# Too many LINCS Warnings in MARTINI Lipid Bilayer simulations **URL:** \ **Category:** User discussions\ **Created:** [September 14, 2024, 5:29am UTC](https://gromacs.bioexcel.eu/t/too-many-lincs-warnings-in-martini-lipid-bilayer-simulations/10157 "2024-09-14T05:29:49Z")\ **Posts on this page:** 20\ **Page:** 1
**Author:** ![abelxf](https://avatars.discourse-cdn.com/v4/letter/a/cdc98d/32.png) [@abelxf](https://gromacs.bioexcel.eu/u/abelxf)\ **Post date:** [September 14, 2024, 5:29am UTC](https://gromacs.bioexcel.eu/t/too-many-lincs-warnings-in-martini-lipid-bilayer-simulations/10157/1 "2024-09-14T05:29:49Z")
GROMACS version:2022 GROMACS modification: No Dear GROMACS users, I have a coarse grained lipid bilayer(MARTINI 2.2), containing DPPC, DOPC and CHOL. I have managed to minimize the system in two steps, first to a tolerance of 1000 kJ/nm/mol followed by another step with a tolerance of 500 kJ/nm/mol. Both minimizations were done using the steepest descent algorithm with a step size of 0.015 and 0.005 respectively. Now while performing equilibriation runs, I am getting the following errors. Fatal error: Too many LINCS warnings (1214) If you know what you are doing you can adjust the lincs warning threshold in your mdp file or set the environment variable GMX\_MAXCONSTRWARN to -1, but normally it is better to fix the problem The angle deviations shown in the log file are 179.8-180 degrees, but the maximum threshold seems to be 30 degrees. Can any one tell me how I can resolve this? Thank you for patience. ---
**Author:** ![MagnusL](https://dub1.discourse-cdn.com/flex017/user_avatar/gromacs.bioexcel.eu/magnusl/32/2380_2.png) [@MagnusL](https://gromacs.bioexcel.eu/u/MagnusL)\ **Post date:** [September 16, 2024, 7:02am UTC](https://gromacs.bioexcel.eu/t/too-many-lincs-warnings-in-martini-lipid-bilayer-simulations/10157/2 "2024-09-16T07:02:31Z")
I assume that you are already applying restraints during EM and the crashing equilibration stage. You could see if it helps running a first (short) equilibration simulation with a shorter integration time step. Try `dt = 0.001` (yes even with Martini). ---
**Author:** ![abelxf](https://avatars.discourse-cdn.com/v4/letter/a/cdc98d/32.png) [@abelxf](https://gromacs.bioexcel.eu/u/abelxf)\ **Post date:** [September 16, 2024, 7:36am UTC](https://gromacs.bioexcel.eu/t/too-many-lincs-warnings-in-martini-lipid-bilayer-simulations/10157/3 "2024-09-16T07:36:46Z")
Dear MagnusL, Thank you for your input. I actually have multiple equilibration steps, starting with an integration time step of `dt = 0.002`, and gradually increasing it all the way to `dt = 0.02` These are some example mdp files ```auto ;define = -DBILAYER_LIPIDHEAD_FC=200 integrator = md tinit = 0.0 dt = 0.002 nsteps = 500000 nstlog = 1000 nstenergy = 1000 nstxout-compressed = 1000 compressed-x-precision = 100 cutoff-scheme = Verlet nstlist = 20 ns_type = grid pbc = xyz verlet-buffer-tolerance = 0.005 epsilon_r = 15 coulombtype = reaction-field rcoulomb = 2 vdw_type = cutoff vdw-modifier = Potential-shift-verlet rvdw = 2 tcoupl = v-rescale tc-grps = membrane W tau_t = 1.0 1.0 ref_t = 303.15 303.15 ; Pressure coupling: Pcoupl = berendsen Pcoupltype = semiisotropic tau_p = 5.0 compressibility = 3e-4 3e-4 ref_p = 1.0 1.0 ; GENERATE VELOCITIES FOR STARTUP RUN: gen_vel = yes gen_temp = 303.15 gen_seed = 2009092072 refcoord_scaling = all ``` ```auto define = -DBILAYER_LIPIDHEAD_FC=10 integrator = md tinit = 0.0 dt = 0.020 nsteps = 50000 nstlog = 1000 nstenergy = 1000 nstxout-compressed = 1000 compressed-x-precision = 100 cutoff-scheme = Verlet nstlist = 20 ns_type = grid pbc = xyz verlet-buffer-tolerance = 0.005 epsilon_r = 15 coulombtype = reaction-field rcoulomb = 2 vdw_type = cutoff vdw-modifier = Potential-shift-verlet rvdw = 2 tcoupl = v-rescale tc-grps = membrane W tau_t = 1.0 1.0 ref_t = 303.15 303.15 ; Pressure coupling: Pcoupl = berendsen Pcoupltype = semiisotropic tau_p = 5.0 compressibility = 3e-4 3e-4 ref_p = 1.0 1.0 ; GENERATE VELOCITIES FOR STARTUP RUN: gen_vel = no refcoord_scaling = all ``` P.S, I have commented the restrains on the lipid heads as my system is generated using PACKMOL. Some of the packing defects were not closing in earlier attempts, but closed up after removing restrains on the lipid heads ---
**Author:** ![abelxf](https://avatars.discourse-cdn.com/v4/letter/a/cdc98d/32.png) [@abelxf](https://gromacs.bioexcel.eu/u/abelxf)\ **Post date:** [September 16, 2024, 7:41am UTC](https://gromacs.bioexcel.eu/t/too-many-lincs-warnings-in-martini-lipid-bilayer-simulations/10157/4 "2024-09-16T07:41:16Z")
P.S, Further minimization runs were also performed and the system was minimized to a tolerance of 200 kJ/mol/nm ---
**Author:** ![MagnusL](https://dub1.discourse-cdn.com/flex017/user_avatar/gromacs.bioexcel.eu/magnusl/32/2380_2.png) [@MagnusL](https://gromacs.bioexcel.eu/u/MagnusL)\ **Post date:** [September 16, 2024, 8:33am UTC](https://gromacs.bioexcel.eu/t/too-many-lincs-warnings-in-martini-lipid-bilayer-simulations/10157/5 "2024-09-16T08:33:24Z")
So, did the simulations crash in the first or the second of those two example equilibration runs? Have you tried restraining all of the lipids? It seems like PACKMOL does not give very stable configurations since many people try to work-around using different methods. So, if you apply restraints, make sure that you do not always use the PACKMOL coordinates as reference. It is better to use the output from the previous simulation instead. But that might also mean that you need more simulations, e.g.: 1. EM, restrain whole lipids 2. Equilibration NVT, dt = 0.001, restrain whole lipids 3. Equilibration NVT, dt = 0.002, restrain whole lipids 4. Equilibraiton NVT, dt = 0.004, restrain whole lipids 5. Equilibration NVT, dt = 0.005, restrain lipid heads 6. Equilibration NPT, dt = 0.010, restrain lipid heads 7. Equilibration NPT, dt = 0.020, restrain lipid heads 8. Equilibration NPT, dt = 0.020, no restraints If it turns out that the simulations crash with dt = 0.020 you might have to settle for 0.015 or even 0.010. ---
**Author:** ![abelxf](https://avatars.discourse-cdn.com/v4/letter/a/cdc98d/32.png) [@abelxf](https://gromacs.bioexcel.eu/u/abelxf)\ **Post date:** [September 17, 2024, 8:36am UTC](https://gromacs.bioexcel.eu/t/too-many-lincs-warnings-in-martini-lipid-bilayer-simulations/10157/6 "2024-09-17T08:36:53Z")
Dear MagnusL, The LINCS error comes up in the very first equilibriation run using a time step of `dt = 0.002`. The example mdp files that I had posted in my previous reply was for the first and last equilibration steps respectively. I have 6 such steps, not including minimization, with the following parameters 1. Minimization 1, steepest descent with a tolerance of 1000 kJ/mol/nm 2. Minimization 2, steepest descent with a tolerance of 500 kJ/mol/nm 3. Minimization 3, steepest descent with a tolerance of 300 kJ/mol/nm 4. Minimization 1, steepest descent with a tolerance of 200 kJ/mol/nm 5. Equilibration 1, NPT, dt = 0.002, no restrains 6. Equilibration 2, NPT, dt = 0.005, no restrains 7. Equilibration 3, NPT, dt = 0.010, no restrains 8. Equilibration 4, NPT, dt = 0.015, no restrains 9. Equilibration 5, NPT, dt = 0.020, no restrains 10. Production Run, NPT, dt = 0.020, no restrains I have managed to minimize the system to below a tolerance of 200 kJ/mol/nm, but in the very first equilibration run, I am getting LINCS errors. I will try out your procedure as well. Thank you for your inputs ---
**Author:** ![abelxf](https://avatars.discourse-cdn.com/v4/letter/a/cdc98d/32.png) [@abelxf](https://gromacs.bioexcel.eu/u/abelxf)\ **Post date:** [September 17, 2024, 8:58am UTC](https://gromacs.bioexcel.eu/t/too-many-lincs-warnings-in-martini-lipid-bilayer-simulations/10157/7 "2024-09-17T08:58:50Z")
I have attempted to run the equilibration runs as specified by you, but with not luck. I am still receiving LINCS errors in the very first step. I also tried reducing the value of dt to `dt = 0.0005` and `dt = 0.0001` with no luck. Do you think running further steps of energy minimization could help? ---
**Author:** ![MagnusL](https://dub1.discourse-cdn.com/flex017/user_avatar/gromacs.bioexcel.eu/magnusl/32/2380_2.png) [@MagnusL](https://gromacs.bioexcel.eu/u/MagnusL)\ **Post date:** [September 17, 2024, 9:32am UTC](https://gromacs.bioexcel.eu/t/too-many-lincs-warnings-in-martini-lipid-bilayer-simulations/10157/8 "2024-09-17T09:32:56Z")
I think one stage of EM with the default tolerance is almost always enough (5000 to 50000 steps depending on the system). How many EM steps are performed before your EM simulations finish? I remember some cases when EM finished prematurely when run on GPU. Are your simulations running on GPU? If so, does it make a difference if you run with `-nb cpu`? ---
**Author:** ![abelxf](https://avatars.discourse-cdn.com/v4/letter/a/cdc98d/32.png) [@abelxf](https://gromacs.bioexcel.eu/u/abelxf)\ **Post date:** [September 17, 2024, 9:39am UTC](https://gromacs.bioexcel.eu/t/too-many-lincs-warnings-in-martini-lipid-bilayer-simulations/10157/9 "2024-09-17T09:39:42Z")
Dear MagnusL, These are the minimization outputs for the different systems I have. The minimization runs were done on a CPU machine and the equilibration runs are being carried out on GPU. ![Screenshot from 2024-09-17 15-05-05](https://europe1.discourse-cdn.com/flex017/uploads/bioexcel1/original/2X/4/4581c0c14086d0b319e9e6bdeb7e09da9c34e6dd.png) ![Screenshot from 2024-09-17 15-05-13](https://europe1.discourse-cdn.com/flex017/uploads/bioexcel1/original/2X/c/cab94cf23e7488c7b13a1a5a5830352cc1d6ac1e.png) ![Screenshot from 2024-09-17 15-05-17](https://europe1.discourse-cdn.com/flex017/uploads/bioexcel1/original/2X/c/c995ef6556acb004ef16a8c28b7c2aadec127f64.png) ![Screenshot from 2024-09-17 15-05-25](https://europe1.discourse-cdn.com/flex017/uploads/bioexcel1/original/2X/9/91e3247312f7e748a3119da4fda3615a251e7efc.png) ---
**Author:** ![abelxf](https://avatars.discourse-cdn.com/v4/letter/a/cdc98d/32.png) [@abelxf](https://gromacs.bioexcel.eu/u/abelxf)\ **Post date:** [September 17, 2024, 9:41am UTC](https://gromacs.bioexcel.eu/t/too-many-lincs-warnings-in-martini-lipid-bilayer-simulations/10157/10 "2024-09-17T09:41:09Z")
I have received LINCS errors for all systems, bar the 5\_LPE one which has already ran for about 880ns with `dt = 0.02`. ---
**Author:** ![MagnusL](https://dub1.discourse-cdn.com/flex017/user_avatar/gromacs.bioexcel.eu/magnusl/32/2380_2.png) [@MagnusL](https://gromacs.bioexcel.eu/u/MagnusL)\ **Post date:** [September 17, 2024, 9:58am UTC](https://gromacs.bioexcel.eu/t/too-many-lincs-warnings-in-martini-lipid-bilayer-simulations/10157/11 "2024-09-17T09:58:33Z")
I must admit I don’t know what could be the cause of these problems. Four rounds of EM should definitely be (more than?) enough. It might be worth switching to one temperature coupling group, but I don’t think that will make any significant difference. If NVT equilibration with restraints and `dt = 0.0001` does not work after EM there must be something strange going on. ---
**Author:** ![MagnusL](https://dub1.discourse-cdn.com/flex017/user_avatar/gromacs.bioexcel.eu/magnusl/32/2380_2.png) [@MagnusL](https://gromacs.bioexcel.eu/u/MagnusL)\ **Post date:** [September 17, 2024, 9:59am UTC](https://gromacs.bioexcel.eu/t/too-many-lincs-warnings-in-martini-lipid-bilayer-simulations/10157/12 "2024-09-17T09:59:08Z")
Does it help running the equilibrations on CPU only? ---
**Author:** ![MagnusL](https://dub1.discourse-cdn.com/flex017/user_avatar/gromacs.bioexcel.eu/magnusl/32/2380_2.png) [@MagnusL](https://gromacs.bioexcel.eu/u/MagnusL)\ **Post date:** [September 17, 2024, 10:00am UTC](https://gromacs.bioexcel.eu/t/too-many-lincs-warnings-in-martini-lipid-bilayer-simulations/10157/13 "2024-09-17T10:00:54Z")
Or does it work in GROMACS 2024.3? ---
**Author:** ![abelxf](https://avatars.discourse-cdn.com/v4/letter/a/cdc98d/32.png) [@abelxf](https://gromacs.bioexcel.eu/u/abelxf)\ **Post date:** [September 17, 2024, 11:34am UTC](https://gromacs.bioexcel.eu/t/too-many-lincs-warnings-in-martini-lipid-bilayer-simulations/10157/14 "2024-09-17T11:34:53Z")
I tried doing this as well. It still throws the same LINCS error. I am unsure of what to try at this point. During my minimization runs, the first minimization was done as a soft-core minimization, and all the following steps were not. Do you think this could be causing a problem? ---
**Author:** ![MagnusL](https://dub1.discourse-cdn.com/flex017/user_avatar/gromacs.bioexcel.eu/magnusl/32/2380_2.png) [@MagnusL](https://gromacs.bioexcel.eu/u/MagnusL)\ **Post date:** [September 17, 2024, 11:50am UTC](https://gromacs.bioexcel.eu/t/too-many-lincs-warnings-in-martini-lipid-bilayer-simulations/10157/15 "2024-09-17T11:50:13Z")
Can you generate the starting configurations without using PACKMOL? ---
**Author:** ![abelxf](https://avatars.discourse-cdn.com/v4/letter/a/cdc98d/32.png) [@abelxf](https://gromacs.bioexcel.eu/u/abelxf)\ **Post date:** [September 17, 2024, 12:22pm UTC](https://gromacs.bioexcel.eu/t/too-many-lincs-warnings-in-martini-lipid-bilayer-simulations/10157/16 "2024-09-17T12:22:00Z")
No, I cannot. I have an initial phase-separated system which requires packing of different sets of lipids in different regions of the membrane. ---
**Author:** ![abelxf](https://avatars.discourse-cdn.com/v4/letter/a/cdc98d/32.png) [@abelxf](https://gromacs.bioexcel.eu/u/abelxf)\ **Post date:** [September 17, 2024, 12:25pm UTC](https://gromacs.bioexcel.eu/t/too-many-lincs-warnings-in-martini-lipid-bilayer-simulations/10157/17 "2024-09-17T12:25:31Z")
![Double_Raft](https://europe1.discourse-cdn.com/flex017/uploads/bioexcel1/original/2X/6/6e82a43fa8e4995cfc9b1288e358e92dce4c381f.jpeg) I have very different compositions in the middle as compared to the rest of the membrane. To my knowledge, I cannot build such a system on any other platform than PACKMOL ---
**Author:** ![MagnusL](https://dub1.discourse-cdn.com/flex017/user_avatar/gromacs.bioexcel.eu/magnusl/32/2380_2.png) [@MagnusL](https://gromacs.bioexcel.eu/u/MagnusL)\ **Post date:** [September 17, 2024, 12:34pm UTC](https://gromacs.bioexcel.eu/t/too-many-lincs-warnings-in-martini-lipid-bilayer-simulations/10157/18 "2024-09-17T12:34:42Z")
Then I’m afraid I can’t come up with any other suggestions right now. ---
**Author:** ![MagnusL](https://dub1.discourse-cdn.com/flex017/user_avatar/gromacs.bioexcel.eu/magnusl/32/2380_2.png) [@MagnusL](https://gromacs.bioexcel.eu/u/MagnusL)\ **Post date:** [September 17, 2024, 12:38pm UTC](https://gromacs.bioexcel.eu/t/too-many-lincs-warnings-in-martini-lipid-bilayer-simulations/10157/19 "2024-09-17T12:38:19Z")
Or one more thing, what radius are you using for the Martini beads in PACKMOL? ---
**Author:** ![abelxf](https://avatars.discourse-cdn.com/v4/letter/a/cdc98d/32.png) [@abelxf](https://gromacs.bioexcel.eu/u/abelxf)\ **Post date:** [September 17, 2024, 12:43pm UTC](https://gromacs.bioexcel.eu/t/too-many-lincs-warnings-in-martini-lipid-bilayer-simulations/10157/20 "2024-09-17T12:43:11Z")
I have not set any radii… I have taken the already martinized PDBs from the CHARMM-GUI MARTINI Maker and used the APL values given there to construct the system on PACKMOL [Next page](https://gromacs.bioexcel.eu/t/too-many-lincs-warnings-in-martini-lipid-bilayer-simulations/10157.md?page=2)