(1) Are the protein and peptide really never interacting at d=7 nm? I presume you've got a peptide that would be maybe 5 nm long when fully extended, and your dG minimum is at 1.5 nm, so giving half the peptide length that would imply possible contact at 4 nm, so I expect 7 nm is sufficient, but gmx mindist can tell you for certain. For example, if the protein forms an overhanging pocket around the binding site, then it is quite possible that an unfolded peptide would be sometimes (even rarely) contacting a 568 residue protein even at a COM-COM distance of 7 nm.
(2) net charge positive on one and negative on the other? (3) unconverged? Did you try eliminating the first half of your production sampling and see if this fixes the issue? (4) did you do wham properly? Your mdp file indicate that your windows are not *exactly* at 0.1 and 0.2 nm increments (use of pull_coord1_start=yes), which is fine but only as long as wham doesn't think your windows are exactly at these increments. There may be some entropy change as the peptide becomes unbound and can then sample full X and Y, but on its own that should not continue to impact the sampling after contact is permanently broken. ________________________________________ From: gromacs.org_gmx-users-boun...@maillist.sys.kth.se <gromacs.org_gmx-users-boun...@maillist.sys.kth.se> on behalf of Lukas Zimmermann <luk.zi...@gmail.com> Sent: 15 June 2016 12:45:51 To: gmx-us...@gromacs.org Subject: [gmx-users] PMF steadily increasing Dear GROMACS community, I performed umbrella sampling study to estimate the binding free energy between a globular protein with 568 residues and a small peptide with 13 residues. I use the pull code with k = 800 and rate = 0.005 to generate the initial conformations over the time course of 1200 ps. The center of masses distance between the pull groups ranges from 1.4 nm ad 7.8 nm in the pull trajectory. I then extract conformations from pull.xtc with a spacing of 0.1 nm until 3 nm distance and a spacing of 0.2 nm for the remainder, yielding 38 windows in total. After having equilibrated each window with NVT and NPT under full position restraints, I conducted production simulation under NPT ensemble for 14 ns for each window. Finally, gmx wham computed the PMF curve which you can see here: https://www.dropbox.com/s/fp7ol41qoyokmjm/profile.xvg?dl=0 and this is the associated histogram: https://www.dropbox.com/s/bp6obymjc2qeu37/histo.xvg?dl=0 Please find here my MDP pull parameters: pull = yes pull_ngroups = 2 pull_ncoords = 1 pull_group1_name = chainB ; Protein pull_group2_name = chainC ; Peptide pull_coord1_type = umbrella pull_coord1_geometry = distance pull_coord1_groups = 1 2 pull_coord1_dim = N N Y pull_coord1_rate = 0.0 pull_coord1_k = 800 pull_coord1_start = yes I would now be very interested why the curve does not become flat beyond some certain distance, but rather seems to increase in a linear fashion, though the distance between the pull groups is sufficiently large. Could this be related to entropic effects? Is there a way to have the PMF properly normalized? The force field here is GROMOS 53a6 with SPC water. Temperature is coupled to 310 K and pressure to 1 bar. Cutoffs are 1.4 nm, longe range ES are resolved with PME and DispCorr is set to EnerPress. Bonds are constrained with LINCS. The Protein is prevented from rotation by having three CA atoms restrained with FC 1000. Thank you very much, I appreciate any help and suggestions. Lukas -- Gromacs Users mailing list * Please search the archive at http://www.gromacs.org/Support/Mailing_Lists/GMX-Users_List before posting! * Can't post? Read http://www.gromacs.org/Support/Mailing_Lists * For (un)subscribe requests visit https://maillist.sys.kth.se/mailman/listinfo/gromacs.org_gmx-users or send a mail to gmx-users-requ...@gromacs.org. -- Gromacs Users mailing list * Please search the archive at http://www.gromacs.org/Support/Mailing_Lists/GMX-Users_List before posting! * Can't post? Read http://www.gromacs.org/Support/Mailing_Lists * For (un)subscribe requests visit https://maillist.sys.kth.se/mailman/listinfo/gromacs.org_gmx-users or send a mail to gmx-users-requ...@gromacs.org.
