# Umberlla sampling for protein ligand complex **URL:** \ **Category:** User discussions\ **Created:** [November 23, 2024, 7:45am UTC](https://gromacs.bioexcel.eu/t/umberlla-sampling-for-protein-ligand-complex/10617 "2024-11-23T07:45:25Z")\ **Posts on this page:** 20\ **Page:** 1
**Author:** ![NMDr](https://avatars.discourse-cdn.com/v4/letter/n/dec6dc/32.png) [@NMDr](https://gromacs.bioexcel.eu/u/NMDr)\ **Post date:** [November 23, 2024, 7:45am UTC](https://gromacs.bioexcel.eu/t/umberlla-sampling-for-protein-ligand-complex/10617/1 "2024-11-23T07:45:25Z")
GROMACS version: GROMACS modification: Yes/No Here post your question umberlla sampling performed for protein ligand complex While performing gmx grompp -f md\_pull.mdp -c npt.gro -p topol.top -r npt.gro -n index.ndx -t npt.cpt -o pull.tpr i got following error Distance between pull groups 1 and 2 (5.818415 nm) is larger than 0.49 times the box size (5.818141). help me to solve this issue. Thanks in advance ---
**Author:** ![MagnusL](https://dub1.discourse-cdn.com/flex017/user_avatar/gromacs.bioexcel.eu/magnusl/32/2380_2.png) [@MagnusL](https://gromacs.bioexcel.eu/u/MagnusL)\ **Post date:** [November 25, 2024, 9:43am UTC](https://gromacs.bioexcel.eu/t/umberlla-sampling-for-protein-ligand-complex/10617/2 "2024-11-25T09:43:55Z")
Have a look at [Molecular dynamics parameters (.mdp options) - GROMACS 2024.4 documentation](https://manual.gromacs.org/current/user-guide/mdp-options.html#mdp-pull-coord1-geometry). What `pull-coord1-geometry` are you using? ---
**Author:** ![NMDr](https://avatars.discourse-cdn.com/v4/letter/n/dec6dc/32.png) [@NMDr](https://gromacs.bioexcel.eu/u/NMDr)\ **Post date:** [November 26, 2024, 5:15am UTC](https://gromacs.bioexcel.eu/t/umberlla-sampling-for-protein-ligand-complex/10617/3 "2024-11-26T05:15:43Z")
yes here i copy the 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 = lig pull\_coord1\_type = umbrella ; harmonic potential pull\_coord1\_geometry = distance ; simple distance increase pull\_coord1\_dim = N N Y pull\_coord1\_groups = 1 2 pull\_coord1\_start = yes ; define initial COM distance \> 0 pull\_coord1\_rate = 0.01 ; 0.01 nm per ps = 10 nm per ns pull\_coord1\_k = 1000 ; kJ mol^-1 nm^-2 pull-group1-pbcatom =24 pull-pbc-ref-prev-step-com = yes ---
**Author:** ![NMDr](https://avatars.discourse-cdn.com/v4/letter/n/dec6dc/32.png) [@NMDr](https://gromacs.bioexcel.eu/u/NMDr)\ **Post date:** [November 26, 2024, 5:17am UTC](https://gromacs.bioexcel.eu/t/umberlla-sampling-for-protein-ligand-complex/10617/4 "2024-11-26T05:17:28Z")
i increased the box size 16 16 16 again i got the error Distance between pull groups 1 and 2 (7.773946 nm) is larger than 0.49 times the box size (7.773864). using the same pull code help me to solve this issue ---
**Author:** ![MagnusL](https://dub1.discourse-cdn.com/flex017/user_avatar/gromacs.bioexcel.eu/magnusl/32/2380_2.png) [@MagnusL](https://gromacs.bioexcel.eu/u/MagnusL)\ **Post date:** [November 26, 2024, 8:08am UTC](https://gromacs.bioexcel.eu/t/umberlla-sampling-for-protein-ligand-complex/10617/5 "2024-11-26T08:08:42Z")
Yes, with `distance` and `direction` you can’t pull further than half (0.49 times) the box size. Try `direction-periodic`, but read the instruction of it first. How far do you need to pull the molecules apart? Are you sure you need to separate them this much? ---
**Author:** ![NMDr](https://avatars.discourse-cdn.com/v4/letter/n/dec6dc/32.png) [@NMDr](https://gromacs.bioexcel.eu/u/NMDr)\ **Post date:** [November 26, 2024, 12:01pm UTC](https://gromacs.bioexcel.eu/t/umberlla-sampling-for-protein-ligand-complex/10617/6 "2024-11-26T12:01:04Z")
Thank you , i will try and let u know, I am beginner to this study. At what basis , how far have to pull the ligand from the protein ? How to reduce this separation distance ? if i am wrong correct me pls . Thank you ---
**Author:** ![MagnusL](https://dub1.discourse-cdn.com/flex017/user_avatar/gromacs.bioexcel.eu/magnusl/32/2380_2.png) [@MagnusL](https://gromacs.bioexcel.eu/u/MagnusL)\ **Post date:** [November 26, 2024, 12:50pm UTC](https://gromacs.bioexcel.eu/t/umberlla-sampling-for-protein-ligand-complex/10617/7 "2024-11-26T12:50:56Z")
I don’t know the what you are studying, but I assume you want to pull the ligand away from the protein into a pure aqueous environment. When the protein is outside (with some margin) the cutoff of the LJ and Coulomb interactions the interactions between the ligand and the protein can be considered negligible. Currently you are using the COM distances, which means that there is no easy way to estimate how far away the ligand is from the closest protein residues, but if you have a look at the trajectories when you pull you can get an idea about this. I don’t see any reason to pull much further than the cutoff + 1 nm away from the protein (not the COM of the protein). But in the end it is you who must decide how far you need to pull. ---
**Author:** ![NMDr](https://avatars.discourse-cdn.com/v4/letter/n/dec6dc/32.png) [@NMDr](https://gromacs.bioexcel.eu/u/NMDr)\ **Post date:** [November 27, 2024, 12:00pm UTC](https://gromacs.bioexcel.eu/t/umberlla-sampling-for-protein-ligand-complex/10617/8 "2024-11-27T12:00:58Z")
i dnt want to pull the ligand 0.49 times the box size . how to change the distance or reduce the pulling distance can you clarify me pls ---
**Author:** ![MagnusL](https://dub1.discourse-cdn.com/flex017/user_avatar/gromacs.bioexcel.eu/magnusl/32/2380_2.png) [@MagnusL](https://gromacs.bioexcel.eu/u/MagnusL)\ **Post date:** [November 27, 2024, 12:17pm UTC](https://gromacs.bioexcel.eu/t/umberlla-sampling-for-protein-ligand-complex/10617/9 "2024-11-27T12:17:48Z")
You can lower the pull rate and/or lower the number of steps (the simulation time). ---
**Author:** ![NMDr](https://avatars.discourse-cdn.com/v4/letter/n/dec6dc/32.png) [@NMDr](https://gromacs.bioexcel.eu/u/NMDr)\ **Post date:** [November 28, 2024, 10:49am UTC](https://gromacs.bioexcel.eu/t/umberlla-sampling-for-protein-ligand-complex/10617/10 "2024-11-28T10:49:48Z")
reduced the pull rate, can you check is it correct ; Run parameters integrator = md dt = 0.002 tinit = 0 nsteps = 1000000 ; 2 ns nstcomm = 10 ; 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 = lig pull\_coord1\_type = umbrella ; harmonic potential pull\_coord1\_geometry = distance; simple distance increase pull\_coord1\_dim = N N Y pull\_coord1\_groups = 1 2 pull\_coord1\_start = yes ; define initial COM distance \> 0 pull\_coord1\_rate = 0.001 ; 0.001 nm per ps = 1 nm per ns pull\_coord1\_k = 1000 ; kJ mol^-1 nm^-2 pull-group1-pbcatom =24 pull-pbc-ref-prev-step-com = yes ---
**Author:** ![MagnusL](https://dub1.discourse-cdn.com/flex017/user_avatar/gromacs.bioexcel.eu/magnusl/32/2380_2.png) [@MagnusL](https://gromacs.bioexcel.eu/u/MagnusL)\ **Post date:** [November 28, 2024, 11:03am UTC](https://gromacs.bioexcel.eu/t/umberlla-sampling-for-protein-ligand-complex/10617/11 "2024-11-28T11:03:00Z")
With those settings you will pull the ligand (or its target position) 2 nm in the Z direction from its initial position. If that is what you want, it looks correct. ---
**Author:** ![NMDr](https://avatars.discourse-cdn.com/v4/letter/n/dec6dc/32.png) [@NMDr](https://gromacs.bioexcel.eu/u/NMDr)\ **Post date:** [November 28, 2024, 12:42pm UTC](https://gromacs.bioexcel.eu/t/umberlla-sampling-for-protein-ligand-complex/10617/12 "2024-11-28T12:42:09Z")
can you guide me pls… again i got the error after changing the pull rate Distance between pull groups 1 and 2 (7.772358 nm) is larger than 0.49 times the box size (7.771990). how to fix this , can you refer some basic about protein ligand umbrella sampling ---
**Author:** ![MagnusL](https://dub1.discourse-cdn.com/flex017/user_avatar/gromacs.bioexcel.eu/magnusl/32/2380_2.png) [@MagnusL](https://gromacs.bioexcel.eu/u/MagnusL)\ **Post date:** [November 28, 2024, 12:51pm UTC](https://gromacs.bioexcel.eu/t/umberlla-sampling-for-protein-ligand-complex/10617/13 "2024-11-28T12:51:32Z")
What’s the initial distance between the groups? ---
**Author:** ![NMDr](https://avatars.discourse-cdn.com/v4/letter/n/dec6dc/32.png) [@NMDr](https://gromacs.bioexcel.eu/u/NMDr)\ **Post date:** [November 29, 2024, 6:58am UTC](https://gromacs.bioexcel.eu/t/umberlla-sampling-for-protein-ligand-complex/10617/14 "2024-11-29T06:58:17Z")
gmx\_mpi distance -f md.xtc -s md.tpr -n prolig.ndx -oav distave.xvg -oall dist.xvg -oxyz distxyz.xvg -oh disthist.xvg -oallstat diststat.xvg -select -tu ns -dt 50 i select the option 22 protein ligand but its not working can you correct my mistake ---
**Author:** ![NMDr](https://avatars.discourse-cdn.com/v4/letter/n/dec6dc/32.png) [@NMDr](https://gromacs.bioexcel.eu/u/NMDr)\ **Post date:** [November 29, 2024, 7:02am UTC](https://gromacs.bioexcel.eu/t/umberlla-sampling-for-protein-ligand-complex/10617/15 "2024-11-29T07:02:54Z")
here i copy the my pull\_md.mdp file title = Umbrella pulling simulation define = -DPOSRES\_B ; Run parameters integrator = md ; Start time and Timestep in ps dt = 0.002 tinit = 0 nsteps = 250000 ; 500 ps nstcomm = 10 ; Output parameters nstxout = 5000 ; every 10 ps nstvout = 5000 nstfout = 500 nstxtcout = 500 ; every 1 ps nstenergy = 500 ; Bond parameters constraint\_algorithm = lincs constraints = all-bonds continuation = yes ; continuing from NPT ; Single-range cutoff scheme cutoff-scheme = Verlet nstlist = 20 ns\_type = grid rlist = 1.4 rcoulomb = 1.4 rvdw = 1.4 ; PME electrostatics parameters coulombtype = PME fourierspacing = 0.12 fourier\_nx = 0 fourier\_ny = 0 fourier\_nz = 0 pme\_order = 4 ewald\_rtol = 1e-5 optimize\_fft = yes ; Berendsen temperature coupling is on in two groups Tcoupl = V-rescale tc\_grps = Protein\_lig Water\_and\_ions tau\_t = 1.0 1.0 ref\_t = 310 310 ; Pressure coupling is on Pcoupl = Parrinello-Rahman pcoupltype = isotropic tau\_p = 1.0 compressibility = 4.5e-5 ref\_p = 1.0 refcoord\_scaling = com ; Generate velocities is off gen\_vel = no ; Periodic boundary conditions are on in all directions pbc = xyz ; Long-range dispersion correction DispCorr = EnerPres ; Pull code pull = yes pull\_ncoords = 1 ; only one reaction coordinate pull\_ngroups = 2 ; two groups defining one reaction coordinate pull\_group1\_name = lig pull\_group2\_name = Protein pull\_coord1\_type = umbrella ; harmonic potential pull\_coord1\_geometry = distance; simple distance increase pull\_coord1\_dim = N N Y pull\_coord1\_groups = 1 2 pull\_coord1\_start = yes ; define initial COM distance \> 0 pull\_coord1\_rate = 0.01 ; 0.01 nm per ps = 10 nm per ns pull\_coord1\_k = 1000 ; kJ mol^-1 nm^-2 pull-group1-pbcatom =24 pull-pbc-ref-prev-step-com = yes using this gmx mdrun -deffnm pull -pf pullf.xvg -px pullx.xvg was worked [pullf.xvg](https://gromacs.bioexcel.eu/uploads/short-url/r6qxBhJ3zqBjDuXsTEWNguofyHZ.xvg) (82.9 KB) [pullx.xvg](https://gromacs.bioexcel.eu/uploads/short-url/lzGGV095Z1b86WPguhmuVt6LLcT.xvg) (82.7 KB) here with i attached pullf.xvg and pullx.xvg , is it correct way to perform , if am wrong correct it pls ---
**Author:** ![MagnusL](https://dub1.discourse-cdn.com/flex017/user_avatar/gromacs.bioexcel.eu/magnusl/32/2380_2.png) [@MagnusL](https://gromacs.bioexcel.eu/u/MagnusL)\ **Post date:** [November 29, 2024, 7:41am UTC](https://gromacs.bioexcel.eu/t/umberlla-sampling-for-protein-ligand-complex/10617/16 "2024-11-29T07:41:15Z")
The difference I see between your mdp settings is that you have changed the order of the pull groups. This is of course fine - should not make any difference (with this pull geometry). What you have done, though, is that you are now using atom 24 of the ligand as the reference to define the center of mass of the ligand, and no PBC atom for the protein. Before you used atom 24 of the protein to define the center of mass of the protein, but you did not specify an atom for the ligand. The ligand is so small (I guess) that you don’t really need a `pbcatom`, but you probably need one for the protein, especially since you are using `pull-pbc-ref-prev-step-com = yes`. If atom 24 was not correct before, the distances might have been wrong. With the new settings you are pulling 5 nm from the initial distance. Before, you only pulled 2 nm. How long do you want to pull? You may be lucky that it works in the second case, but that mostly indicates an error in the first case. I would suggest: ```auto ; 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 = lig pull_coord1_type = umbrella ; harmonic potential pull_coord1_geometry = distance; simple distance increase pull_coord1_dim = N N Y pull_coord1_groups = 1 2 pull_coord1_start = yes ; define initial COM distance > 0 pull_coord1_rate = 0.001 ; 0.001 nm per ps = 1 nm per ns pull_coord1_k = 1000 ; kJ mol^-1 nm^-2 pull-group1-pbcatom = pull-pbc-ref-prev-step-com = yes ``` I don’t promise it will help your problem, but it’s almost certainly more correct. To get the initial distances, just look at the first line of the pullx.xvg file. ---
**Author:** ![NMDr](https://avatars.discourse-cdn.com/v4/letter/n/dec6dc/32.png) [@NMDr](https://gromacs.bioexcel.eu/u/NMDr)\ **Post date:** [November 29, 2024, 9:04am UTC](https://gromacs.bioexcel.eu/t/umberlla-sampling-for-protein-ligand-complex/10617/17 "2024-11-29T09:04:22Z")
Thank you , in the pullx.xvg initial distance 0.0000 (ps) 0.362757 (nm) in the previous case its worked for nsteps = 250000 ; 500 ps then increased the nsteps = 500000 ; 1000 ps (1ns) its not working , i get the same error Fatal error: Distance between pull groups 1 and 2 (6.703034 nm) is larger than 0.49 times the box size (6.702723). is it correct if i run pull.tpr for nsteps = 250000 ; 500 ps is it correct. how to confirm this ---
**Author:** ![MagnusL](https://dub1.discourse-cdn.com/flex017/user_avatar/gromacs.bioexcel.eu/magnusl/32/2380_2.png) [@MagnusL](https://gromacs.bioexcel.eu/u/MagnusL)\ **Post date:** [November 29, 2024, 9:13am UTC](https://gromacs.bioexcel.eu/t/umberlla-sampling-for-protein-ligand-complex/10617/18 "2024-11-29T09:13:04Z")
If you start at 0.36 nm and pull for 500 ps at 10 nm/ns, you should end at a distance of 5.36 nm, which is below 6.7 nm, which is why it works. It’s no surprise that it does not work when you pull for 1 ns (with the same pull rate), as you will then end at 10.36 nm. It is difficult to say if it’s correct or not. If you generate the conformations you are interested in, as input to the umbrella sampling, you can do almost whatever you want. Remember, you are still just trying to generate input conformations to the actual simulations. But keep in mind, if you pull quickly (what is quick is system dependent, but for simplicity let’s say \> 1 nm/ns) or the umbrella sampling starting conformations are generated far from equilibrium in other ways, you will probably have to run the umbrella sampling windows longer and discard more data in the beginning of them. ---
**Author:** ![NMDr](https://avatars.discourse-cdn.com/v4/letter/n/dec6dc/32.png) [@NMDr](https://gromacs.bioexcel.eu/u/NMDr)\ **Post date:** [November 29, 2024, 9:51am UTC](https://gromacs.bioexcel.eu/t/umberlla-sampling-for-protein-ligand-complex/10617/19 "2024-11-29T09:51:05Z")
thank you so much. You mean , if i reduce the pull rate may be it will work longer nsteps = 500000 ; 1000 ps (1ns). but dnt use the pull rate \> 10 nm . is it right ---
**Author:** ![MagnusL](https://dub1.discourse-cdn.com/flex017/user_avatar/gromacs.bioexcel.eu/magnusl/32/2380_2.png) [@MagnusL](https://gromacs.bioexcel.eu/u/MagnusL)\ **Post date:** [November 29, 2024, 9:53am UTC](https://gromacs.bioexcel.eu/t/umberlla-sampling-for-protein-ligand-complex/10617/20 "2024-11-29T09:53:36Z")
Yes, with lower pull rate you will not pull as far in the same period of time (same number of steps). [Next page](https://gromacs.bioexcel.eu/t/umberlla-sampling-for-protein-ligand-complex/10617.md?page=2)