As Bogdan suggested this could be an issue with the sign of the distance. When you run grompp you get
>Pull group natoms pbc atom distance at start reference at t=0 > 0 994 497 > 1 2 46347 1.224 1.950 which suggests your starting structure is at 1.22 nm but it could be +/- 1.224 nm you might want to manually check the distance for your starting structure. Gromacs restraints does not distinguish the sign of the distance so when u started running the simulation, since you have a huge force constant of 10000 kJ/mol, the pulling group is pulled to the window distance of 1.95 except that it could be -1.95 instead of +1.95 resulting in a COM distance around 2.7 nm. I had a similar problem when I tried pulling a peptide into the bilayer with the reference group being the center of mass of the bilayer. For the window at 0.5 nm, my starting structure had the pull group around -0.5 nm which I did not notice was off by 1 nm since I had used g_dist to check the distances. However the simulations ran fine with the pullx file listing distances around -0.5 nm. This should not ideally happen if the restraining section of gromacs distinguishes postive and negative distances because if it did, then the peptide would have been pulled to the window distance of 0.5 nm within the first few ps of the simulation. I used 'pull_geometry = distance' and I think gromacs restrains the pull_group at the magnitude of the distance although the pullx file lists negative distances. My understanding is that it will make no difference whether you give +1.95 or -1.95 for the pull_init1 unless your starting conformations have the distances at +1.95 and -1.95 respectively. Also another problem I had with negative distances is that g_wham does not distinguish the sign of the distances so even if the pullx file has negative values the pmf will be given only for the magnitude of the distances which is not what you want. So it will be best if you can choose a reference group such that the distance between your pull group and the reference is always positive. For my case I used the center of mass of the phosphate atoms in the lower leaflet of the bilayer as the reference group to pull the peptide into the bilayer instead of using the center of mass of the bilayer. The important thing is also to manually check the distance between ur pull and ref groups for the starting structures and start from a structure very close to the window position especially with large force constants so you can avoid sudden pulling for the pull groups. Please note, I used Gromacs 4.0.5 for my umbrella sampling simulations and I am not sure if what I observed is applicable to version 4.5.3. Sheeba ----- Sheeba J. Irudayam Postdoctoral Researcher Department of Chemistry University of North Carolina at Chapel Hill isheeba[at]email.unc.edu -- View this message in context: http://gromacs.5086.n6.nabble.com/Umbrella-Sampling-Pull-code-Problem-tp5000715p5000725.html Sent from the GROMACS Users Forum mailing list archive at Nabble.com. -- gmx-users mailing list gmx-users@gromacs.org http://lists.gromacs.org/mailman/listinfo/gmx-users * Please search the archive at http://www.gromacs.org/Support/Mailing_Lists/Search before posting! * Please don't post (un)subscribe requests to the list. Use the www interface or send it to gmx-users-requ...@gromacs.org. * Can't post? Read http://www.gromacs.org/Support/Mailing_Lists