a few questions come to mind-
- how strong is the real-world binding of the peptide?
Unfortunately, we don’t have experimental data on Kd but my guess would be the protease-substrate complex is not a long-lived state. Yet I wouldn’t expect to see dissociation in 20 ns.
- are all of the heavy atoms well determined in your initial structure?
The initial structure is a 1.6 A x-ray structure. The electron density is very clear for all the heavy atoms except for 5 residues. But those 5 residues are very far away from the binding site (22 A) and are solvent-exposed. The temperature factors are also low for the residues around the binding site including the peptide.
- are there any uncertainties about protonation states?
I assigned the protonation states using the program Epik from Schrodinger.
- is it possible that your equilibration protocol was not gentle enough?
Here’s my minimization/equilibration protocol:
Added the hydrogens, did a restrained minimization, where all the heavy atoms are restrained with a harmonic weight of 1000 kcal/mol/A2 in vacuum non-periodic boundary conditions until the maximum gradient fell below 0.1 kcal/mol/A. Further relaxed the hydrogens by 1000 steps of steepest descent minimization.
Added waters and counterions, did 1000 steps of steepest descent energy minimization with a restraint of 1000 kcal/mol/A2 on the nonhydrogen atoms of the protease-substrate complex.
Optimized the positions of the side chains for 250 steps of steepest descent minimization followed by 4750 steps of conjugate gradient minimization. During this process, the backbone nonhydrogen atoms were restrained by a harmonic weight of 1000 kcal/mol/A2.
The restraint on the protein backbone was gradually decreased (500, 125, 25 kcal/mol/A2), each time for 250 cycles of steepest descent followed by 4750 cycles of conjugate gradient minimization.
Finally the whole system was relaxed for 5000 cycles (250 cycles of steepest descent and 4750 cycles of conjugate gradient) or until the threshold of 0.01 kcal/mol/A2 was reached.
Heated the system gradually in NVT from 50K to 300K over 50 ps with a time step of 0.001 psec with a 10 kcal/mol/A2 restraint on the heavy atoms.
Followed MD in the NPT without any harmonic restraints.
I'd appreciate any comments on my protocol.
with regard to your question, you can use restraints where the enzyme is in
the "fitmask" and the peptide is in the "rmsmask" and it will best-fit the
enzyme and keep the peptide in the same location relative to the enzyme as
it was at the start. check the manual for details.
Thanks for the suggestion. I understand I have two options: (1) targeted MD keeping initial and final the peptide location same relative to the enzyme and (2) steered MD (as suggested by Dmitry Nilov) applying harmonic restraint on the distance between substrate and binding site residues.
Is it reasonable to expect both to give the same conformational ensemble for the complex given that the simulation is long enough?
Thanks!
Aysegul
________________________________________
From: Carlos Simmerling [carlos.simmerling.gmail.com]
Sent: Thursday, July 14, 2011 5:22 PM
To: AMBER Mailing List
Subject: Re: [AMBER] Restrained MD
a few questions come to mind-
- how strong is the real-world binding of the peptide?
- are all of the heavy atoms well determined in your initial structure?
- are there any uncertainties about protonation states?
- is it possible that your equilibration protocol was not gentle enough?
with regard to your question, you can use restraints where the enzyme is in
the "fitmask" and the peptide is in the "rmsmask" and it will best-fit the
enzyme and keep the peptide in the same location relative to the enzyme as
it was at the start. check the manual for details.
On Thu, Jul 14, 2011 at 12:10 PM, Ozen, Aysegul
<Aysegul.Ozen.umassmed.edu>wrote:
> Hello,
>
> I'm trying to set up an MD simulation using Amber 10 for a
> protein-substrate complex starting from an x-ray structure. The problem is
> that the protein has a very shallow binding site and the substrate is a
> 6-amino acid peptide and in about 20 ns, the peptide starts diffusing away
> from the binding site. The goal of this simulation is to get information
> about the local conformational space spanned by the substrate when it's
> bound to the enzyme. I would like to impose a reasonable/minimal restraint
> on the peptide just enough to prevent it from drifting away from the active
> site.
>
> I typically use restrained minimizations where the coordinates are
> harmonically restrained with respect to a reference set of coordinates.
> However, in this case, the reference is the enzyme and its coordinates are,
> of course, changing throughout the production phase. Could you have any
> suggestions on how to define this restraint on the substrate wrt the protein
> binding site region? Or any smarter way to set up this simulation?
>
> Thanks much!
>
> Aysegul Ozen
> PhD Student
> Biochemistry and Molecular Pharmacology
> University of Massachusetts Medical School
> 364 Plantation Street, LRB 827
> Worcester, MA 01605
> ph: (508) 856-2636
>
>
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> AMBER mailing list
> AMBER.ambermd.org
> http://lists.ambermd.org/mailman/listinfo/amber
>
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Received on Fri Jul 15 2011 - 11:00:03 PDT
