Hello everyone,
I have a question regarding the correct protocol for generating independent replicas of a membrane-protein simulation.
I am simulating a heterogeneous lipid membrane with an embedded transmembrane protein. So far, I have completed: Energy minimization, 100 ps NVT equilibration followed 400 ns NPT equilibration. My goal is to generate five independent production replicas.
My current approach is as follows:
Take the final configuration (.gro) from the 400 ns NPT equilibration.
Generate five production .tpr files using the production NVT/MD .mdp file, changing the random seed (gen_seed) for each replica.
Start the production runs directly for all five replicas.
I would like to know whether this is the correct approach.
Specifically, I have the following questions:
Is it acceptable to generate replicas from the final configuration of the fully equilibrated 400 ns NPT simulation, or should the replicas instead be generated immediately after energy minimization (or after the initial equilibration)?
If I generate replicas from the final equilibrated structure, do I need to perform any additional equilibration for each replica, or is it appropriate to start the production runs directly after assigning different random seeds?
Are there any recommended best practices for generating statistically independent replicas of membrane-protein systems in GROMACS?
I would appreciate any advice or suggestions. Thank you!
If you consider the system as equilibrated, then in a sense you can “just” generate new velocities, as the new trajectories will diverge exponentially and strictly speaking I would not care about having another equilibration time after this. This is what I would do for example for a simple globular protein in water without ligands, and just generate replicas from the first simulation where I removed the last restraint on the protein.
However, I would argue that in systems with lipids (and in others as well) the inertia from the bilayer itself is huge. Lipids mix very slowly and they can be trapped (or keep your protein trapped) in local metastable states that might have a veeeery long relaxation time. So, for example, I would expect that even after hundreds of nanoseconds the final positions of the lipids will still be highly correlated between the replicas, this being particularly true for mixed bilayers. And it’s even worse if you have very large chunky lipids, like with large sugars as head groups or with several (>2) tails. As such, if you care about being a bit more consistent I would at least restart every replica from the end of the energy minimization, or, even better, rebuild N times the box so that the starting positions are truly independent. This last option I feel it’s by far the best, especially if you care about lipid-protein interactions in your analysis.
As stated above, the replicas will likely never be independent over reachable simulation times as the timescales for the slowest conformational changes in both protein and bilayer are often longer than simulation times. Thus generating new random velocities and setting a new seed before production is sufficient. Even less is sufficient in practice, but this is the standard procedure.
Thank you both for your valuable insights and recommendations.
Based on your comments, I understand that although such replicas cannot be considered completely independent because of the intrinsically slow conformational dynamics of both the membrane and the protein, generating independent velocity distributions from a well-equilibrated configuration is the standard and accepted approach for producing production replicas in GROMACS. I also appreciate the suggestion that rebuilding multiple independent membrane systems would provide a more rigorous level of statistical independence, although this comes with a substantially higher computational cost.
Thank you once again for your time and for helping clarify the recommended protocol. It really means a lot.