Equilibrating Endstates in ABFE Calculations (Tyk2) =================================================== | Zeke A. Piskulich\ :sup:`1`, Patricio Barletta\ :sup:`1`, Ryan Snyder\ :sup:`1`, and Darrin M. York\ :sup:`1` | :sup:`1`\ Laboratory for Biomolecular Simulation Research, Institute | for Quantitative Biomedicine and Department of Chemistry and Chemical | Biology, Rutgers University, Piscataway, NJ 08854, USA Learning objectives ------------------- .. start-learning-objectives - Learn how to equilibrate the endstates in preparation for running absolute binding free energy calculations (ABFE). .. end-learning-objectives Relevant literature ------------------- - Coming Soon! Tutorial -------- In this Tutorial, you will learn how to equilibrate the endstates in preparation for running ABFE calculations for TYK2. .. contents:: :local: :depth: 4 .. start-tutorial Setting Up and Running End-State Equilibration ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ The starting point for this tutorial is the topology (parm7) and coordinate (rst7) files for the complex and aqueous endstates. The files for this tutorial are located: - :download:`Download the tutorial files ` Once you have downloaded the files, extract them into a directory where you will be working on this tutorial. .. code-block:: bash tar -xvzf equil_tyk2.tar.gz cd equil_tyk2 mkdir working_dir cd working_dir The files you should see in the top level directory are parm7 and rst7 files for both the complex and aqueous end states. The parm7 files contain the hmr tag indicating that hydrogen mass repartitioning has been used to increase the timestep of the simulations to 4 fs. .. note:: While this tutorial focuses on equilibrating for ABFE calculations, these are generally good practices for equilibrating any protein system using Amber. Equilibrating the Aqueous Endstates ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ To equilibrate the aqueous endstates, we will be using a number of steps to gradually relax the system, the MD input files are included below, and again can be found in the tutorial files you downloaded above. .. tab-set:: .. tab-item:: Minimization .. literalinclude:: /_static/files/ModularTutorials/Alchemical/ABFE_of_Tyk2/abfe_tyk2_tutorial/equil_files/binder_mdins/minimization.mdin .. tab-item:: Heating .. literalinclude:: /_static/files/ModularTutorials/Alchemical/ABFE_of_Tyk2/abfe_tyk2_tutorial/equil_files/binder_mdins/heat.mdin .. tab-item:: Density 1 .. literalinclude:: /_static/files/ModularTutorials/Alchemical/ABFE_of_Tyk2/abfe_tyk2_tutorial/equil_files/binder_mdins/density1.mdin .. tab-item:: Density 2 .. literalinclude:: /_static/files/ModularTutorials/Alchemical/ABFE_of_Tyk2/abfe_tyk2_tutorial/equil_files/binder_mdins/density2.mdin .. tab-item:: Lower Protein Restraints 1 .. literalinclude:: /_static/files/ModularTutorials/Alchemical/ABFE_of_Tyk2/abfe_tyk2_tutorial/equil_files/binder_mdins/lower_restraints1.mdin .. tab-item:: Lower Protein Restraints 2 .. literalinclude:: /_static/files/ModularTutorials/Alchemical/ABFE_of_Tyk2/abfe_tyk2_tutorial/equil_files/binder_mdins/lower_restraints2.mdin .. tab-item:: Lower Protein Restraints 3 .. literalinclude:: /_static/files/ModularTutorials/Alchemical/ABFE_of_Tyk2/abfe_tyk2_tutorial/equil_files/binder_mdins/lower_restraints3.mdin .. tab-item:: Lower Protein Restraints 4 .. literalinclude:: /_static/files/ModularTutorials/Alchemical/ABFE_of_Tyk2/abfe_tyk2_tutorial/equil_files/binder_mdins/lower_restraints4.mdin .. tab-item:: Lower Protein Restraints 5 .. literalinclude:: /_static/files/ModularTutorials/Alchemical/ABFE_of_Tyk2/abfe_tyk2_tutorial/equil_files/binder_mdins/lower_restraints5.mdin .. tab-item:: Backbone Binder .. literalinclude:: /_static/files/ModularTutorials/Alchemical/ABFE_of_Tyk2/abfe_tyk2_tutorial/equil_files/binder_mdins/backbone.mdin .. tab-item:: Unrestrained .. literalinclude:: /_static/files/ModularTutorials/Alchemical/ABFE_of_Tyk2/abfe_tyk2_tutorial/equil_files/binder_mdins/free.mdin There are a total of 11 steps to equilibrate the aqueous endstate; however, many of these steps can be grouped together into general goals. These goals are: - Minimize the system to remove bad contacts (step 1) - Heat the system to the target temperature while restraining the solute (step 2) - Equilibrate the density of the system while restraining the solute (steps 3 and 4) - Gradually relax the solute restraints (steps 5-10) - Run a short unrestrained simulation to relax the system (step 11) Below, we have included the commands to run each of these steps using amber. .. code-block:: bash pmemd.cuda -O -i ../binder_mdins/minimization.mdin -c ../binder_ejm_31.rst7 -p ../binder_ejm_31_hmr.parm7 -x binder_ejm_31_minimization.nc -r binder_ejm_31_minimization.rst7 -o binder_ejm_31_minimization.mdout -ref ../binder_ejm_31.rst7 pmemd.cuda -O -i ../binder_mdins/heat.mdin -c binder_ejm_31_minimization.rst7 -p ../binder_ejm_31_hmr.parm7 -x binder_ejm_31_heat.nc -r binder_ejm_31_heat.rst7 -o binder_ejm_31_heat.mdout -ref ../binder_ejm_31.rst7 pmemd.cuda -O -i ../binder_mdins/density1.mdin -c binder_ejm_31_heat.rst7 -p ../binder_ejm_31_hmr.parm7 -x binder_ejm_31_density1.nc -r binder_ejm_31_density1.rst7 -o binder_ejm_31_density1.mdout -ref ../binder_ejm_31.rst7 pmemd.cuda -O -i ../binder_mdins/density2.mdin -c binder_ejm_31_density1.rst7 -p ../binder_ejm_31_hmr.parm7 -x binder_ejm_31_density2.nc -r binder_ejm_31_density2.rst7 -o binder_ejm_31_density2.mdout -ref ../binder_ejm_31.rst7 pmemd.cuda -O -i ../binder_mdins/lower_restraints1.mdin -c binder_ejm_31_density2.rst7 -p ../binder_ejm_31_hmr.parm7 -x binder_ejm_31_lower_restraints1.nc -r binder_ejm_31_lower_restraints1.rst7 -o binder_ejm_31_lower_restraints1.mdout -ref ../binder_ejm_31.rst7 pmemd.cuda -O -i ../binder_mdins/lower_restraints2.mdin -c binder_ejm_31_lower_restraints1.rst7 -p ../binder_ejm_31_hmr.parm7 -x binder_ejm_31_lower_restraints2.nc -r binder_ejm_31_lower_restraints2.rst7 -o binder_ejm_31_lower_restraints2.mdout -ref ../binder_ejm_31.rst7 pmemd.cuda -O -i ../binder_mdins/lower_restraints3.mdin -c binder_ejm_31_lower_restraints2.rst7 -p ../binder_ejm_31_hmr.parm7 -x binder_ejm_31_lower_restraints3.nc -r binder_ejm_31_lower_restraints3.rst7 -o binder_ejm_31_lower_restraints3.mdout -ref ../binder_ejm_31.rst7 pmemd.cuda -O -i ../binder_mdins/lower_restraints4.mdin -c binder_ejm_31_lower_restraints3.rst7 -p ../binder_ejm_31_hmr.parm7 -x binder_ejm_31_lower_restraints4.nc -r binder_ejm_31_lower_restraints4.rst7 -o binder_ejm_31_lower_restraints4.mdout -ref ../binder_ejm_31.rst7 pmemd.cuda -O -i ../binder_mdins/lower_restraints5.mdin -c binder_ejm_31_lower_restraints4.rst7 -p ../binder_ejm_31_hmr.parm7 -x binder_ejm_31_lower_restraints5.nc -r binder_ejm_31_lower_restraints5.rst7 -o binder_ejm_31_lower_restraints5.mdout -ref ../binder_ejm_31.rst7 pmemd.cuda -O -i ../binder_mdins/backbone.mdin -c binder_ejm_31_lower_restraints5.rst7 -p ../binder_ejm_31_hmr.parm7 -x binder_ejm_31_backbone.nc -r binder_ejm_31_backbone.rst7 -o binder_ejm_31_backbone.mdout -ref ../binder_ejm_31.rst7 pmemd.cuda -O -i ../binder_mdins/free.mdin -c binder_ejm_31_backbone.rst7 -p ../binder_ejm_31_hmr.parm7 -x binder_ejm_31_unrestrained.nc -r binder_ejm_31_unrestrained.rst7 -o binder_ejm_31_unrestrained.mdout -ref ../binder_ejm_31.rst7 .. note:: **On Running Amber commands** The above commands use the pmemd.cuda executable to run the simulations on a GPU. If you are running on a CPU, you can replace pmemd.cuda with pmemd.MPI or sander as appropriate. Every command uses a number of common flags, which we will break down below for the first pmemd.cuda command. .. code-block:: bash pmemd.cuda -O -i ../binder_mdins/minimization.mdin -c ../binder_ejm_31.rst7 -p ../binder_ejm_31_hmr.parm7 -x binder_ejm_31_minimization.nc -r binder_ejm_31_minimization.rst7 -o binder_ejm_31_minimization.mdout -ref ../binder_ejm_31.rst7 The flags used here are: - -O: Overwrite output files if they already exist - -i: Input mdin file - -c: Input coordinate file (rst7) - -p: Input topology file (parm7) - -x: Output trajectory file (nc) - -r: Output restart file (rst7) - -o: Output mdout file (text) - -ref: Reference coordinate file for restraints In the mdin files, the restraints are defined using the reference coordinate file provided with the -ref flag. We are currently using Root Mean Square (RMS) restrains on the ligand heavy atoms to keep them close to this reference structure during equilibration. Equilibrating the Complex Endstates ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ To equilibrate the complex endstates, we will follow essentially the same steps as we did for the aqueous endstate, with the same general goals. The primary difference between these mdins and the previous is the protein restraint mask 1-289, which restrains the protein residues in addition to the ligand. The mdin files are included below, and again can be found in the tutorial files you downloaded above. .. warning:: Note that the mdin restrains here have been tailored to the TYK2 system, and should be adjusted if you are working with a different system. The key change to make is to adjust the protein residue range in the restraint mask (e.g. 1-289) to match the protein you are working with, plus 1 to account for the ligand being the first residue. .. tab-set:: .. tab-item:: Minimization .. literalinclude:: /_static/files/ModularTutorials/Alchemical/ABFE_of_Tyk2/abfe_tyk2_tutorial/equil_files/complex_mdins/minimization.mdin .. tab-item:: Heating .. literalinclude:: /_static/files/ModularTutorials/Alchemical/ABFE_of_Tyk2/abfe_tyk2_tutorial/equil_files/complex_mdins/heat.mdin .. tab-item:: Density 1 .. literalinclude:: /_static/files/ModularTutorials/Alchemical/ABFE_of_Tyk2/abfe_tyk2_tutorial/equil_files/complex_mdins/density1.mdin .. tab-item:: Density 2 .. literalinclude:: /_static/files/ModularTutorials/Alchemical/ABFE_of_Tyk2/abfe_tyk2_tutorial/equil_files/complex_mdins/density2.mdin .. tab-item:: Lower Protein Restraints 1 .. literalinclude:: /_static/files/ModularTutorials/Alchemical/ABFE_of_Tyk2/abfe_tyk2_tutorial/equil_files/complex_mdins/lower_restraints1.mdin .. tab-item:: Lower Protein Restraints 2 .. literalinclude:: /_static/files/ModularTutorials/Alchemical/ABFE_of_Tyk2/abfe_tyk2_tutorial/equil_files/complex_mdins/lower_restraints2.mdin .. tab-item:: Lower Protein Restraints 3 .. literalinclude:: /_static/files/ModularTutorials/Alchemical/ABFE_of_Tyk2/abfe_tyk2_tutorial/equil_files/complex_mdins/lower_restraints3.mdin .. tab-item:: Lower Protein Restraints 4 .. literalinclude:: /_static/files/ModularTutorials/Alchemical/ABFE_of_Tyk2/abfe_tyk2_tutorial/equil_files/complex_mdins/lower_restraints4.mdin .. tab-item:: Lower Protein Restraints 5 .. literalinclude:: /_static/files/ModularTutorials/Alchemical/ABFE_of_Tyk2/abfe_tyk2_tutorial/equil_files/complex_mdins/lower_restraints5.mdin .. tab-item:: Backbone Binder .. literalinclude:: /_static/files/ModularTutorials/Alchemical/ABFE_of_Tyk2/abfe_tyk2_tutorial/equil_files/complex_mdins/backbone.mdin .. tab-item:: Unrestrained .. literalinclude:: /_static/files/ModularTutorials/Alchemical/ABFE_of_Tyk2/abfe_tyk2_tutorial/equil_files/complex_mdins/free.mdin The commands to run each of these steps using amber are included below. .. code-block:: bash pmemd.cuda -O -i ../complex_mdins/minimization.mdin -c ../target_ejm_31.rst7 -p ../target_ejm_31_hmr.parm7 -x complex_ejm_31_minimization.nc -r complex_ejm_31_minimization.rst7 -o complex_ejm_31_minimization.mdout -ref ../target_ejm_31.rst7 pmemd.cuda -O -i ../complex_mdins/heat.mdin -c complex_ejm_31_minimization.rst7 -p ../target_ejm_31_hmr.parm7 -x complex_ejm_31_heat.nc -r complex_ejm_31_heat.rst7 -o complex_ejm_31_heat.mdout -ref ../target_ejm_31.rst7 pmemd.cuda -O -i ../complex_mdins/density1.mdin -c complex_ejm_31_heat.rst7 -p ../target_ejm_31_hmr.parm7 -x complex_ejm_31_density1.nc -r complex_ejm_31_density1.rst7 -o complex_ejm_31_density1.mdout -ref ../target_ejm_31.rst7 pmemd.cuda -O -i ../complex_mdins/density2.mdin -c complex_ejm_31_density1.rst7 -p ../target_ejm_31_hmr.parm7 -x complex_ejm_31_density2.nc -r complex_ejm_31_density2.rst7 -o complex_ejm_31_density2.mdout -ref ../target_ejm_31.rst7 pmemd.cuda -O -i ../complex_mdins/lower_restraints1.mdin -c complex_ejm_31_density2.rst7 -p ../target_ejm_31_hmr.parm7 -x complex_ejm_31_lower_restraints1.nc -r complex_ejm_31_lower_restraints1.rst7 -o complex_ejm_31_lower_restraints1.mdout -ref ../target_ejm_31.rst7 pmemd.cuda -O -i ../complex_mdins/lower_restraints2.mdin -c complex_ejm_31_lower_restraints1.rst7 -p ../target_ejm_31_hmr.parm7 -x complex_ejm_31_lower_restraints2.nc -r complex_ejm_31_lower_restraints2.rst7 -o complex_ejm_31_lower_restraints2.mdout -ref ../target_ejm_31.rst7 pmemd.cuda -O -i ../complex_mdins/lower_restraints3.mdin -c complex_ejm_31_lower_restraints2.rst7 -p ../target_ejm_31_hmr.parm7 -x complex_ejm_31_lower_restraints3.nc -r complex_ejm_31_lower_restraints3.rst7 -o complex_ejm_31_lower_restraints3.mdout -ref ../target_ejm_31.rst7 pmemd.cuda -O -i ../complex_mdins/lower_restraints4.mdin -c complex_ejm_31_lower_restraints3.rst7 -p ../target_ejm_31_hmr.parm7 -x complex_ejm_31_lower_restraints4.nc -r complex_ejm_31_lower_restraints4.rst7 -o complex_ejm_31_lower_restraints4.mdout -ref ../target_ejm_31.rst7 pmemd.cuda -O -i ../complex_mdins/lower_restraints5.mdin -c complex_ejm_31_lower_restraints4.rst7 -p ../target_ejm_31_hmr.parm7 -x complex_ejm_31_lower_restraints5.nc -r complex_ejm_31_lower_restraints5.rst7 -o complex_ejm_31_lower_restraints5.mdout -ref ../target_ejm_31.rst7 pmemd.cuda -O -i ../complex_mdins/backbone.mdin -c complex_ejm_31_lower_restraints5.rst7 -p ../target_ejm_31_hmr.parm7 -x complex_ejm_31_backbone.nc -r complex_ejm_31_backbone.rst7 -o complex_ejm_31_backbone.mdout -ref ../target_ejm_31.rst7 pmemd.cuda -O -i ../complex_mdins/free.mdin -c complex_ejm_31_backbone.rst7 -p ../target_ejm_31_hmr.parm7 -x complex_ejm_31_unrestrained.nc -r complex_ejm_31_unrestrained.rst7 -o complex_ejm_31_unrestrained.mdout -ref ../target_ejm_31.rst7 Again, run these, and you will be left with equilibrated endstate files for both the complex and aqueous endstates, which can be used to start the production ABFE calculations. You can repeat these steps for each of the remaining three ligands; the mdin files are included in the tutorial files you downloaded above. .. end-tutorial