Pulling chain B from protein Dimer through gromacs using Justin Lemkul tutorial

GROMACS version: 4.6.6
GROMACS modification: Yes/No no
Here post your question :
We are trying to study the effect mutation on protein dimerization. So we are pulling chain B from chain A of a protein dimer with a pulling force of 0.01, spring constant of 1000 and restrained chain B for 500PS . We have done 5 simulations of same dimer with the same parameters (mdp is attached) and observed that in three simulations, part of chain B was started getting open from its terminal end during the simulation (as shown in movie and figure) while rest of the two simulations have not shown any such problem and detached from the chain A, normally. So, I want to know that what may be the possible reason behind such random pulling (unfolding of chain B from the terminal end) behavior.

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If you’re applying restraints to chain B and it is still unfolding, then you’re likely not applying them correctly.

Dear Sir, thank you for your prompt reply. your tutorial has been a great help for learning MD simulation and in my research.

As we mentioned earlier that we are pulling chain B from chain A and applying restrained on chain A in md.mdp file (). We have tried different pulling force (0.001, 0.005, 0.01, 0.05) with spring constant 1000. we have observed that chain B unfold (as in attached in figure

). Some time if we replicate chain B detached smoothly (same simulation same files). please help !!!

here is my md.mdp file
title = Umbrella pulling simulation
define = -DPOSRES_A
; Run parameters
integrator = md
nsteps = 250000 ; 500 ps
dt = 0.002
; Output parameters
nstxout = 5000 ; suppress .trr output 100ps
nstvout = 5000 ; suppress .trr output 100ps
nstenergy = 500 ; save energies every 10.0 ps
nstlog = 5000 ; update log file every 10.0 ps
nstxtcout = 500 ; write .xtc trajectory every 20.0 ps
; Bond parameters
continuation = yes ; first dynamics run
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
; Single-range cutoff scheme
cutoff-scheme = Verlet
ns_type = grid ; search neighboring grid cells
nstlist = 10 ; 20 fs, largely irrelevant with Verlet
rcoulomb = 1.0 ; short-range electrostatic cutoff (in nm)
rvdw = 1.0 ; short-range van der Waals cutoff (in nm)
; PME electrostatics parameters
coulombtype = PME
fourierspacing = 0.16
pme_order = 4
; Berendsen temperature coupling is on in two groups
Tcoupl = V-rescale
tc_grps = Protein Non-Protein
tau_t = 1.0 1.0
ref_t = 300 300
; Pressure coupling is on
Pcoupl = Parrinello-Rahman
pcoupltype = isotropic
tau_p = 2.0 ; time constant, in ps
ref_p = 1.0 ; reference pressure, in bar
compressibility = 4.5e-5 ; isothermal compressibility of water, bar^-1

refcoord_scaling = com
; Periodic boundary conditions
pbc = xyz ; 3-D PBC
; Dispersion correction
DispCorr = EnerPres ; account for cut-off vdW scheme
; Velocity generation
gen_vel = no ; assign velocities from Maxwell distribution

; Pull code
pull = umbrella
pull_geometry = distance
pull_dim = N N Y
pull_start = yes ; define initial COM distance > 0
pull_ngroups = 1
pull_group0 = chainA
pull_group1 = chainB
pull_rate1 = 0.01 ; 0.01
pull_k1 = 1000 ; kJ mol^-1 nm^-2

Protein dimerization and dissociation may not proceed as if the proteins are rigid bodies (in fact, I’d wager it almost never is). Some structural perturbation may be observed when pulling the complex apart. Individual trajectories may vary, which is why repetition of calculations is essential to describe the ensemble behavior.

Hi all,
I’m experiencing the same observation on the dissociation of our dimer (consisting of Chain A and Chain B) too.

We’re pulling Chain B away from Chain A: position-restraint on Chain A, using COM-pulling, pulling mode as direction. We aim to study only the dissociation, not the unfolding of chain B. But through more than 60 simulations of different combinations of the pulling rate (v) and the pulling const (k), we see that in some simulations, the dissociation occured, while in others, only unfolding occured. They are not consistent. Should it be ok, as a fact of the process of dissociation?

Or, should there be any restraint that we should include in our simulation to obtain a consistent result?

Anh Mai

Hi Dr. Lemkul,
So, does it mean that we can restraint chain B from unfolding while pulling it away from chain A?
Could you please elaborate on how to do it?

Thanks a lot,
Anh Mai

exactly we face the same issue. have you resolved the problem. please let me know if you come up with a solution

Partial unfolding is often observed in dissociation processes. You can’t assume that two proteins dissociate or associate as rigid bodies, as I mentioned above.

Thank you, Dr. Lemkul.

Yes. When we figure it out, I’ll share our solution with you.

Hi akhil,
I didn’t get to see the videos of your pulling simulations.
Our chain A and chain B have about 200 residues each. Based on the picture of your protein, I guess ours are about the same size with your protein.

In our simulations, at first, we observed some unfolding occuring at the COM of our Chain B. It should not be artificially pulled that way. So, we adjust the two parameters: pulling rate v and force constant k, to find a consistency between all the simulations.

For now, we see that while in many simulations, there was a “clean” dissociation (I mean, as “no unfolding”), in others, there was still unfolding at the N-terminus of our chain B (which was binding to chain A). We see that it’s good enough, and makes sense, since the protein should not be rigid, and there should be partial unfolding simultaneously occurring along the dissociation. Hopefully, when we conduct the umbrella sampling along this initial trajectory (from the pulling), the production at each location should provide enough time for the structure to equilibrate back to its native conformation (no unfolding).

In short, we just experimented with different sets of k and v to find the consistency. We pulled along the z-direction.

Hope it helps a bit.
Anh Mai