	cp	mult
   15     1     9    28     9
   23     1     9    28     9
    9     1    15    14     9
    9     1    15    33     9
   23     1    15    33     9
   17     2     3    24     9
   17     2     3    25     9
   21     2     3    24     9
   21     2     3    25     9
    3     2    17     5     9
    3     2    17    18     9
    3     2    21    16     9
    4     3     2    17     9
    4     3     2    21     9
    2     3     4    19     9
    2     3     4    20     9
   19     4     3    24     9
   19     4     3    25     9
   20     4     3    24     9
   20     4     3    25     9
   11     5     6    26     9
   17     5     6    26     9
    6     5    11    10     9
    6     5    11    30     9
   17     5    11    30     9
    6     5    17    18     9
   11     5    17    18     9
    7     6     5    11     9
    7     6     5    17     9
    5     6     7     8     9
    5     6     7    27     9
   26     6     7    27     9
    8     7     6    26     9
    6     7     8    10     9
    6     7     8    22     9
   10     8     7    27     9
   22     8     7    27     9
    7     8    10    11     9
    7     8    10    29     9
   22     8    10    29     9
   12     9     1    15     9
   12     9     1    23     9
    1     9    12    13     9
    1     9    12    31     9
   28     9    12    31     9
   11    10     8    22     9
    8    10    11    30     9
   29    10    11    30     9
   10    11     5    17     9
    5    11    10     8     9
    5    11    10    29     9
   13    12     9    28     9
    9    12    13    14     9
    9    12    13    16     9
   14    13    12    31     9
   16    13    12    31     9
   12    13    14    15     9
   12    13    14    32     9
   16    13    14    32     9
   12    13    16    18     9
   12    13    16    21     9
   14    13    16    18     9
   14    13    16    21     9
   15    14    13    16     9
   13    14    15    33     9
   32    14    15    33     9
   14    15     1    23     9
    1    15    14    13     9
    1    15    14    32     9
   13    16    18    17     9
    5    17     2    21     9
   18    17     2    21     9
    2    17     5     6     9
    2    17     5    11     9
    2    17    18    16     9
    5    17    18    16     9
   17    18    16    21     9
   16    21     2    17     9
    2    21    16    13     9
    2    21    16    18     9

[ dihedrals ]
; ai	aj	ak	al	funct	q0	cq
    4    20    19     3     2

[ virtual_sites3 ]
; Site   from               funct theta d
   34    22     8     7     3   180.00    0.164

[ exclusions ]
;   ai    aj
   34    22     1
   34     8     1
   34     7     1
   34     6     1
   34    27     1
   34    10     1
   34    11     1
   34    29     1

#ifdef POSRES
[ position_restraints ]
    1     1    POSRES_FC_BB    POSRES_FC_BB    POSRES_FC_BB   
    2     1    POSRES_FC_BB    POSRES_FC_BB    POSRES_FC_BB   
    3     1    POSRES_FC_BB    POSRES_FC_BB    POSRES_FC_BB   
    4     1    POSRES_FC_BB    POSRES_FC_BB    POSRES_FC_BB   
    5     1    POSRES_FC_BB    POSRES_FC_BB    POSRES_FC_BB   
    6     1    POSRES_FC_BB    POSRES_FC_BB    POSRES_FC_BB   
    7     1    POSRES_FC_BB    POSRES_FC_BB    POSRES_FC_BB   
    8     1    POSRES_FC_BB    POSRES_FC_BB    POSRES_FC_BB   
    9     1    POSRES_FC_BB    POSRES_FC_BB    POSRES_FC_BB   
   10     1    POSRES_FC_BB    POSRES_FC_BB    POSRES_FC_BB   
   11     1    POSRES_FC_BB    POSRES_FC_BB    POSRES_FC_BB   
   12     1    POSRES_FC_BB    POSRES_FC_BB    POSRES_FC_BB   
   13     1    POSRES_FC_BB    POSRES_FC_BB    POSRES_FC_BB   
   14     1    POSRES_FC_BB    POSRES_FC_BB    POSRES_FC_BB   
   15     1    POSRES_FC_BB    POSRES_FC_BB    POSRES_FC_BB   
   16     1    POSRES_FC_BB    POSRES_FC_BB    POSRES_FC_BB   
   17     1    POSRES_FC_BB    POSRES_FC_BB    POSRES_FC_BB   
   18     1    POSRES_FC_BB    POSRES_FC_BB    POSRES_FC_BB   
   19     1    POSRES_FC_BB    POSRES_FC_BB    POSRES_FC_BB   
   20     1    POSRES_FC_BB    POSRES_FC_BB    POSRES_FC_BB   
   21     1    POSRES_FC_BB    POSRES_FC_BB    POSRES_FC_BB   
   22     1    POSRES_FC_BB    POSRES_FC_BB    POSRES_FC_BB   
#endif

So one weird thing I found is that when I commented out the entire [ exclusions ] directive, the error was gone. If I kept only one line and commented out the rest, the warning came back, regardless of which line I kept. I tried to compare the output of gmx dump for the case with and without the exclusion directives as shown below, but it is still not clear to my why this is the case:

<    ld-seed                        = -1451233606
---
>    ld-seed                        = -228074497
188c188
<    buffer size = 8271471
---
>    buffer size = 8271399
322663,322664c322663,322664
<          numElements=372
<          excls[0][num=12]={0, 8, 11, 12, 13, 14, 22, 27, 30, 31, 32, 33}
---
>          numElements=354
>          excls[0][num=11]={0, 8, 11, 12, 13, 14, 22, 27, 30, 31, 32}
322671,322674c322671,322673
<          excls[5][num=14]={1, 4, 5, 6, 7, 9, 10, 16, 17, 21, 25, 26, 29, 
<             33}
<          excls[6][num=12]={4, 5, 6, 7, 9, 10, 16, 21, 25, 26, 28, 33}
<          excls[7][num=12]={4, 5, 6, 7, 9, 10, 21, 25, 26, 28, 29, 33}
---
>          excls[5][num=13]={1, 4, 5, 6, 7, 9, 10, 16, 17, 21, 25, 26, 29}
>          excls[6][num=11]={4, 5, 6, 7, 9, 10, 16, 21, 25, 26, 28}
>          excls[7][num=11]={4, 5, 6, 7, 9, 10, 21, 25, 26, 28, 29}
322676,322678c322675,322676
<          excls[9][num=12]={4, 5, 6, 7, 9, 10, 16, 21, 26, 28, 29, 33}
<          excls[10][num=14]={1, 4, 5, 6, 7, 9, 10, 16, 17, 21, 25, 28, 29, 
<             33}
---
>          excls[9][num=11]={4, 5, 6, 7, 9, 10, 16, 21, 26, 28, 29}
>          excls[10][num=13]={1, 4, 5, 6, 7, 9, 10, 16, 17, 21, 25, 28, 29}
322695c322693
<          excls[21][num=9]={5, 6, 7, 9, 10, 21, 26, 28, 33}
---
>          excls[21][num=8]={5, 6, 7, 9, 10, 21, 26, 28}
322700c322698
<          excls[26][num=9]={4, 5, 6, 7, 9, 21, 25, 26, 33}
---
>          excls[26][num=8]={4, 5, 6, 7, 9, 21, 25, 26}
322702c322700
<          excls[28][num=9]={4, 6, 7, 9, 10, 21, 28, 29, 33}
---
>          excls[28][num=8]={4, 6, 7, 9, 10, 21, 28, 29}
322707c322705
<          excls[33][num=10]={0, 5, 6, 7, 9, 10, 21, 26, 28, 33}
---
>          excls[33][num=1]={33}

For convenience here I am attaching the figure of the ligand with atom labels.

  • The two black atoms are the two atoms involved in the warning, i.e. atoms 8722 (C1 atom, which is the left one and has an index of 1 in the topology file) and 8755 (virtual site, which is the right one and has an index of 34 in the topology file).
  • The three blue atoms are the parent atoms of the virtual site, i.e. indices 7, 8 and 22 in the topology file.
  • The four orange atoms are the atoms involved with index 34 in the [ pairs ] directive, i.e., indices 6, 11, 27, and 29.
  • All atoms above are in the [ exclusions ] directive, in which there is one additionally defined exclusion between indices 34 and 10 The atom corresponding to index 10 is in gray.

@hess do you perhaps have some insights into this issue? It is not even clear to me wether the issue lies in incorrect topology file or that GROMACS incorrectly identifies exclusions between atoms that are not even bonded neighbors. Any suggestions would be appreciated!

Have a look at [ exclusions ] in File formats - GROMACS 2024.4 documentation. I think you would want:

[ exclusions ]
;   ai    aj   ...
   34    22
   34     8
   34     7
   34     6
   34    27
   34    10
   34    11
   34    29

or even

[ exclusions ]
;   ai    aj   ...
   34    22   8   7   6   27   10   11   29

Hi @MagnusL thanks a lot for your reply!

I initially assumed that the last column of 1 was the functional type (and I should have checked!) Now I see the issue comes from the fact that GROMACS sees the 1’s as atom indices to be considered for exclusion (as it should). I don’t know why CHARMM-GUI assigned 1 for each line in [ exclusions ] directive though, since I don’t think that’s correct in my case. (And removing the column did resolve the issue.) It seems that CHARMM does that for other cases as well, as indicated by the OP. Not entirely sure if this is a bug but I’ll report to CHARMM.

Again thank you so much!

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