How to setup protein-ligand simulation using CHARMM-GUI for ligand preparation

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1

GROMACS version: 2025.3
GROMACS modification: No

Goal:

I would like help getting the protein-ligand tutorial working with GROMACS 2025.3 and using CHARMM-GUI to produce the ligand parameters. I am stuck at the step of combining the ligand parameters with the protein setup.

What I’ve done so far:

Used the CHARMM-GUI Ligand Reader & Modeller to produce ligand parameters from a ligand .pdb file with hydrogens added. The output of that includes outputs gromacs/topol.top, gromacs/LIG.itp, gromacs/charmm36.itp, and ligandrm.pdb (transcribed at bottom).

I can then use gmx editconf to produce a corresponding .gro file for the ligand.

Following the instructions in the tutorial, I have created a 3HTB_processed.gro file, along with corresponding posres.itp and topol.top files.

Where I have issues:

When I try to combine the output of CHARMM-GUI with the protein files, attempting the solvation fails during the first call to gmx editconf, with the message:

Fatal error:

Invalid line in system.gro for atom 2615

Query:

How should I combine the ligand parameters output by CHARMM-GUI with the protein parameters produced by GROMACS?

My thanks to everyone reading.

gromacs/topol.top

;;
;; Generated by CHARMM-GUI (url removed) v1.7
;;
;; psf2itp_mol.py
;;
;; Correspondance:
;; jul316@lehigh.edu or wonpil@lehigh.edu
;;
;; The main GROMACS topology file
;;

; Include forcefield parameters
#include “charmm36.itp”
#include “LIG.itp”

[ system ]
; Name
Title

[ molecules ]
; Compound #mols
LIG 1

gromacs/LIG.itp

;;
;; Generated by CHARMM-GUI (url removed) v1.7
;;
;; psf2itp_mol.py
;;
;; Correspondance:
;; jul316@lehigh.edu or wonpil@lehigh.edu
;;
;; GROMACS topology file for LIG
;;

[ moleculetype ]
; name nrexcl
LIG 3

[ atoms ]
; nr type resnr residu atom cgnr charge mass
1 CG331 1 LIG C1 1 -0.275 12.0110 ; qtot -0.275
2 HGA3 1 LIG H1 2 0.090 1.0080 ; qtot -0.185
3 HGA3 1 LIG H2 3 0.090 1.0080 ; qtot -0.095
4 HGA3 1 LIG H3 4 0.090 1.0080 ; qtot -0.005
5 CG2R61 1 LIG C2 5 -0.107 12.0110 ; qtot -0.112
6 HGR61 1 LIG H4 6 0.115 1.0080 ; qtot 0.003
7 CG2R61 1 LIG C3 7 -0.114 12.0110 ; qtot -0.111
8 HGR61 1 LIG H5 8 0.115 1.0080 ; qtot 0.004
9 CG2R61 1 LIG C4 9 -0.105 12.0110 ; qtot -0.101
10 HGR61 1 LIG H6 10 0.115 1.0080 ; qtot 0.014
11 CG2R61 1 LIG C5 11 0.109 12.0110 ; qtot 0.123
12 CG2R61 1 LIG C6 12 -0.117 12.0110 ; qtot 0.006
13 HGR61 1 LIG H7 13 0.115 1.0080 ; qtot 0.121
14 CG2R61 1 LIG C7 14 0.001 12.0110 ; qtot 0.122
15 CG321 1 LIG C8 15 -0.195 12.0110 ; qtot -0.073
16 HGA2 1 LIG H8 16 0.090 1.0080 ; qtot 0.017
17 HGA2 1 LIG H9 17 0.090 1.0080 ; qtot 0.107
18 CG321 1 LIG C9 18 -0.176 12.0110 ; qtot -0.069
19 HGA2 1 LIG H10 19 0.090 1.0080 ; qtot 0.021
20 HGA2 1 LIG H11 20 0.090 1.0080 ; qtot 0.111
21 OG311 1 LIG O 21 -0.531 15.9994 ; qtot -0.420
22 HGP1 1 LIG H12 22 0.420 1.0080 ; qtot -0.000

[ bonds ]
; ai aj funct b0 Kb
1 2 1
1 3 1
1 4 1
1 18 1
5 6 1
5 7 1
5 12 1
7 8 1
7 9 1
9 10 1
9 11 1
11 14 1
11 21 1
12 13 1
12 14 1
14 15 1
15 16 1
15 17 1
15 18 1
18 19 1
18 20 1
21 22 1

[ pairs ]
; ai aj funct c6 c12
1 14 1
1 16 1
1 17 1
2 15 1
2 19 1
2 20 1
3 15 1
3 19 1
3 20 1
4 15 1
4 19 1
4 20 1
5 10 1
5 11 1
5 15 1
6 8 1
6 9 1
6 13 1
6 14 1
7 13 1
7 14 1
7 21 1
8 10 1
8 11 1
8 12 1
9 12 1
9 15 1
9 22 1
10 14 1
10 21 1
11 13 1
11 16 1
11 17 1
11 18 1
12 16 1
12 17 1
12 18 1
12 21 1
13 15 1
14 19 1
14 20 1
14 22 1
15 21 1
16 19 1
16 20 1
17 19 1
17 20 1

[ angles ]
; ai aj ak funct th0 cth S0 Kub
2 1 3 5
2 1 4 5
2 1 18 5
3 1 4 5
3 1 18 5
4 1 18 5
6 5 7 5
6 5 12 5
7 5 12 5
5 7 8 5
5 7 9 5
8 7 9 5
7 9 10 5
7 9 11 5
10 9 11 5
9 11 14 5
9 11 21 5
14 11 21 5
5 12 13 5
5 12 14 5
13 12 14 5
11 14 12 5
11 14 15 5
12 14 15 5
14 15 16 5
14 15 17 5
14 15 18 5
16 15 17 5
16 15 18 5
17 15 18 5
1 18 15 5
1 18 19 5
1 18 20 5
15 18 19 5
15 18 20 5
19 18 20 5
11 21 22 5

[ dihedrals ]
; ai aj ak al funct phi0 cp mult
2 1 18 15 9
2 1 18 19 9
2 1 18 20 9
3 1 18 15 9
3 1 18 19 9
3 1 18 20 9
4 1 18 15 9
4 1 18 19 9
4 1 18 20 9
6 5 7 8 9
6 5 7 9 9
6 5 12 13 9
6 5 12 14 9
7 5 12 13 9
7 5 12 14 9
8 7 5 12 9
9 7 5 12 9
5 7 9 10 9
5 7 9 11 9
8 7 9 10 9
8 7 9 11 9
7 9 11 14 9
7 9 11 21 9
10 9 11 14 9
10 9 11 21 9
9 11 14 12 9
9 11 14 15 9
9 11 21 22 9
14 11 21 22 9
5 12 14 11 9
5 12 14 15 9
13 12 14 15 9
12 14 11 21 9
15 14 11 21 9
11 14 12 13 9
11 14 15 16 9
11 14 15 17 9
11 14 15 18 9
12 14 15 16 9
12 14 15 17 9
12 14 15 18 9
14 15 18 19 9
14 15 18 20 9
16 15 18 19 9
16 15 18 20 9
17 15 18 19 9
17 15 18 20 9
1 18 15 14 9
1 18 15 16 9
1 18 15 17 9

gromacs/charmm36.itp

;;
;; Generated by CHARMM-GUI (url removed) v1.7
;;
;; psf2itp_mol.py
;;
;; Correspondance:
;; jul316@lehigh.edu or wonpil@lehigh.edu
;;
;; CHARMM36 FF in GROMACS format
;;

[ defaults ]
; nbfunc comb-rule gen-pairs fudgeLJ fudgeQQ
1 2 yes 1.0 1.0

[ atomtypes ]
; name at.num mass charge ptype sigma epsilon ; sigma_14 epsilon_14
CG2R61 6 12.0110 -0.107 A 3.55005321205e-01 2.928800e-01
CG321 6 12.0110 -0.195 A 3.58141284692e-01 2.343040e-01 ; 3.38541512893e-01 4.184000e-02
CG331 6 12.0110 -0.275 A 3.65268474438e-01 3.263520e-01 ; 3.38541512893e-01 4.184000e-02
HGA2 1 1.0080 0.090 A 2.38760856462e-01 1.464400e-01
HGA3 1 1.0080 0.090 A 2.38760856462e-01 1.004160e-01
HGP1 1 1.0080 0.420 A 4.00013524445e-02 1.924640e-01
HGR61 1 1.0080 0.115 A 2.42003727796e-01 1.255200e-01
OG311 8 15.9994 -0.531 A 3.14487247504e-01 8.037464e-01

[ bondtypes ]
; i j func b0 Kb
CG2R61 CG2R61 1 1.375000e-01 2.552240e+05
CG2R61 CG321 1 1.490000e-01 1.924640e+05
CG2R61 CG331 1 1.490000e-01 1.924640e+05
CG2R61 HGR61 1 1.080000e-01 2.845120e+05
CG2R61 OG311 1 1.411000e-01 2.797422e+05
CG321 CG321 1 1.530000e-01 1.861880e+05
CG321 CG331 1 1.528000e-01 1.861880e+05
CG321 HGA2 1 1.111000e-01 2.585712e+05
CG321 OG311 1 1.420000e-01 3.581504e+05
CG331 CG331 1 1.530000e-01 1.861880e+05
CG331 HGA3 1 1.111000e-01 2.694496e+05
CG331 OG311 1 1.420000e-01 3.581504e+05
OG311 HGP1 1 9.600000e-02 4.560560e+05

[ pairtypes ]
; i j func sigma1-4 epsilon1-4
CG2R61 CG321 1 3.46773417049e-01 1.10698234855e-01
CG2R61 CG331 1 3.46773417049e-01 1.10698234855e-01
CG321 CG321 1 3.38541512893e-01 4.18400000000e-02
CG321 CG331 1 3.38541512893e-01 4.18400000000e-02
CG321 HGA2 1 2.88651184677e-01 7.82754725313e-02
CG321 HGA3 1 2.88651184677e-01 6.48182492821e-02
CG321 HGP1 1 1.89271432669e-01 8.97368027066e-02
CG321 HGR61 1 2.90272620344e-01 7.24690057887e-02
CG321 OG311 1 3.26514380198e-01 1.83381431383e-01
CG331 CG331 1 3.38541512893e-01 4.18400000000e-02
CG331 HGA2 1 2.88651184677e-01 7.82754725313e-02
CG331 HGA3 1 2.88651184677e-01 6.48182492821e-02
CG331 HGP1 1 1.89271432669e-01 8.97368027066e-02
CG331 HGR61 1 2.90272620344e-01 7.24690057887e-02
CG331 OG311 1 3.26514380198e-01 1.83381431383e-01

[ angletypes ]
; i j k func th0 cth S0 Kub
CG2R61 CG2R61 CG2R61 5 1.2000000e+02 3.3472000e+02 2.4162000e-01 2.9288000e+04
CG2R61 CG2R61 CG321 5 1.2000000e+02 3.8325440e+02 0.0000000e+00 0.0000000e+00
CG2R61 CG2R61 CG331 5 1.2000000e+02 3.8325440e+02 0.0000000e+00 0.0000000e+00
CG2R61 CG2R61 HGR61 5 1.2000000e+02 2.5104000e+02 2.1525000e-01 1.8409600e+04
CG2R61 CG2R61 OG311 5 1.2000000e+02 3.7823360e+02 0.0000000e+00 0.0000000e+00
CG2R61 CG321 CG2R61 5 1.0750000e+02 4.3346240e+02 0.0000000e+00 0.0000000e+00
CG2R61 CG321 CG321 5 1.0750000e+02 4.3346240e+02 0.0000000e+00 0.0000000e+00
CG2R61 CG321 CG331 5 1.0750000e+02 4.3346240e+02 0.0000000e+00 0.0000000e+00
CG2R61 CG321 HGA2 5 1.0750000e+02 4.1254240e+02 0.0000000e+00 0.0000000e+00
CG2R61 CG321 OG311 5 1.1010000e+02 6.3345760e+02 0.0000000e+00 0.0000000e+00
CG2R61 CG331 HGA3 5 1.0750000e+02 4.1254240e+02 0.0000000e+00 0.0000000e+00
CG2R61 OG311 HGP1 5 1.0800000e+02 5.4392000e+02 0.0000000e+00 0.0000000e+00
CG321 CG321 CG321 5 1.1360000e+02 4.8827280e+02 2.5610000e-01 9.3386880e+03
CG321 CG321 CG331 5 1.1500000e+02 4.8534400e+02 2.5610000e-01 6.6944000e+03
CG321 CG321 HGA2 5 1.1010000e+02 2.2175200e+02 2.1790000e-01 1.8853104e+04
CG321 CG321 OG311 5 1.1010000e+02 6.3345760e+02 0.0000000e+00 0.0000000e+00
CG321 CG331 HGA3 5 1.1010000e+02 2.8953280e+02 2.1790000e-01 1.8853104e+04
CG321 OG311 HGP1 5 1.0600000e+02 4.1840000e+02 0.0000000e+00 0.0000000e+00
CG331 CG321 CG331 5 1.1400000e+02 4.4643280e+02 2.5610000e-01 6.6944000e+03
CG331 CG321 HGA2 5 1.1010000e+02 2.8953280e+02 2.1790000e-01 1.8853104e+04
CG331 CG321 OG311 5 1.1010000e+02 6.3345760e+02 0.0000000e+00 0.0000000e+00
CG331 CG331 HGA3 5 1.1010000e+02 3.1380000e+02 2.1790000e-01 1.8853104e+04
CG331 OG311 HGP1 5 1.0600000e+02 4.8116000e+02 0.0000000e+00 0.0000000e+00
HGA2 CG321 HGA2 5 1.0900000e+02 2.9706400e+02 1.8020000e-01 4.5187200e+03
HGA3 CG331 HGA3 5 1.0840000e+02 2.9706400e+02 1.8020000e-01 4.5187200e+03
OG311 CG321 HGA2 5 1.0889000e+02 3.8409120e+02 0.0000000e+00 0.0000000e+00
OG311 CG331 HGA3 5 1.0889000e+02 3.8409120e+02 0.0000000e+00 0.0000000e+00

[ dihedraltypes ]
; i j k l func phi0 cp mult
CG2R61 CG2R61 CG2R61 CG2R61 9 1.800000e+02 1.297040e+01 2
CG2R61 CG2R61 CG2R61 CG321 9 1.800000e+02 1.297040e+01 2
CG2R61 CG2R61 CG2R61 CG331 9 1.800000e+02 1.297040e+01 2
CG2R61 CG2R61 CG2R61 HGR61 9 1.800000e+02 1.757280e+01 2
CG2R61 CG2R61 CG2R61 OG311 9 1.800000e+02 1.297040e+01 2
CG2R61 CG2R61 CG321 CG2R61 9 1.800000e+02 9.623200e-01 2
CG2R61 CG2R61 CG321 CG321 9 1.800000e+02 9.623200e-01 2
CG2R61 CG2R61 CG321 CG331 9 1.800000e+02 9.623200e-01 2
CG2R61 CG2R61 CG321 HGA2 9 0.000000e+00 8.368000e-03 6
CG2R61 CG2R61 CG321 OG311 9 0.000000e+00 0.000000e+00 2
CG2R61 CG2R61 CG331 HGA3 9 0.000000e+00 8.368000e-03 6
CG2R61 CG2R61 OG311 HGP1 9 1.800000e+02 4.142160e+00 2
CG2R61 CG321 CG321 CG321 9 0.000000e+00 1.673600e-01 3
CG2R61 CG321 CG321 CG331 9 0.000000e+00 1.673600e-01 3
CG2R61 CG321 CG321 CG331 9 0.000000e+00 1.673600e-01 3
CG2R61 CG321 CG321 HGA2 9 0.000000e+00 1.673600e-01 3
CG2R61 CG321 CG321 OG311 9 0.000000e+00 8.368000e-01 3
CG2R61 CG321 CG331 HGA3 9 0.000000e+00 1.673600e-01 3
CG2R61 CG321 OG311 HGP1 9 0.000000e+00 3.096160e+00 3
CG2R61 CG321 OG311 HGP1 9 0.000000e+00 5.857600e+00 2
CG2R61 CG321 OG311 HGP1 9 0.000000e+00 8.786400e+00 1
CG321 CG2R61 CG2R61 CG331 9 1.800000e+02 1.004160e+01 2
CG321 CG2R61 CG2R61 HGR61 9 1.800000e+02 1.004160e+01 2
CG321 CG2R61 CG2R61 OG311 9 1.800000e+02 1.004160e+01 2
CG321 CG321 CG321 CG321 9 0.000000e+00 2.698680e-01 2
CG321 CG321 CG321 CG321 9 0.000000e+00 3.957227e-01 4
CG321 CG321 CG321 CG321 9 0.000000e+00 4.707418e-01 5
CG321 CG321 CG321 CG321 9 1.800000e+02 6.265540e-01 3
CG321 CG321 CG321 CG331 9 0.000000e+00 4.528762e-01 4
CG321 CG321 CG321 CG331 9 0.000000e+00 6.297338e-01 2
CG321 CG321 CG321 CG331 9 0.000000e+00 8.531594e-01 5
CG321 CG321 CG321 CG331 9 1.800000e+02 3.402847e-01 3
CG321 CG321 CG321 HGA2 9 0.000000e+00 8.158800e-01 3
CG321 CG321 CG321 OG311 9 0.000000e+00 8.158800e-01 3
CG321 CG321 CG331 HGA3 9 0.000000e+00 6.694400e-01 3
CG321 CG321 OG311 HGP1 9 0.000000e+00 5.857600e-01 2
CG321 CG321 OG311 HGP1 9 0.000000e+00 1.004160e+00 3
CG321 CG321 OG311 HGP1 9 0.000000e+00 4.727920e+00 1
CG331 CG2R61 CG2R61 CG331 9 1.800000e+02 1.004160e+01 2
CG331 CG2R61 CG2R61 HGR61 9 1.800000e+02 1.004160e+01 2
CG331 CG2R61 CG2R61 OG311 9 1.800000e+02 1.004160e+01 2
CG331 CG321 CG321 CG331 9 0.000000e+00 1.597870e-01 2
CG331 CG321 CG321 CG331 9 1.800000e+02 1.329675e-01 6
CG331 CG321 CG321 HGA2 9 0.000000e+00 7.531200e-01 3
CG331 CG321 CG321 OG311 9 0.000000e+00 8.158800e-01 3
CG331 CG321 CG331 HGA3 9 0.000000e+00 6.694400e-01 3
CG331 CG321 OG311 HGP1 9 0.000000e+00 5.857600e-01 2
CG331 CG321 OG311 HGP1 9 0.000000e+00 1.004160e+00 3
CG331 CG321 OG311 HGP1 9 0.000000e+00 4.727920e+00 1
HGA2 CG321 CG321 HGA2 9 0.000000e+00 9.204800e-01 3
HGA2 CG321 CG331 HGA3 9 0.000000e+00 6.694400e-01 3
HGA2 CG321 OG311 HGP1 9 0.000000e+00 7.531200e-01 3
HGA3 CG331 CG331 HGA3 9 0.000000e+00 6.485200e-01 3
HGA3 CG331 OG311 HGP1 9 0.000000e+00 7.531200e-01 3
HGR61 CG2R61 CG2R61 HGR61 9 1.800000e+02 1.004160e+01 2
OG311 CG2R61 CG2R61 HGR61 9 1.800000e+02 1.004160e+01 2
OG311 CG2R61 CG2R61 OG311 9 1.800000e+02 1.414527e+01 2
OG311 CG321 CG321 HGA2 9 0.000000e+00 8.158800e-01 3
OG311 CG321 CG321 OG311 9 0.000000e+00 8.368000e-01 3
OG311 CG321 CG331 HGA3 9 0.000000e+00 6.694400e-01 3

[ dihedraltypes ]
; i j k l func q0 cq
CG2R61 CG2R61 CG2R61 OG311 2 0.000000e+00 1.255200e+02

ligandrm.pdb

REMARK GENERATE LIGAND
REMARK DATE: 10/ 1/25 16:55:28 CREATED BY USER: charmm-gui
ATOM 1 C1 LIG L 1 24.305 -24.087 -0.006 1.00 0.00 LIG
ATOM 2 H1 LIG L 1 23.647 -24.500 0.789 1.00 0.00 LIG
ATOM 3 H2 LIG L 1 24.180 -22.983 -0.031 1.00 0.00 LIG
ATOM 4 H3 LIG L 1 25.359 -24.317 0.255 1.00 0.00 LIG
ATOM 5 C2 LIG L 1 21.531 -27.253 -4.159 1.00 0.00 LIG
ATOM 6 H4 LIG L 1 21.072 -28.228 -4.096 1.00 0.00 LIG
ATOM 7 C3 LIG L 1 22.026 -26.786 -5.384 1.00 0.00 LIG
ATOM 8 H5 LIG L 1 21.948 -27.401 -6.268 1.00 0.00 LIG
ATOM 9 C4 LIG L 1 22.631 -25.524 -5.460 1.00 0.00 LIG
ATOM 10 H6 LIG L 1 23.026 -25.165 -6.400 1.00 0.00 LIG
ATOM 11 C5 LIG L 1 22.748 -24.730 -4.315 1.00 0.00 LIG
ATOM 12 C6 LIG L 1 21.652 -26.460 -3.009 1.00 0.00 LIG
ATOM 13 H7 LIG L 1 21.295 -26.833 -2.060 1.00 0.00 LIG
ATOM 14 C7 LIG L 1 22.258 -25.197 -3.078 1.00 0.00 LIG
ATOM 15 C8 LIG L 1 22.504 -24.431 -1.799 1.00 0.00 LIG
ATOM 16 H8 LIG L 1 21.791 -24.783 -1.020 1.00 0.00 LIG
ATOM 17 H9 LIG L 1 22.314 -23.350 -1.968 1.00 0.00 LIG
ATOM 18 C9 LIG L 1 23.950 -24.701 -1.360 1.00 0.00 LIG
ATOM 19 H10 LIG L 1 24.113 -25.804 -1.298 1.00 0.00 LIG
ATOM 20 H11 LIG L 1 24.660 -24.313 -2.128 1.00 0.00 LIG
ATOM 21 O LIG L 1 23.391 -23.478 -4.416 1.00 0.00 LIG
ATOM 22 H12 LIG L 1 23.432 -23.119 -3.524 1.00 0.00 LIG
TER 23 LIG 1
END

2

check if your complex.gro (when you combine protein and ligand gro file there are no. of atoms written on top of both file so when you combine both you need to add those atoms also so that no. of atoms written and no. of atoms present in complex gro file are equal) file have atom equal (top) to what how many atoms are present in gro or not.

3

I have produced a combined .gro file named system.gro from the ligandrm.gro file (created from ligandrm.pdb using gmx editconf) and the 3HTB_processed.gro file. system.gro is attached with .txt extension:

system.gro.txt (115.9 KB)

Is this system.gro file correct or are there further issues that need to be resolved here?

4

see this system.txt and what modification i have done at ligand 1st atom position, please try it, i hope this will not raise any error.

system.txt.txt (118.5 KB)
i have used txt file just to explain please modify in your gro file.

1 Like
5

Removing the blank line and aligning the columns has resolved the initial issue. I believe this can be attributed to removing the blank line in the system .gro file, but I have not tested this specifically.

Now I am able to complete the system setup step and do the solvation.

In case it is of interest, I set up the topol.top to have the inclusions of the ligand force field definitions and the ligand definition after the inclusion of the general force field and prior to the definition of the protein in the topology.

I do run into an issue during the add ions step where it complains about either a duplicate defaults directive, or errors and warnings about duplicate definitions depending on what I do next. If I instead remove the general forcefield definition, I instead hit a no definition for NH3 error.

However, I believe I can fix this by the following algorithm:

  1. Have included the general forcefield, the ligand force field, and the ligand definition in that order.
  2. Attempt the steps that lead to generation of the errors related to duplicate definitions
  3. Delete the offending defninitions from the ligand force field file
  4. Repeat from 2 until there are no duplicate definitions and all steps succeed

I will come back and add more detail once I have make a few more attempts.

6

Hello again,

I was able to use the CHARMM-GUI generated files in combination with the general CHARMM36 force field by commenting out lines in the generated force-field parameters that duplicated items already present in the CHARMM36 force-field files. I believe CHARMM-GUI outputs CHARMM36 parameters, so I think that is consistent.

From there, I was able to go all the way to starting the production molecular dynamics run. As of the time of writing I am waiting for this to complete.

I did have to ignore a warning during the first part of equilibration (NVT I think) about the thermostat not being recommended in order to complete the tutorial as written. I ignored that warning by setting maxwarn to 2.

That warning was:

The Berendsen barostat does not generate any strictly correct ensemble,

and should not be used for new production simulations (in our opinion).

We recommend using the C-rescale barostat instead.

I hope this is helpful.

7

Hello @Caterpillar

Why two maxwarns?

The C-rescale is a barostat, so I do not think you got that error during the NVT, but rather during the NPT. I think that the equilibration procedure of CHARMM-GUI still has berendsen both as thermostat and barostat, and then switches to probably Nose-Hoover and Parrinello-Rahaman or C-rescale. In newer GROMACS this results in a critical warning, as you noticed.

GROMACS’s suggestion is very good, so stick to v-rescale and c-rescale if you can during both equilibration and production. Just double check that the two maxwarns are needed for the thermostat/barostat combination and not for other reasons.

8

I probably could have done -maxwarn 1 because the warning I mention is the only one I get. I didn’t want to check the documentation for whether the -maxwarn flag indicates “at most (inclusive)” or “less than (exclusive)”

I used the mdp files from the tutorial, and they mention V-rescale as a modified Berendsen thermostat. The file from the NPT step has an argument pcoupl given a value of Berendsen, so that might be where the barostat, and thus the warning message, is coming from.

Are there are other parameters I would need to modify in order to use the C-rescale barostat instead?

Additionally, the production run finished some minutes after I last posted a message. While I was not able to do the exact analysis steps called for by the tutorial because of an issue where something that was expected to be paired wasn’t, I was able to view the re-centered trajectory in PyMOL.

EDIT: Adding link to animation uploaded to youtube.

https://youtu.be/Rhgn-htdO_M

9

Ok, nice that you do not have other warnings.

Are there are other parameters I would need to modify in order to use the C-rescale barostat instead?

Not really, as far as I remember the standard CHARMM-GUI parameters in the mdp files are okay, setting the thermostat and barostat to v-rescale and c-rescale is more than sufficient.

10

I can confirm that changing the pcoupl argument to C-rescale prevents the warning previously raised by using the Berendsen barostat during the NPT equilibration stage.

I was able to run the simulation using the results of the equilibration done with the C-rescale barostat.

After I ran the simulation, I did the re-centering as instructed by the tutorial.

At that point, I deviated from the instructions and used gmx hbond

gmx hbond -f md_0_10_center.xtc -s md_0_10.tpr -dist hdist.xvg -ang hand.xvg -num hnum.xvg -dan hdan.xvg -o hbond.ndx

And gave the Protein group for the first static index group and JZ4 (the ligand) for the second static index group.

This succeeds, and the number of hydrogen bonds, the angles, and the distances involved all match what the tutorial says should happen (xmgrace figures at bottom).

I think at this point I can consider the tutorial completed.

h_dist
h_dist792×612 4.06 KB

h_ang
h_ang792×612 5.52 KB