# Umbrella sampling of Lipid Protein system using Martini FF **URL:** \ **Category:** User discussions\ **Created:** [December 8, 2020, 11:42pm UTC](https://gromacs.bioexcel.eu/t/umbrella-sampling-of-lipid-protein-system-using-martini-ff/1232 "2020-12-08T23:42:18Z")\ **Posts on this page:** 2\ **Page:** 1
**Author:** ![qaiser\_fatmi](https://dub1.discourse-cdn.com/flex017/user_avatar/gromacs.bioexcel.eu/qaiser_fatmi/32/535_2.png) [@qaiser\_fatmi](https://gromacs.bioexcel.eu/u/qaiser_fatmi)\ **Post date:** [December 8, 2020, 11:42pm UTC](https://gromacs.bioexcel.eu/t/umbrella-sampling-of-lipid-protein-system-using-martini-ff/1232/1 "2020-12-08T23:42:18Z")
GROMACS version: 2020.4 GROMACS modification: No Hello, I am calculating potential of mean force (PMF) for a protein-lipid bilayer system using Coarse Grained Martini force field and Gromacs 2020.4. In steered MD simulation I applied force to get a reaction coordinates in z-direction, where protein moved closer to the lipid bilayer and reached to the bilayer center (using pull code of Gromacs). I generated a series of initial configurations, each corresponding to a location where the protein was harmonically restrained at decreasing center-of­-mass (COM) distance from the lipid molecule (from 3.9 nm to 0.5 nm) using an umbrella biasing potential following Dr. Justin A. Lemkul tutorial. For Umbrella sampling (US), I generated a total of 37 different starting configurations along the z-reaction coordinate. Each configurations were independently equilibrated followed by the US for 10ns using following pull code: ; Pull code pull = yes pull\_ncoords = 1 ; only one reaction coordinate pull\_ngroups = 2 ; two groups defining one reaction coordinate pull\_group1\_name = PROTEIN pull\_group2\_name = MEMBRANE pull\_coord1\_type = umbrella ; harmonic potential pull\_coord1\_geometry = distance ; simple distance increase pull\_coord1\_dim = N N Y ; pull along z pull\_coord1\_groups = 1 2 ; groups 1 and 2 define the reaction coordinate pull\_coord1\_start = yes ; define initial COM distance \> 0 pull\_coord1\_rate = 0.0 ; 0.001 nm per ps = 1 nm per ns pull\_coord1\_k = 1000 ; kJ mol^-1 nm^-2 pull\_coord1\_init = 0.2 pull\_group1\_pbcatom = 20 pull-pbc-ref-prev-step-com = yes This is generating 37 trajectories, each of 10 ns. To use in WHAM I am using the last 5ns from each trajectory. Everything seems to be running fine, however, I am getting histogram, which seems not to be correct (see attachment please), as each window is not overlapping. I have three questions: Q.1 Why my COM distance is increased form 0.5 nm to over 1nm during production run despite using pulling force? Q.2 Why windows in my histogram is not overlapping? If I use small force constant, the COM starts increasing. Q.3 How can I resolve it? Many thanks for taking time and reading the message. Highly appreciated. Looking forward for the solution. ![histo_dist_US](https://europe1.discourse-cdn.com/flex017/uploads/bioexcel1/original/1X/e0c6b44de7355979c1a5b39ed84543bfe49c5086.jpeg) ---
**Author:** ![alevilla](https://dub1.discourse-cdn.com/flex017/user_avatar/gromacs.bioexcel.eu/alevilla/32/439_2.png) [@alevilla](https://gromacs.bioexcel.eu/u/alevilla)\ **Post date:** [December 11, 2020, 9:24am UTC](https://gromacs.bioexcel.eu/t/umbrella-sampling-of-lipid-protein-system-using-martini-ff/1232/2 "2020-12-11T09:24:56Z")
Hi, The lack of overlapping between each window means that you did not reach convergence. A option is to expend the run and check for convergence. Concerning your second issue, according to mdp file you define the distance as COM of starting conformation at t=0 + 0.2 nm) pull\_coord1\_start = yes =\> add the COM distance of the starting conformation to pull-coord1-init pull\_coord1\_init = 0.2 I am not sure that it is what you want. Using this setting, to have to sure what is the distance at t=0. One possible alternative is to use pull\_coord1\_start = no and define pull\_coord1\_init = selected distance. Kind regards Alessandra