LINCS WARNINGs in minimization of a membrane-protein simulation

User discussions
1

GROMACS version: gromacs-2022
GROMACS modification: Yes/No

Hi,
I have two questions but I will open different topic for the other one, just so it will be organized.
I am using CHARMM-GUI generated system of a transporter protein in a membrane bilayer. During minimization the system gives many LINCS WARNINGs in the beginning (from step 11), almost every 2-3 steps, until around step 108, and then it stabilizes and has 5 warnings total until step 270, and from there it goes smoothly until the end (step 467) to reach this note:

"Energy minimization has stopped, but the forces have not converged to the
requested precision Fmax < 1000 (which may not be possible for your system).
It stopped because the algorithm tried to make a new step whose size was too
small, or there was no change in the energy since last step. Either way, we
regard the minimization as converged to within the available machine
precision, given your starting configuration and EM parameters.

Double precision normally gives you higher accuracy, but this is often not
needed for preparing to run molecular dynamics.
You might need to increase your constraint accuracy, or turn
off constraints altogether (set constraints = none in mdp file)

writing lowest energy coordinates.

Steepest Descents converged to machine precision in 468 steps,
but did not reach the requested Fmax < 1000.
Potential Energy = -4.9085356e+05
Maximum force = 2.1745186e+04 on atom 7141
Norm of force = 1.3814017e+02"

All the atoms in the LINCS WARNINGs are related to the membrane molecules (none from the protein), as well as the maximum force parameter. When I continue to equilibration, there are no warnings (if there can even be any at this phase), and production looks good (Although I watch the protein and not the membrane). I have done around 4-5 tries with different combinations (with or without ligand, homologs etc) and it was a pattern.
If the pressure, temperature and potential curves are stable, is it fine to assume the system is valid?
I will try a water environment version (after the other runs will finish) because it all indicates that the membrane is the problem but in the end I want the protein to be in a membrane.

Thank you,
David.

2

Hi David!
How did you create your initial membrane topology? It sounds like it’s in a sub-optimal initial configuration and the membrane atoms needed to move more than what LINCS would allow without warning you about it. This is not necessarily bad, unless you really wanted to simulate something very similar to your initial configuration. If the potential energy looks good, and the system looks good to you (but please have a look at the membrane too!), I don’t think you need to worry. Just remember that membranes can take a while to properly equilibrate.

3

Initial membrane positioning was made by CHARMM-GUI following positioning of the protein in the membrane by the PPM 2.0 option (OPM). The overall potential looks good(10^ -5 and lower). I checked the membrane and I think it is fine as well, although the membrane lipids near the box edges are being “pushed” a bit outside (not into the water). However I will have a look at it in other frames and not just in the last frame.

Thank you!