1. Double-click the Maestro icon
   • (No icon? See Starting Maestro)

 

Figure 1-1. Change Working Directory option.

 

2. Go to File > Change Working Directory
3. Find your directory, and click Choose
4. Pre-generated input and results files are included for running jobs or examining output. Download the zip file here: https://www.schrodinger.com/sites/default/files/s3/release/current/Tutorials/zip/membrane-bound_fep_a2a.zip
5. After downloading the zip file, unzip the contents in your Working Directory for ease of access throughout the tutorial.

Figure 1-2. Save Project panel.

6. Go to File > Save Project As
7. Change the File name to Membrane-Bound_FEP_A2A, click Save
   • The project is now named Membrane-Bound_FEP_A2A.prj

Note: Banners appear when files have been imported, jobs incorporated into the Entry Lista simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion, or to prompt a common next step.

Note: By default the structure corresponding to the imported file is both included the entry is represented in the Workspace, the circle in the In column is bluein the Workspace the 3D display area in the center of the main window, where molecular structures are displayedand selected(1) the atoms are chosen in the Workspace. These atoms are referred to as "the selection" or "the atom selection". Workspace operations are performed on the selected atoms. (2) The entry is chosen in the Entry List (and Project Table) and the row for the entry is highlighted. Project operations are performed on all selected entries in the Entry Lista simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion. Please refer to the Glossary of Terms for the difference between includedthe entry is represented in the Workspace, the circle in the In column is blue and selected(1) the atoms are chosen in the Workspace. These atoms are referred to as "the selection" or "the atom selection". Workspace operations are performed on the selected atoms. (2) The entry is chosen in the Entry List (and Project Table) and the row for the entry is highlighted. Project operations are performed on all selected entries.

Figure 2-1. Importing the prepared protein structure.

1. Go to File > Import Structures
2. Select the file 4EIY_OPM_prepared.maegz
3. Click Open
   • The prepared 4EIY_OPM_prepared protein is loaded into the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed

Figure 2-2. Selecting a ligand using the QuickSelect Menu.

4. Includethe entry is represented in the Workspace, the circle in the In column is blue the 4EIY_OPM_prepared entry then click L form the Quick Select menu to select only the bound ligand
   • The atoms of the bound ligand in the 4EIY_OPM_prepared protein binding pocket is highlighted with blue squares

Figure 2-3. Extracting a selection as a New Entry.

5. Right-click on one of the ligand atoms and from the menu that appears choose Create New Entry by > Extracting Selected Atoms
   • A new entry as added to the Entry Lista simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion
   • A banner appears at the top of the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed stating that a New Entry has been created

Figure 2-4. Renaming a New Entry.

6. In the banner at the top of the Workspace, rename the new entry 4EIY_cognate_ligand and click the green check
   • A new entry named 4EIY_cognate_ligand is added to the Entry Lista simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion

Note: If you dismiss the “New entry created” banner you can also rename an Entry by double-clicking on its name in the Entry List to the left of the Workspace.

Figure 2-5. Importing the prepared A2A ligand series.

1. Go to File > Import Structures
2. Select the file A2A_prepared_FEP_ligands.maegz
3. Click Open
   • A new group called A2A_prepared_docked_ligands containing a series of 7 ligands from the reference paper is loaded into the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed

Figure 2-6. Including multiple ligand entries.

4. To compare the 4EIY cognate ligand and the series of 7 ligands, hold ctrl and click to includethe entry is represented in the Workspace, the circle in the In column is blue all of the ligand entries in the Entry Lista simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion
   • The 4EIY cognate ligand as well as the series of 7 A2A ligands are displayed in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed and show good alignment

Figure 2-7. Workspace Layout tiling for comparison of multiple structures.

5. Click on the + symbol at the lower-right of the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed to open the Workspace Toggles menu
6. Under Workspace Layout, click Tile by > Entry
   • Each entry is displayed in an individual tile; the ligands can be inspected side by side
7. Toggle off the Tile display by clicking Tile again to return to the standard view.

Figure 2-8. Including multiple entries in the Workspace.

8. Double-click to includethe entry is represented in the Workspace, the circle in the In column is blue and lock the 4EIY_OPM_prepared entry in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed and then click to includethe entry is represented in the Workspace, the circle in the In column is blue the first ligand in the A2A_prepared_docked_ligands group, cmp_001
   • The 4EIY protein and cmp_001 in its binding pocket are displayed in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed

 

 

Figure 2-9. Interactions Toggle.

9. Make sure Interactions in the Workspace Configuration Toolbar at the lower-right of the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed is toggled on so that the key interactions between the ligand and the 4EIY binding pocket (such as the yellow dashed lines in Figure 2-8) can be seen
10. In turn, includethe entry is represented in the Workspace, the circle in the In column is blue and inspect the remaining A2A ligand poses, noting that the hydrogen bonds and pi-pi interactions seen in Figure 2-8 are also present for the other ligands in the series

Figure 3-1. Selecting multiple entries from the Entry List.

1. Click to select(1) the atoms are chosen in the Workspace. These atoms are referred to as "the selection" or "the atom selection". Workspace operations are performed on the selected atoms. (2) The entry is chosen in the Entry List (and Project Table) and the row for the entry is highlighted. Project operations are performed on all selected entries the 4EIY_OPM_prepared entry and hold ctrl to also select(1) the atoms are chosen in the Workspace. These atoms are referred to as "the selection" or "the atom selection". Workspace operations are performed on the selected atoms. (2) The entry is chosen in the Entry List (and Project Table) and the row for the entry is highlighted. Project operations are performed on all selected entries the A2A_prepared_docked_ligands group
   • The protein and 7 ligands we will use for the FEP+ calculation are selected

Figure 3-2. FEP+ in Tasks.

2. Go to Tasks > Browse > Free Energy Perturbation > FEP+
   • The FEP+ panel opens

Figure 3-3. Import selected entries for FEP+.

3. For Import structures or perturbation map from, click Project Table (8 selected entries)
4. Click Import
   • A new group called FEP+: 4EIY_OPM_prepared is added to the Entry Lista simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion
   • Health checks for the inputs are performed

Figure 3-4. Select Relative FEP+.

Note: Warning triangles indicate that potential issues have been detected. For this receptor, these include steric clashes between crystal mates (not biologically relevant here) and minor irregularities relating to atoms that are not near to the binding pocket so we will ignore them. The 7 ligands are currently missing torsion parameters (unless you have already incorporated them into your Maestro Preferences) and this will be resolved in the next step.

5. Next to Calculate Binding Free Energy for, click Relative
   • The FEP+ panel Overview tab opens

Figure 3-5. FEP+ Panel Settings.

Within the FEP+ panel warning triangles are displayed for each of the ligands to signify that torsion parameters are missing. In this tutorial, we have provided the torsion parameters with the tutorial files, so you just need to load them in.

6. Click Settings
   • The FEP+ Panel Settings dialog opens
7. Under Custom OPLS4 Settings check Use customized version and then click on the … option to choose the custom parameters location

Note: For your own ligands, you can either use the Force Field builder to parametrize the missing torsions or check the “Generate missing parameters with Force Field Builder” option here to have that done automatically.

Figure 3-6. Select Custom Parameters Location.

8. Select the directory named ffb_A2A_oplsdir which was downloaded with the tutorial files at the beginning of this tutorial and click Select

 

Figure 3-7. Set Custom Parameters Location.

9. Once the location of the ffb_A2A_oplsdir directory has been selected click OK
   • The pre-generated custom torsion parameters for the ligands in this tutorial are located for use in the FEP+ job

Figure 3-8. Add membrane using the FEP+ Settings.

This is also where you can set up the membrane

10. In the FEP+ Settings, click the System Builder shortcut or scroll down to that section.
11. Check the Run membrane protocol box
    • A POPC membrane will be added and placed automatically when  FEP+ job is submitted
    • The system is oriented in the membrane according to the OPM convention: the membrane is placed perpendicular to the z axis with the coordinate origin at the center of the membrane
12. Click OK

Note: See the FEP+ Advanced Options Dialog Box documentation page for a description of the various options. 

Figure 3-9. FEP+ ligand quality and Map.

13. Inspect the Quality column. The warning triangles have now been replaced with green checks indicating that the custom torsion parameters have been correctly located and there is now sufficient force field information for each of the ligands
14. Click the Map tab at the top of the FEP+ panel
    • The Map tab opens

Figure 3-10. Add experimental affinities for FEP+.

15. In order to run Relative Binding FEP+, experimental binding affinity data for at least one ligand is required. In this tutorial, we have experimental data for all ligands in this series and we can use this to assess the accuracy of the FEP+ method. Starting with cp001,  double-click under the Exp. Affinity column to edit and add the known binding affinity reported in Figure 2 of the reference paper (also listed in the next step).

Figure 3-11. Generate Map.

16. Double-click to add the experimental binding affinity and the experimental error for each of the 7 ligands as shown in Figure 3-10
17. Click Generate Map
    • The Map Options dialog opens

Figure 3-12. Generate an Optimal Map.

18. Next to Map topology type select Optimal from the drop-down menu
19. Click Generate Map
    • After a minute or two arrows will be added forming a map connecting the various ligands based on similarity

Figure 3-13. Display similarity scores.

20. From the Display perturbation properties dropdown menu, choose Similarity scores
    • Similarity scores are displayed on each of the edges linking the ligands

Figure 3-14. Drag to rearrange the map layout.

21. In order to recreate the map used in the reference paper, click and drag each of the numbered ligands in the map view to arrange them into the shape displayed in Figure 3-13 and Figure S14 from the reference paper Supplementary Information.

Note: for an example of Map Generation using ligand bias see the BACE1 Inhibitor Design Using Free Energy Perturbation tutorial.

Figure 3-15. Add a new connection and select it to view the properties.

22. Click on the Add new connection icon and draw a line between ligand 4 (cmp008) and 2 (cmp002)
    • A new edge is added
23. Click on this new connection to select it
    • The selected edge is highlighted yellow
    • The details of the mutation are displayed on the left side of the panel

Note: Inspect the atom properties such as Common Core, Atom Mapping, and Hot Atoms for a selected edge in the Show atom properties drop-down menu.

Figure 3-16. Adding additional edges to recreate the reference map.

24. Repeat step 19 to add new connections for the following ligands to fully recreate the map used in the reference paper6 (cmp013) and 4 (cmp008)6 (cmp013) and 7 (cmp017)1 (cmp001) and 7 (cmp017)7 (cmp017) and 4 (cmp008)5 (cmp011) to 4 (cmp008)3 (cmp003) to 7 (cmp017)

Note: Use the magnifier icon to zoom in and view the ligand structure and label that corresponds to the ligand number if desired.

Figure 3-17. View the FEP+ map health.

25. Click on Info in the lower-right corner for the FEP+ panel to get a quick look at the properties and status of information in the map
    • The Status is displayed as OK for Similarity Score, Hot Atoms and Map Connections
    • The No FMPdb File warning can be ignored as this map has not run

Figure 3-18. Rearrange the map layout and change the job name.

26. (Optional) Click Auto-layout to rearrange the map
27. Change the Job name to fep_a2a_membrane-bound
28. Click Run Settings (cog)

Figure 3-19. Set hosts and write the FEP+ job.

29. Choose your CPU Host and GPU Host

Note: Ensure Maximum simultaneous subjobs is set to 0. This removes the limit on the number of subjobs, so they are all submitted to the subjob host queue. If you do not have license checking enabled, set the number of subjobs to ensure that you do not exhaust your licenses.

30. Click OK as we will write out these files
    • To launch the job from your computer instead, click Run
31. Click the Run Settings dropdown
32. Choose Write
    • This will write out the necessary input files and script to launch the job to your Working Directory
    • Use this to run the job on a remote cluster
    • This job requires significant GPU resources to run, so we will look at pre-generated results

Figure 4-1. FEP+ in Tasks.

1. Go to Tasks > Browse > Free Energy Perturbation > FEP+
   • FEP+ panel opens

Figure 4-2. Import a perturbation map results file.

2. For Import structures or perturbation map from, choose File
3. Click Browse
4. Choose fep_a2a_membrane-bound_out.fmp and click Open
   • The results are imported and the Receptor, Membrane, and Total Ligands information is populated
5. Click Next
   • The tables and map are populated

Figure 4-3. Reorder FEP+ results by Predicted Error.

6. In the Overview table click Pred. Error twice
   • The table is reordered with the highest values at the top
   • Values are shown in kcal/mol

Note: Predicted Error values of <0.5 kcal/mol are good, values >1 kcal/mol indicate that there are some inconsistencies in the map

7. Click Plot
   • The Correlation Plot (FEP+) panel opens

Figure 4-4. FEP+ Correlation Plot.

8. Hover over a data point
   • A tooltip with ligand information appears
9. Check Best Fit
   • A best fit line is added to the plot

Note: The dark gray error band highlights ligands with error less than 1 kcal/mol from the correlation line. The light gray error band highlights ligands with error between 1-2 kcal/mol.

Note: Click Save As to copy a .png of the Correlation Plot to your Working Directorythe location that files are saved

10. Click Close

Figure 4-5. Display perturbation properties for a map to analyze the results for the edges.

11. In the FEP+ panel, click the Map tab
12. Click Display perturbation properties
13. Chose the top three properties
    • Properties are displayed along the edges

Figure 4-6. Inspect the Hysteresis.

 

14. Click the Hysteresis tab
15. Click the Hysteresis column header twice
    • The table is reordered with the bad hysteresis cycles at the top
16. Click through the cycles in turn
    • The selected cycle is highlighted in the map as shown in Figure 4-6

Note: The value is colored according to how well it has converged and the threshold depends on the length N of the loop. Less than 0.5√N is good (green), between 0.5√N and 0.8√N is acceptable (yellow), and greater than 0.8√N is poor (red).

Figure 4-7. Optional: display bad perturbations.

Note: If there are red hysteresis cycles, click Display perturbation properties and choose Bad perturbations. Any perturbations with poor convergence are highlighted in red and labeled as a Bad Perturbation. The brighter the shade of red, the worse it is.

Figure 4-8. Analyze an individual edge.

Note: Although the Hysteresis tab shows that the cmp017-cmp008-cmp011-cmp003 cycle has the highest hysteresis there are no bad perturbations in this map (unlike in the original reference paper) and this edge is considered OK (colored yellow).

17. Right-click the cmp003 - cmp011 edge
18. Choose Analyze
    • The Analysis: cmp003 - cmp011 panel opens

Figure 4-9. Analysis of Rotatable Bond sampling.

19. In the Analysis panel, click the Ligand Details tab
20. Click the Rotatable Bonds tab
    • Torsions appear to be adequately sampled

Note: Hover over torsion sampling to highlight the atoms involved in the structures. The gray bars indicate the initial value of the rotatable bond and the strain in kcal/mol is reported below the plot for the complex and solvent legs.

Figure 4-10. Analysis of the ligand RMSD.

21. Under Ligand Details, click the Properties tab
    • Ligand 1 fluctuates a little more than Ligand 2
    • Both ligands remain within ~3 angstroms of the starting position over the course of the simulation
22. Hover the cursor at a point of interest within the plot
    • A red vertical line displays the position of the cursor
    • Frame time and RMSD readouts for each ligand at the selected time point are displayed

Figure 4-11. Analysis of the Convergence for the solvent and complex legs.

23. Click the Convergence tab

Note: The y-axis of the convergence plots will adjust to fit the scale. Pay attention to the y-axis values as good convergence may appear poor if the scale is very small.

Note: The total free energy differences between the two ligands (∆G in kcal/mol) in solvent and complex legs are plotted as a function of time - forward, reverse, and sliding window. The tables report the associated bootstrap and analytical error estimates from the corresponding simulation legs.

24. Close the Analysis panel

Figure 4-12. Optional: delete an edge.

 

Note: If desired, or in the case of an edge being labeled a bad perturbation, you may wish to delete an edge. Right-click on the relevant connection and choose Delete. Click on Plot (follow steps 7-10) to inspect the updated results. Error bars are often reduced when bad perturbations are deleted.

Figure 4-13. Inspecting Activity Cliffs.

25. In the FEP+ panel, click the Activity Cliffs tab
26. Set the Reference Ligand to cmp017
27. Click on the data point for cmp011, shown in Figure 4-13
    • The ligands are highlighted in the table
    • The perturbation of this amide (isomeric flip) results in an increase in affinity

Note: Hover over data points to see which ligand they represent. This graph gives information on which changes in the structure, as compared to the reference ligand, cause the largest changes in potency.

Figure 4-14. Reorder by predicted FEP.

 

1. Click the Analysis tab
2. Click the FEP column twice
   • The table is reordered by the predicted Bennett Free energy difference (ΔΔG) on top
   • Errors for predictions are also shown

Note: If the error for an edge is >0.3, please see Troubleshooting Common Issues for more information.

Figure 4-15. Hover over descriptions to see more information.

3. Hover over the Good, Fair, and Bad descriptions to get more information

Figure 4-16. Display the complex trajectory.

 

4. In the Complex Trajectory row for the cmp003 - cmp011 edge, click 5.0 ns
   • This entry has bad convergence
   • Viewing the trajectory could give more information
   • A new group with entries for each ligand is added to the Entry Lista simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion

Note: See the Analysis Tab help for more information about options in this tab.

 

 

Figure 4-17. Load complex trajectory for the edge.

5. In the Entry List group Trajectories, includethe entry is represented in the Workspace, the circle in the In column is blue cmp003
6. Click the T or right-click and choose Load Trajectory
   • The Trajectory Viewer loads into the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed
   • The A2A receptor is embedded in a POPC bilipid membrane as per the FEP+ setup

Figure 4-18. Undisplay the solvent.

7. In Quick Select, click S
   • The solvent atoms are selected(1) the atoms are chosen in the Workspace. These atoms are referred to as "the selection" or "the atom selection". Workspace operations are performed on the selected atoms. (2) The entry is chosen in the Entry List (and Project Table) and the row for the entry is highlighted. Project operations are performed on all selected entries in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed
8. Open the Style toolbox and click Undisplay selected atoms
   • The solvent is hidden

Figure 4-19. Adjust the trajectory playback settings.

9. In the Trajectory Viewer, click Playback Settings
10. Next to Speed, reduce to 80%
11. Check Beyond binding site to hide these atoms

Figure 4-20. Align playback on the protein.

12. Click the View Position tab
13. Next to Align on, choose Protein
14. Click in the empty Workspace to close Playback Settings
15. Type L to zoom to the ligand

Figure 4-21. Play the complex trajectory.

16. In the Trajectory Viewer, click Play
    • The frames from the 5 ns complex simulation are shown
    • The orientation of the phenyl ring and the triazole ring both twist during the simulation

Figure 4-22. The Ligand has different orientations in frames 2 (top) and 22 (bottom).

17. Click Move one step forward to manually view the frames
    • The phenyl ring moves back and forth
    • The orientation of the triazole ring twists
    • Some protein-ligand interactions (e.g. pi-pi stacking interactions, blue dashed lines, and hydrogen bonds, yellow dashed lines) appear to be present throughout the simulation whereas others only in some of the frames

 

Note: Toggle on the Interactions in the Workspace Configuration Toolbar at the lower-right of the of the Workspace to assist with interaction visualization

Figure 4-23. Plot the Pi-Pi Stacking interactions.

18. In the Workspace, click Plot and choose Interaction Counts > Pi-Pi Stacking
    • The Plot Computed Values Over Time panel opens

 

Note: Toggle on the Interactions in the Workspace Configuration Toolbar to assist with interaction visualization.

Figure 4-24. Inspect the Pi-Pi Stacking interactions at various time points.

19. In the Pi-Pi Stacking plot, click on a point in the blue line
    • The frame corresponding to the point in the plot is shown in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed

Figure 4-25. Compare multiple Interaction Counts over the course of the simulation.

20. Click Plot and choose Interaction Counts > Hydrogen Bonds
    • A new plot is added to the Plot Computed Values Over Time panel
    • We can see that the number of hydrogen bonds fluctuates between 2 and 4 for cmp003 over the course of the simulation

 

Note: Minimize or remove plots within the Plot Computed Values Over Time panel using the _ and X buttons above each plot.

 

 

Figure 4-26. Compare interactions for different ligands over the course of the simulation.

21. Close or Minimize the cmp003 Hydrogen Bonds plot
22. In the Entry List group Trajectories, includethe entry is represented in the Workspace, the circle in the In column is blue cmp011
23. Click Plot and choose Interaction Counts > Pi-Pi Stacking
    • A Pi-Pi Stacking plot for cmp011 is added to the Plot Computed Values Over Time panel below the cmp003 Pi-Pi Stacking plot