A Receptor Preparation Tool with a Graphical User Interface

AutoGridFR v1.1: a receptor preparation tool with a Graphical User Interface

Introduction

AutoGridFR (AGFR) is a software program for preparing a receptor for docking with AutoDockFR v1.1 . Its supports the preparation of rigid receptors as well as receptors with flexible side chains. It also supports preparing receptors forMGLTools2 covalent -docking.1.1/bin/agfr It includes the AutoSite [1] software package that can be used to identifyMGLTools2 potential-1.1 /bin/agfrguibinding pockets automatically. AGFR can be used from the command line ( ) or through its graphical user interface (GUI shown below) ( ) for defining the docking-box and for computing affinity maps for that box.

agfr agfrgui MGLTools2-1.1/bin/adfr The target files (.trg) produced by and can be used directly as an input to AutoDockFR ( ). They can also be unzipped to obtain maps files usable by AutoDock4. AGFR currently only operates on molecules in the PDBQT file format. The minimum input is a PDBQT file for a receptor. Optionally, a PDBQT file for a ligand molecule can be specified and can be used to initialize the docking-box position and size, and to set initial map types to be computed. Alternatively, the GUI can load an existing target file allowing its inspection and modification. [1] Pradeep Anand Ravindranath Michel F. Sanner. AutoSite: an automated approach for pseudo-ligands prediction—from ligand-binding sites identification to predicting key ligand atoms.Bioinformatics, Volume 32, Issue 20, 15 October 2016, Pages 3142–3149, https://doi.org/10.1093/bioinformatics/btw367 MGLTools2-1.1/bin/agfrgui.

This tutorial is focusing on the Graphical User Interface We assume have downloaded and installed MGLTools2 v1.1. The following provides a short overview of the GUI and five use cases that demonstrate the preparation of a receptor for docking in different scenarios. Overview

The GUI is divided in 3 sections: 1) a panel of widgets for operating the software (left); 2) a tool-bar for the 3D Viewer (middle); 3) the 3D viewer providing visual feedback (right). A status bar located at the bottom of the GUI runs across these 3 panels. The left side of the status bar displays messages directing the useri.e. and the right side presents 4 status lights informing the user of the state of the GUI. The “generate target filee.g. ...” button will only be enabled once all requirements are fulfilled, all enabled status lights are green. Some status lights might be disabled depending on the type of docking, the pocket-definition light will be disabled when preparing a ligand for covalent docking. The control panel is ifurther subdivided in to 6 sections for: ) specifyingii the receptor optionally a ligand; ) placing the docking box onto the receptoriii using various techniques; ) optionally selecting receptor side-chainsiv to be made flexible during docking; ) optionally specifying receptor ivatoms for covalently attaching a ligand; ) identifying binding pockets using AutoSite and vi selecting one or more to specify the regions where AutoDockFR should try to dock the ligand; and finally ) a section for computing and saving the affinity maps. Use case 1: Pocket defined by a known ligand in a rigid receptor

MGLTools2-1.1/bin/prepare_receptor MGLTools2- 1.1In this/bin/prepare_ligand scenario we have a receptor and known ligand, which we have been prepared for docking with AutoDockFR ( and ). In this example, we will use the cyclic dependent kinase protein 2 CDK2 (pdb:4EK3) and one of its ligands (pdb:4EK4). The files 4EK3_rec.pdbqt and 4EK4_lig.pdbqt are available in the data associate with this tutorial. Step 1: Click on the button to load 4EK3_rec.pdbqt

The receptor molecule is loaded and displayed as line representing atomic bonds colored by atom type with carbon atoms shown in the color cyan. The default docking-box covers the entire receptor with the default padding (4.0 Angstroms) added to each side.

NOTE: amino acids located ini.e. the docking box with no flexible side- chains ( glycine, alanine and proline) are displayed dimmed down.

NOTE: several buttons in the control section and the tool-bar are now enabled.

The 3D scene can be rotated, translated and scaled using the 3 mouse buttons:

Mouse button Action Depth-cueing can be turned on and off by pressing the Left Rotate keyboard key ‘d’ while the mouse pointer is in the 3D Middle Translate view. Right Scale Step 2: Click on the button to load 4EK4_lig.pdbqt.

The ligand molecule appears in the licorice representation with carbon atoms displayed in yellow.

NOTE: more buttons are enabled by this operation both in the control panel and in the tool-bar.

Step 3: Click on the button to make docking box cover the ligand

The docking box is now centered on the ligand and scaled to have a 4.0 angstroms padding from the ligand atoms. The padding can be adjusted using the spin box in the “docking box” section. NOTE: that amino acids outside the docking box are dimmed down. Also, amino acids within the docking box with no flexible side- chains (i.e. glycine, alanine and proline) are displayed dimmed down. This facilitates the visual identification of the amino acids in the docking box that can potentially be made flexible during the docking calculation.

Click on to focus the 3D scene on the box. Click the button to display the “box parameters” user interface. This interface provides widgets to modify the box center coordinates, and box size along the 3 dimensions. NOTE: the mouse scroll wheel can be used to alter values while the mouse pointer is over the widget displaying numerical values. The spacing widget sets the distance between the grid points at which affinity values are calculated. The smoothing widget allows the specification of the smoothing factor used for calculating affinity values. cmd These values are initialized with AutoGrid’s default values. The typein widget allows the command-line specification of the docking box. For instance: center 10 11 12.3 will center the docking-box on (10.0, 11.0, 12.3). Multiple commands can be entered on a single line separated by a semi colon character ‘;’ (e.g. “center 12.34 34.12 56.56; size 26 26 26 . Step 4: Click on the “compute pockets” button

Ligand binding pockets are computed using AutoSite. The pocket with the largest AutoSite score is selected by defaults, and displayed as a set of green dots. Binding pockets are defined as subsets of high affinity grid points calculated on a 1 Angstrom resolution grid. These points are clustered. Each cluster is called a fill and is assigned a rank. Usually, high ranking clusters are likely to be ligand binding pockets. The fill points (green dots) will be used by ADFR to seed the initial population of solutions of the genetic algorithm during docking. NOTE: the button labeled “generate target file…” is now enabled. This is because we have ai.e. valid docking box and a valid pocket definition ( a set of fill points that overlap with the docking box). Step 5: Check the “for all atom types” button

Loading the ligand initialized the list of maps to be computed to the list of AutoDock atom types found in the ligand, in this case: “A C Cl HD N NA OA”. Computing maps for all atoms types will use a little more disk space but the resulting target (.trg) file can be used for any ligand and is highly recommended for virtual screening of libraries of ligands. Alternatively, the “edit …” button will display an interface for manually specifying the list of atoms types for which affinity maps should be generated. Step 6: Click on the “generate target file…” button to calculate affinity maps and save them as a target file

A dialog will allow you to specify the location and name of the target file to be created. The target file will contain all the requested affinity maps along with the fill points. As such this file provides a complete description of the receptor for docking with AtuoDockFR. In addition, the target file contains a PDBQT file of the receptor, the grid parameter file used to run AutoGrid and the AutoGrid log file, and meta data such as the time and computer NativeCDK2BindingSite_rigidarchitecture on which the maps were computed, the docking-box parameters, the grid parameter file, etc. It is recommended to use names that are descriptive. In this case we use as we defined the docking box using the native ligand and we did not specify any flexible receptor side chains. Pressing the “OK” button will start the calculation in a separate thread, leaving the graphical user interface active. The progress bar below the button will indicate the level of completion of the calculation. about MGLTools2-1.1/bin/about NOTE: after the calculation completed and you quite the GUI it is possible to inspect the content of a target file using the command ( ) Use case 2: Pocket defined by some receptor amino acids in a rigid receptors

In this scenario we have a receptor with a known active site but no ligand in the active site. We will illustrate this use case using the Cyclin-dependent kinase protein 2 (CDK2, pdb:4EK3). The prepared receptor file (4EK3_rec.pdbqt) is available in the data associate with this tutorial. Step 1: Click on the button to load 4EK3_rec.pdbqt

See Step 1 of Use case 1 for details Step 2: Click on the button to select receptor amino acids: ILE10, PHE80, PHE82 and LYS89

An interface is displayed for selecting receptor amino acids. The amino acids are organized by chain in a tree widget.

NOTE: the docking box scales to encompass the selected amino acids as they get selected.

Click on to focus the 3D scene on the box.

The top portion of the residue selection widget interface allows selecting amino acids located within a distance cutoff of a ligand molecule. This interface is currently disabled as no ligand has been loaded. Close the side chain selection widget by destroying the window, using the button in the corner of the window. Step 3: Click on the “compute pockets” button

For this docking box AutoSite identifiedsmallClusterCutOff’ 4 binding pockets comprised of more than ‘ points and created a fill containing all other smallClusterCutOffhigh affinity points, = which 10. did not cluster. By default

The binding pockets 1-4 are ranked by decreasing AutoSite score with higher scores reflecting a higher predicted probability for the pocket to be a ligand-binding pocket. Every binding pocket can be selected by checking the button in the ”fills” column. The fill points of all selected (i.e. displayed) binding pockets will be used by ADFR as potential initial potions of the ligand during the search. In this example pick fill #1. All selected (and displayed) pockets will be written as fill points into the target file. Step 4: Click on the “generate target file …” button to calculate affinity maps

Affinity maps will be generated for this docking box, and saved under the name you specify along with the receptor and binding-pocket fill points in a target file directly usable as input for AutoDockFR. See Step 5 of Use case 1 for details Use case 3: Pockets identified by AutoSite in a rigid receptor

In this scenario we have a receptor but no ligand binding site information. We will illustrate this use case using Cyclin-dependent kinase protein 2 (CDK2, pdb:4EK3). The files prepared receptor (4EK3_rec.pdbqt) is available in the data associate with this tutorial. Step 1: Click on the button to load 4EK3_rec.pdbqt

See Step 1 of Use case 1 for details Step 2: Click on the “compute pockets” button

AutoSite runs for a docking box covering thesmallClusterCutOff entire receptor and identifies 53 binding pockets comprised of more than ‘ ’ points. The first and highest scoring fill is selected (and shown) by default.

Step 3: Click on the button to define the docking box using the top ranking fill

The box is sized and moved to cover the fill using the current padding value (4.0 in this case). Step 4: Click on the “generate target file ...” button to calculate affinity maps

See Step 5 of Use case 1 for details. Save the target file as 4EK3_mainSite.trg. Step 5: Uncheck pocket 1, check pocket 5, and click on the button to define the docking box

For the sake of illustration we assume that visual inspection of the other fills reveals that fill #5 provides an interesting alternate binding pocket (different pocket than from fill #1) for which we would like to perform a docking experiment. Hence we position the box on the fill and compute and save affinity grids for this binding pocket as well. Step 6: Click on the “generate target file…” button to calculate affinity maps

See Step 5 of Use case 1 for details. Save the targetMGLTools2 file as 4EK3_alternateSite.-1.1/bin/agfr

NOTE: the command line version of the program ( ) provides option to automated the creation of target files for each Use case 4: Receptor with flexible side-chains

In this scenario we have a receptor and known ligand, which we have prepared for docking with AutoDockFR. In this example we will use the cyclic dependent kinase protein 2 CDK2 (PDB ID 4EK3) and one of its ligands (PDB ID 1YKR). The files 4EK3_rec.pdbqt and 1YKR_lig.pdbqt are available in the data associate with this tutorial. Step 1: Click on the button to load 4EK3_rec.pdbqt

Step 2: Click on the button to load 1YKR_lig.pdbqt

Step 3: Click on the button to make docking box cover the ligand and

to focus the view on the docking box

This particular ligand overlaps with side chains of the receptor’s apo conformation. Label thep receptor residues in the docking box ( button in the tool bar) and zoom in and rotate to observe the overlap of lysine 33 and lysine 89 with the ligand. Step 4: Click on the button to set LYS33 and LYS89 as residues to be made flexible during docking

The residue selection window displays all receptor amino acidsi.e. currently in the docking box and having a flexible side chain ( not alanine, glycine or proline). Flexible residue side chains are displayed as orange Sticks & Balls. Receptor side chains to be made flexible can also be selected based on their distance from a ligand by checking the “residue within” button (NOTE: this section of the graphical user interface is only enabled when a ligand has been loaded). Checking the “residues within” button will override the current selection with the set of receptor side chains having at least one moving side chain atom within the current distance cutoff (i.e. 3.0 Angstroms in this case) the ligand. Close the interface by destroying the window, using the button in the corner of the window. Note that the receptor amino acids selected to have flexible side-chains during the docking calculation are now listed in the type-in widget. along withe.g. the number such residues. Flexible residuese.g. can be specified manually by typing their identified directly in this widget. A residue can be specified by its residue number ( “89”), or by its residue type and number ( “LYS89”). Multiple residues must be separated by a comma ‘,’ or space characters, optionally preceded by a chain number (i.e. “A:120,LYS89”). If the e.g.chain id is omitted matching residues from all chains are selected. Residues from different chains can be specified by separating the selection string for each chain by a semi colon ‘;’ “A:10,24,35;B:12,34” Step 5: Append “,88” in type in widget for flexible residues en press

One more residue (lysine 88) is added to the list of flexible residues and the selection string in expanded. NOTE: after executing this command the generate target file button is disabled. The ‘FR’ status button at the lower right corner of the GUI is red, indicating that the flexible residues definition is not valid, because the box does not cover all of them. Step 7: Click on the button to automatically adjust the docking to cover all flexible receptor atoms

The docking box has been extended to fully cover flexible residues 33, 88 and 89 and the button for flexible side chains validity ‘FR’ is now green in the status bar.

Step 8: Click on the “compute pockets” button to calculate binding pockets

AutoSite identifies 7 pockets in the box. The first one is selected by default. Clicking the fill check-button of other pocket will display them and shows that they correspond the small alternative pockets at the edge of the box and therefore not of interest. The binding pocket fill-points validity button ‘Pocket’ is now green in the status bar and all requirements are fulfilled, hence. the “generate target file …” button is now enabled. Step 9: Click on the “generate target file ...” button to calculate affinity maps

See Step 5 of Use case 1 for details. Save the target file as 4EK3_FR33_88_89. Use case 5: Receptor with covalent attachment for ligand

In this scenario we have a receptor with a covalently attached ligand and we will prepare the receptor for docking covalent ligands with AutoDockFR. In this example we will use the 3c9w.pdb. While the file 3c9w.pdbqt is available in the data associate with this tutorial we willCreate also the describe pdbqt how file forthe thepdbqt receptor file was generated.

1 - download 3c9w.pdb from http://www.rcsb.org 2 - extract chain A including the covalent ligand HMY but excluding water molecules using your favorite molecule editor (e.g. pymol)n and save it as 3c9w_A.pdb 3 – addMGLTools2 hydrogen-1.1/MGLToolsPckgs/binaries/reduce atoms using reduce 3c9w_A.pdb > 3c9w_A_H.pdb

4 – generateMGLTools2 the- receptor1.1/bin/prepare_receptor PDBQT file -r 3c9w_A_H.pdb

5 - startupMGLTools2 agfrgui:-1.1/bin/agfrgui

Step 1: Click on the button to load 3c9w_A_H.pdbqt

Step 2: Click on the button to select HMY1 to place the box on the native covalent ligand, increasing padding to 6.0, display residue labels ( ), and click on to center the scene on the docking box

Close the side-chain selection widget by destroying the window. Step 3: Enable covalent docking by checking the check-button.

The covalent ligand specification dialog is displayed. The top row of this dialog displays the 2 atoms that will form the covalent bond between the receptor and the ligand. Initially these fields are empty. The fields can be populated by left clicking on atoms in the viewer. NOTE: when the form is displayed initially, the left mouse click is automatically assigned the function to pick the first atom of the covalent bond. NOTE: atom1 should be on the receptor side and atom2 on the ligand side.

Step 4: Pick the first atom of the covalent bond, A:CYS164:CB

As we left click on the atom in the 3d Viewer, a small sphere is displayed on the picked atom, the picked atom name is displayed in the the atom1 field and the mouse function is set to picking the second atom.

NOTE: left clicking on the arrow button to the right of the atom1/2 fields set the left mouse picking function to pick this atom. Step 5: Pick the second atom of the covalent bond, A:CYS164:SG

As we click on the atom a small sphere is displayed on the picked atom, the picked atom name is displayed in the atom2 field, and the sub-graph rooted at the covalent bond is shown with dashed lines, indicating that these atoms will be removed from the receptor when calculating maps. The list of removed atoms appears in the dialog. The left mouse picking function is now to add/remove atoms to the list of atoms shown with dashed lines. Picking on an atom will add or remove the sub- tree of atoms connected to the picked atom, according to its current state.

NOTE: In some cases the covalent ligand has a second covalent bond with the receptor (due to close contacts) which leads the sub-graph identified as the covalent ligand to include large parts of the receptor. The “limit cov. Lig.” filed in the dialog allows to address this issue. This field can be used to limit the traversal of the molecule graph to certain residues. For instance, typing CYS164,HMY1 in this field will limit the traversal to the ligand and the cys164 residue and prevent the erroneous inclusion of other receptor amino acids.

NOTE: The “Start over” button will clear the dialogue and let you start over

NOTE: If the second atom picket is not covalently bound to the first picked atom, a dialog is displayed asking whether or not to proceed. The dialog show the atoms identity as well as the distance between these atoms helping the user decide whether they picked the right atom or not and whether to proceed or not. i.e. Click OK on the dialog once the proper covalent bond was defined and set of atoms to removed ( the covalent ligand) is correct.

NOTE: the covalent validity light is now green and the pocket validity light is disabled as no fill points are needed for covalent docking. Step 6: Click on the “generate target file ...” button to calculate affinity maps APPENDIX

Toolbar buttons

Focus the view on the current docking box

Show/Hide the receptor line representation. This button cycles through 3 mode: 1) display entire receptor; 2) display receptor atoms inside the current docking box; 3) hide the receptor line representation. Show/Hide the receptor surface representation. This button cycles through 3 modes: 1) display the surface for the entire receptor; 2) display the surface for atoms inside the docking box; 3) hide the surface representation. Show/Hide residue labels. This button cycles through 3 modes: 1) label residues of the entire receptor; 2) label residues that have at least one side chain atom within the box; 3) hide the residue labels. Show/Hide the ligand.

Show/Hide the ligand anchor atom. The anchor atom is the ligand atom that the AutoDockFR will be translate to binding pocket fill points to generate an initial pool of solutions to be optimized by its Genetic Algorithm. Show/Hide the docking box.

Show/Hide atoms forming covalent bond. Parameter panel buttons

Define docking box to enclose the receptor

Define docking box to enclose the ligand

Define docking box to enclose the binding pocket fill points

Define docking box to enclose user selected receptor amino acids

Manually define the docking box and set grid point spacing and smooth factor

Validity status lights (disabled, invalid, valid) i.e.

The box status light is red if the box does not overlap with the receptor, no receptor atom is found to be inside the box. This light is never disabled and the condition applied to any docking calculation.

The “Pocket” validity status light is enabled unless a receptor is prepared for covalent docking, in which case the light becomes disabled. The light will be red if none of the currently selected/displayed fill points is located within the box.

The validity status light for Flexible Residues “FR” is only enabled when receptor amino acid side chain have been selected to be flexible during docking. The light will be red if at least one atom in these side chain is outside the docking-box.

The covalent docking validity status light is only enabled if the covalent docking check button is checked. It will turn green after 2 atoms forming the covalent bond in the receptor have been selected.