# Problem encountered when running the Umbrella sampling tutorial by Justin **URL:** \ **Category:** User discussions\ **Created:** [January 14, 2023, 4:16am UTC](https://gromacs.bioexcel.eu/t/problem-encountered-when-running-the-umbrella-sampling-tutorial-by-justin/5573 "2023-01-14T04:16:12Z")\ **Posts on this page:** 6\ **Page:** 1
**Author:** ![Johnla](https://avatars.discourse-cdn.com/v4/letter/j/57b2e6/32.png) [@Johnla](https://gromacs.bioexcel.eu/u/Johnla)\ **Post date:** [January 14, 2023, 4:16am UTC](https://gromacs.bioexcel.eu/t/problem-encountered-when-running-the-umbrella-sampling-tutorial-by-justin/5573/1 "2023-01-14T04:16:12Z")
Command line: gmx grompp -f md\_pull.mdp -c npt.gro -p topol.top -r npt.gro -n index.ndx -t npt.cpt -o pull.tpr GROMACS version: version 2022.4 GROMACS modification: No Here post your question When I followed Justin’s Gromacs tutorial of umbrella sampling, I encountered the following errors. So what should I look for WARNING 1 [file topol.top, line 56]: The following macros were defined in the ‘define’ mdp field with the -D prefix, but were not used in the topology: POSRES\_B If you haven’t made a spelling error, either use the macro you defined, or don’t define the macro WARNING 2 [file topol.top, line 56]: The GROMOS force fields have been parametrized with a physically incorrect multiple-time-stepping scheme for a twin-range cut-off. When used with a single-range cut-off (or a correct Trotter multiple-time-stepping scheme), physical properties, such as the density, might differ from the intended values. Since there are researchers actively working on validating GROMOS with modern integrators we have not yet removed the GROMOS force fields, but you should be aware of these issues and check if molecules in your system are affected before proceeding. Further information is available at [GROMACS can not reproduce properties with the GROMOS force fields - Redmine #2884 (#2884) · Issues · GROMACS / GROMACS · GitLab](https://gitlab.com/gromacs/gromacs/-/issues/2884), and a longer explanation of our decision to remove physically incorrect algorithms can be found at [On The Importance of Accurate Algorithms for Reliable Molecular Dynamics Simulations | Theoretical and Computational Chemistry | ChemRxiv | Cambridge Open Engage](https://doi.org/10.26434/chemrxiv.11474583.v1) . * * * Program: gmx grompp, version 2022.4 Source file: src/gromacs/gmxpreprocess/readir.cpp (line 3077) Fatal error: Group Chain\_A referenced in the .mdp file was not found in the index file. Group names must match either [moleculetype] names or custom index group names, in which case you must supply an index file to the ‘-n’ option of grompp. ---
**Author:** ![Johnla](https://avatars.discourse-cdn.com/v4/letter/j/57b2e6/32.png) [@Johnla](https://gromacs.bioexcel.eu/u/Johnla)\ **Post date:** [January 15, 2023, 1:00am UTC](https://gromacs.bioexcel.eu/t/problem-encountered-when-running-the-umbrella-sampling-tutorial-by-justin/5573/2 "2023-01-15T01:00:38Z")
Also, I checked the index.ndx file, which was generated from the following command: ```auto gmx make_ndx -f npt.gro ... > r 1-27 > name 19 Chain_A > r 28-54 > name 20 Chain_B > q ``` But still “Chain\_A” was not found in the index.ndx file. So does anyone know what is going on? Thank you. ---
**Author:** ![Johnla](https://avatars.discourse-cdn.com/v4/letter/j/57b2e6/32.png) [@Johnla](https://gromacs.bioexcel.eu/u/Johnla)\ **Post date:** [January 16, 2023, 12:21am UTC](https://gromacs.bioexcel.eu/t/problem-encountered-when-running-the-umbrella-sampling-tutorial-by-justin/5573/3 "2023-01-16T00:21:23Z")
Hello guys, I found gromacs-2021 is free of the problem I encountered. It’s probably due to the bug in the “gmx make\_ndx” command. ---
**Author:** ![jalemkul](https://dub1.discourse-cdn.com/flex017/user_avatar/gromacs.bioexcel.eu/jalemkul/32/18_2.png) [@jalemkul](https://gromacs.bioexcel.eu/u/jalemkul)\ **Post date:** [January 16, 2023, 11:37am UTC](https://gromacs.bioexcel.eu/t/problem-encountered-when-running-the-umbrella-sampling-tutorial-by-justin/5573/4 "2023-01-16T11:37:54Z")
The other issues are also version-specific. Note that the tutorial is designed for 2018.x versions and does explicitly state that use of different versions may lead to problems. There are some additional checks in newer versions that don’t necessarily mean anything is wrong, but that are meant to inform you about syntactical and force field issues. ---
**Author:** ![Johnla](https://avatars.discourse-cdn.com/v4/letter/j/57b2e6/32.png) [@Johnla](https://gromacs.bioexcel.eu/u/Johnla)\ **Post date:** [January 19, 2023, 10:33pm UTC](https://gromacs.bioexcel.eu/t/problem-encountered-when-running-the-umbrella-sampling-tutorial-by-justin/5573/5 "2023-01-19T22:33:49Z")
Thanks a lot Justin. Great tutorial by the way. ---
**Author:** ![sab](https://avatars.discourse-cdn.com/v4/letter/s/7c8e57/32.png) [@sab](https://gromacs.bioexcel.eu/u/sab)\ **Post date:** [May 1, 2023, 1:52pm UTC](https://gromacs.bioexcel.eu/t/problem-encountered-when-running-the-umbrella-sampling-tutorial-by-justin/5573/6 "2023-05-01T13:52:50Z")
Hello, I encountered the same error, when following Umbrella sampling tutorial by Justin with Gromacs version2023. I am beginner user. How do you fix the issue. Thank you.