GROMACS version:2022.4 Hi, I am trying to do 10ns folding simulation for receptor terminals (N and C both). In the output, I observe the structure gets folded initially, but gradually after around 2.5-3ns, the structure starts to rotate and also unfolds in latter frames. Is this a normal occurrence, or 10ns simulation is too long? This is the mdp used for the simulation.

title = N-terminal domain folding simulation ; Run parameters integrator = md ; leap-frog integrator ;nsteps = 1000000 ; 0.002 * 1000000 = 2 ns ;nsteps = 250000 ; 0.002 * 250000 = 0.5 ns nsteps = 5000000 ; 0.002 * 5000000 = 10 ns dt = 0.002 ; 2 fs ; Output control nstxout = 50 ; save coordinates every 0.1 ps nstvout = 1000 ; save velocities every 2 ps nstenergy = 1000 ; save energies every 2 ps nstlog = 1000 ; update log file every 2 ps ; Bond parameters continuation = no ; not a restarted simulation constraint_algorithm = lincs ; holonomic constraints constraints = all-bonds ; all bonds (even heavy atom-H bonds) constrained lincs_iter = 1 ; accuracy of LINCS lincs_order = 4 ; also related to accuracy ; Neighborsearching cutoff-scheme = group ns_type = grid ; search neighboring grid cels nstlist = 5 ; 10 fs rlist = 1.4 ; short-range neighborlist cutoff (in nm) rcoulomb = 1.4 ; short-range electrostatic cutoff (in nm) rvdw = 1.4 ; short-range van der Waals cutoff (in nm) ; Implicit solvent implicit-solvent = GBSA gb-algorithm = Still rgbradii = 1.4 ; Electrostatics coulombtype = Cut-off ; Particle Mesh Ewald for long-range electrostatics pme_order = 4 ; cubic interpolation fourierspacing = 0.12 ; grid spacing for FFT ; Temperature coupling is on tcoupl = v-rescale ; More accurate thermostat tc-grps = Protein ; one coupling group tau_t = 0.1 ; time constant, in ps-1 ref_t = 310 ; reference temperature, one for each group, in K ; Dispersion correction DispCorr = Ener ; account for cut-off vdW scheme ; Velocity generation gen_vel = no ; Velocity generation is off ; COM motion removal ; These options remove motion of the protein/bilayer relative to the solvent/ions nstcomm = 1 comm-mode = Linear comm-grps = Protein ; Scale COM of reference coordinates refcoord_scaling = com

I also tried few by changing comm-mode from linear to angular, in which case the rotation and unfolding does not occur, but there is always a warning before the run about it being an artifact.

WARNING 1 [file …/md_gbsa_2.mdp]: Removing the rotation around the center of mass in a periodic system, this can lead to artifacts. Only use this on a single (cluster of) molecules. This cluster should not cross periodic boundaries.

How should I approach these? Is the rotation and unfolding for 10ns folding simulation common, and if I should choose from the conformation before the rotation starts? Thank you in advance