Ligand Path Finder

In this tutorial, we will show you how to use the Ligand Path Finder app for finding possible ligand unbinding pathways from a protein. The Ligand Path Finder applies the ART-RRT method for finding ligand unbinding pathways from a protein. The ART-RRT method is a combination of the T-RRT method and the ARAP modeling method ¹. The T-RRT method is used for finding possible pathways and the ARAP modeling method is for generating possible ligand motions. Moreover, a constrained minimization is used for adapting the motion of the protein with the ligand.

Requirements

• SAMSON version 2020 R1 or higher
• Ligand Path Finder app
• FIRE state updater

Load the input model

In SAMSON, go to Home > Download and insert https://www.samson-connect.net/documents/b75291cc-8f4d-4119-8aa3-7d547b83818f - this will load a document with this tutorial's sample from SAMSON Connect.

The sample document contains a structural model of Lactose permease with its ligand Thiodigalactosid (TDG). In the Document View (1), you can see the protein under the name Protein_chain_A and the ligand under the name TDG as shown in the picture below. You also can see one conformation named bound_minimized which is the minimized conformation of the protein-ligand complex.

1. Interface menu > Document view or , : Ctrl+1, : Cmd+1

Note

For your own model, you might need to first prepare it (remove alternate locations, add hydrogens) - for that you can use Home > Prepare.

Launch the Ligand Path Finder app

Open the Ligand Path Finder app via Home > Apps > Biology or find it via Find everything.

In the app, you can see two tabs: the Settings tab is for configuring the search and the Results tab is for visualizing results.

Setup interaction and state updater models for energy evaluation

Note

The system needs to be already minimized. You can minimize the system using Edit > Minimize which uses Universal Force Field (UFF).

First, in the Settings tab, we need to specify an interaction model and a state updater which will be used in the computations. For the Interaction model select Universal Force Field (UFF) in the drop-down list, and for the State updater select FIRE. If you do not see the FIRE state updater, please check the requirements section and make sure you installed it from SAMSON Connect.

A window will pop up asking if you want to apply a new model, click Yes.

The Universal Force Field (UFF) setup will then ask whether to use existing bonds - choose to use existing bonds and click OK.

Two windows should appear: the Universal Force Field properties window showing its parameters and UFF energies of the current state of the system and the FIRE Properties window showing the state updater parameters. Set the parameters for FIRE as in the picture below (the step size to 1 fs, the number of steps to 1).

Setup the system

Let's now set up the system. Expand the Set up the system box.

Note

During the setup of the system, a new visual model should appear for showing the sampling box and atom types: blue for passive ARAP atoms, green for active ARAP atoms, and red for fixed atoms.

Define the bound state

Let's choose the starting conformation. Select the bound_minimized conformation in the Document View and then, in the App, click Set as starting conformation. The chosen conformation will be considered as the starting state for the search tree.

Note

Before creating a conformation for your system, make sure the system is oriented/aligned in such a way that the search domain efficiently encapsulates the possible unbinding pathways since the search domain is defined as a box in cartesian coordinates. You can orient the system using Move editors and you can align the system with respect to cartesian coordinates by selecting a structural model and in its context menu going to Move selection > ....

The system in this tutorial sample is already aligned with Z-axis.

Tip

To create a conformation for your system, use Edit > Conformation.

Define the ligand atoms

Now we need to define the ligand. Select TDG in the Document view, this will select all the ligand atoms (see User guide: Selecting).

Then, in the App, click the Set button to set the ligand atoms.

The rest of the atoms will be considered as protein atoms.

In the Advanced information box, a new line should appear: “31 atoms set as ligand atoms”.

Define the active ARAP atoms

Now we need to specify which ligand atoms will be considered by the ARAP method as active atoms used for controlling the ligand motion. The motions of the rest of the ligand atoms (also called passive ARAP atoms) will follow active atoms. Let's choose the Sulfur atom from the TDG ligand: S1. For simplicity, the document contains a group named S1 from TDG that refers to this atom. In the Document view, double-click on this S1 from TDG group. This will select nodes in the group, i.e. the S1 atom.

Tip

For your system, you can select atoms both in the Document view and in the Viewport using the selection editor. See User guide - Selecting.

Then, in the App, click the Add button to set the active ARAP atoms.

Define the fixed ARAP atoms

Finally, we need to specify which protein atoms will be considered by the ARAP method as having a fixed position. This is to ensure that the protein will not drift along with the ligand (note that this choice may influence the resulting unbindings pathways, so in general you should choose atoms from parts of the protein that are expected to be rather static).

Let's choose the CA atom in the Backbone of HIS 205 residue of the protein as the fixed ARAP atom. For simplicity, the Document has a group named CA from HIS 205 that refers to this atom. In the Document view, double-click on this group to select the corresponding atom.

Tip

For your system, you can select atoms both in the Document view and in the Viewport using the selection editor. See User guide - Selecting.

Then, in the App, click the Add button to set the fixed ARAP atom.

In the Advanced information box, you should see the number of added active and fixed ARAP atoms.

You can see which atoms were chosen as ligand, active or fixed ARAP atoms by clicking the corresponding Select buttons. If you are not satisfied with the starting conformation selection, ligand atom selection, or atom type assignment, you can reset your choices by clicking the corresponding Reset () button.

Define the sampling box

Let's now define the sampling region (the sampling box for the active ARAP atoms). Expand the Set the sampling region box.

The sampling box defines the sampling region for the chosen active ARAP atoms. The size and position of the box biases the ligand motion and therefore the resulting unbindings pathways. The App suggests the sampling box size which encloses the ligand and protein atoms.

Let's set the sampling box dimension as shown below - this box dimension biases the ligand motion towards the periplasmic side of the protein:

A green box visualizes the sampling box.

Define the search parameters

Let's now define the parameters. Expand the Set parameters box and set them as in the following figure:

• Use seed: use the specified seed number for the planner and after each run the seed value is incremented by 1. If the box is unchecked, a random seed is used. The specified seed number is needed if you want to reproduce the results later for the same seed.
• Runs = 2: we run the method 2 times to extract a maximum of 2 paths.
• ARAP-modeling iterations = 20: the number of iterations for the ARAP modeling method.
• Minimization iterations = 20: apply 20 steps of constrained minimization with FIRE each time a new state is sampled to minimize it.
• Initial temperature (T) = 0.001 K, Temperature factor = 2, Failures before increase of T = 1 : parameters for T-RRT.
• Max. ligand displacement = 40 A: each run is stopped as soon as the center of the ligand is displaced by 40 angstrom from its original position.
• RRT extension step size = 1 A: the size of the extension step is 1 angstrom. This affects how fast the conformation space is sampled.
• Maximum time per run: each run is stopped as soon as the elapsed time reaches this value.

Run the planner

Once you set up the system, the sampling box, and specified the parameters, you can launch the search for ligand unbinding pathways.

Click the Run button to start the planner.

Note

The search process can be paused by clicking the Pause button and resumed after that by clicking on the Resume button (these buttons are located at the same place as the Run button while the planner is running). To stop the process, click the Stop button.

During the search, in the Advanced information box under the Planning information, you can observe the elapsed time for the current run (Current running time), the elapsed time for all of the runs (Total running time), the number of nodes of the tree in the current run (Nodes), the run number (Run), and the number of paths found (Paths found).

Results

As soon as a path is found, it is added to the list in the Results tab. For example, the following figure shows two paths found.

Each path contains the following fields

• id: the path id
• # states: the number of conformations in the path.
• MinE (kcal/mol): the minimum energy of the path conformations.
• MaxE (kcal/mol): the maximum energy of the path conformations.
• Saddle (kcal/mol): the difference between MaxE and MinE.
• Barrier (kcal/mol): the difference between MaxE and First.
• Time (s): time elapsed for searching this path.
• First (kcal/mol): the energy of the first conformation in the path.
• Last (kcal/mol): the energy of the last conformation in the path.
• Remarks: comments on the path (editable).
• Color: the color of the energy curve for this path.

View conformation energies along the path

To view the energy curve of the path, select a path by clicking on it in the path table. To plot several energy curves, select several paths (Ctrl/Cmd + left-click for multi-selection). After selecting a path, you can move the slider to see a particular conformation in the path as shown in the picture below. The corresponding conformation is reflected in the structural model in SAMSON and its corresponding energy is shown in the Universal Force Field window.

Export the results: paths, conformations, path table content

You can copy the content of the path table to the clipboard by selecting the paths for which you want to export data and pressing Ctrl/Cmd + C or right-clicking and choosing Copy table content. You can also copy path energy values along the selected paths via right-clicking and choosing Copy path energy.

To export paths from the path table into the document as trajectories, select the paths you are interested in and press the Export paths button.

To export conformations along the path into SAMSON, select the paths from the path table, choose the export interval and click the Export button.

Tip

You can double-click on a path in the document to start/stop it.

To access the path controllers, select the path and open the Inspector (1).

If you right-click on a path, you can also access some of its options via Path > ....

1. Interface > Inspector, , : Ctrl+2, : Cmd+2"""

Next steps

Create pathlines

Check the Pathlines tutorial to learn about how to create pathlines to visualize the movement of the center of mass of a ligand.

Improve paths with P-NEB

The resulting paths can be significantly improved with the help of the parallel Nudged Elastic Band (NEB) method implemented in the P-NEB app. Please check out the P-NEB tutorial for more information.

Export atoms trajectories along paths

The resulting paths can be saved in the .sam and .samx formats. If you want to export only trajectories of some atoms along the path, you can use the Export Along Paths app. Please refer to the tutorial on how to use the Export Along Paths app for more information.

If you have any questions or feedback, please use the SAMSON Connect Forum.

References

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1. Nguyen MK, Jaillet L, Redon S. ART-RRT: As-Rigid-As-Possible exploration of ligand unbinding pathways. J Comput Chem. 2018 Apr 30;39(11):665-678. doi: 10.1002/jcc.25132. Epub 2018 Jan 5. PMID: 29315658. ↩