Hey,

This is probably the imaging problem, you can use 'autoimage' command in cpptraj (part of ambertools) to get rid of the imaging artifacts (RMSD plots should not have peaks after this operation) before looking at your results in VMD or cpptraj. After that, the most straightforward way to load a trajectory to VMD is to first load the topology (it contains the atom and connectivity info) and then load 'autoimaged' trajectory (or trajectories) and alinging it according to CA or backbone atoms to remove translation and rotation. I would not load restart files (this is just a single snapshot which also contain velocities which is useful when you want to extend the simulation further) unless you have a specific interest in that.

Hope that'll help.

Best,

Janek

Jan Ludwiczak Thanks so much for your response! I will try that! So the autoimage command basically will make the all the mdcrd files that I load in during cpptraj to align to the first mdcrd file? So when I use the autoimage command, for the parameters should I select my whole complex as the mask, or just the receptor part or just the ligand?

Here are all the commands that I entered in cpptraj:

parm complex3solvated.prmtop

list parm

parminfo 0

trajin prod3.mdcrd

trajin prod3-extend2ns.mdcrd

trajin prod3-extend3ns.mdcrd

trajin prod3-extend4ns.mdcrd

trajin prod3-extend5ns.mdcrd

trajin prod3-extend6ns.mdcrd

trajin prod3-extend7ns.mdcrd

trajin prod3-extend8ns.mdcrd

trajin prod3-extend9ns.mdcrd

trajin prod3-extend10ns.mdcrd

trajin prod3-extend11ns.mdcrd

trajin prod3-extend12ns.mdcrd

trajin prod3-extend13ns.mdcrd

trajin prod3-extend14ns.mdcrd

trajin prod3-extend15ns.mdcrd

trajin prod3-extend16ns.mdcrd

trajin prod3-extend17ns.mdcrd

trajin prod3-extend18ns.mdcrd

trajin prod3-extend19ns.mdcrd

trajin prod3-extend20ns.mdcrd

trajin prod3-extend21ns.mdcrd

trajin prod3-extend22ns.mdcrd

trajin prod3-extend23ns.mdcrd

trajin prod3-extend24ns.mdcrd

trajin prod3-extend25ns.mdcrd

trajin prod3-extend26ns.mdcrd

trajin prod3-extend27ns.mdcrd

trajin prod3-extend28ns.mdcrd

trajin prod3-extend29ns.mdcrd

trajin prod3-extend30ns.mdcrd

trajin prod3-extend31ns.mdcrd

trajin prod3-extend32ns.mdcrd

trajin prod3-extend33ns.mdcrd

trajin prod3-extend34ns.mdcrd

trajin prod3-extend35ns.mdcrd

list trajin

trajout 35ns_com3_CACN.trj

strip :WAT

reference complex3solvated.inpcrd

rms reference out CACN35nscomplex3.rmsd :1-207@CA

rms reference out CACN35nsligand3.rmsd :208

rms reference out CACN35nscomplex3.gnu :1-207@CA

rms reference out CACN35nsligand3.gnu :208

list actions

run

quit

Should I be using the "autoimage" command right before the "trajin prod3.mdcrd" line?

Everything that people measure in the lab are ensemble averages. Molecular dynamics is one approach to estimating the ensemble average numerically; the other is the Monte Carlo method. If you are seeing odd behaviors in your simulations, it is (almost) always your fault. The MD trajectory will not be fluid, because one typically only saves coordinates every picosecond. Hence the VMD view will be jittery. (If you save every frame, the jitter goes away.) If you are seeing large jumps, then that is an unnatural occurrence and needs to be investigated. In my view, the MD method's greatest advantage is that it is easier to debug: if the trajectories don't look right, they aren't right. If the whole protein shifts to the left from one frame to the next, that is unphysical and is the result of you doing something. You (inadvertently) shifted the coordinates or cause the center of mass to move. Unfortunately, this means that you'll have to take the time to understand what all of the myriad parameters in the simulation software mean. You really cannot treat it as a black box.

I don't know what you mean by equilibration but, generally, one starts a simulation with the protein backbone harmonically constrained. This holds everything close to the position that was found in the crystal structure, while the water solvates the protein. When you release the constraints, there is nothing holding the protein in the box other than the water bath. If the protein is drifting out of the box, you may need to increase the size of the box. Remember that the structure was solved in the crystal and that the solution structure may be somewhat different Additionally, in a "diffuse limit" solution, you do not want the protein to interact with periodic images of itself. With a 13 A cutoff for electrostatic interactions, you need at least 7 A of water buffer between your protein and the edge of the box. This means that you will be pushing around a lot of water, more than protein, but that is the current limitation of simulation. Fortunately computers are much faster now.