AGFR v0.1 – Prepare AutoDockFR affinity maps

Overview                                                                                                                                                                                                              3

Use case 1: Docking ligands into a receptor with known ligand                                                    5

Use case 2: Docking ligands to an apo receptor in the proximity of particular receptor amino acids                                                                                                                                                                                                                           9

Use case 3: Blind docking into a receptor                                                                                                                     12

Use case 4: docking with flexible receptor side-chains (AutoDockFR only)                     14                            

APPENDIX                                                                                                                                                                                                                18

Toolbar buttons                                                                                                                                                                                        19

Parameter panel buttons                                                                                                                                                                20

 

Overview

AutoGridFR (AGFR) is a graphical user interface for configuring and computing affinity maps for a receptor molecule to be used for AutoDock4 or AutoDockFR. The AGFR can operate on molecules already prepared for AutoDock or AutoDockFR (i.e. PDBQT files). A zip file generated by AGFR in a previous session can also be opened to load the receptor and the grid box parameters.

 

The graphical user interface is divided in 3 sections: 1) a panel of widgets for operating the software; 2) a tool bar for the 3D Viewer; and 3) the 3D viewer providing visual feedback. The control panel is further subdivided in to 4 sections for a) specifying the receptor and optionally a ligand; b) placing the docking box onto the receptor using various techniques; c) optionally selecting receptor side-chains to be made flexible during docking; d) identifying binding pockets using AutoSite and selecting one or more to specify the regions where AutoDockFR should try to dock the ligand; and finally e) a section for computing and saving the affinity maps.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Use case 1: Docking ligands into a receptor with known ligand

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 Cyclin-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 associated with this tutorial.

Step 1: Click on the  button to load 4EK3_rec.pdbqt

We loaded the receptor molecule, which is displayed as line representing atomic bonds colored by atom type with carbon atoms shown in the color cyan. Note that amino acids in the docking box with no flexible side-chains (i.e. glycine, alanine and proline) are displayed dimmed down. Note that some buttons in the “controls” sections of the interface are now enabled. When a receptor is loaded, the docking box is set to covers the entire receptor. The box size was calculated to be the receptor bounding-box (i.e. the tightest, axis-aligned, box containing all receptor atoms) with some padding (4.0 by default) added in every side of the box. Also note that several buttons in the control section and in the toolbar are now enabled.

 

Depth-cueing can be turned on and off by pressing the keyboard key ‘d’ while the mouse pointer is in the 3D view. The scene can be rotated, scaled, and translated using a mouse.

 

 

Text Box: Mouse button	Action
Left	Rotate
Middle	Translate
Right	Scale

 

 

 

 

 

 

Step 2: Click on the  button to load 4EK4_lig.pdbqt.

 

We loaded the ligand molecule, which appears as licorice representation with carbon atoms displayed in yellow. More buttons are enabled by this operation both in the control panel and in the toolbar.

 

 

 

 

 

 

 

 

 

 

 

 

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

Description: Macintosh HD:Users:sanner:Desktop:Screen Shot 2016-02-12 at 2.19.32 PM.pngThe docking box is now centered on the ligand and scaled to have a 4.0 angstroms padding around the ligand atoms. 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. The padding can be adjusted using the “padding:” spin box in the “docking box” section.

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

NOTE: the  button displays the “box parameters” user interface, which provides widgets to modify manually the box center coordinates and box size in each of the 3 dimensions. 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 a smoothing factor used for calculating affinity maps. These values are initialized with AutoGrid default values. The “cmd:” widget allows the specification of the docking box by typing commands. For instance: typing “center 10 11 12.3<return>” will translate the grid to be centered on (10.0x, 11.0y, 12.3z). 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”). The arrow up and down keys scroll through the history of typed commands.

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 default and displayed as a set of green points. Binding pockets are defined as subsets of high affinity grid points calculated on a 1 Angstrom resolution grid. These points are clustered and each cluster is called a fill and assigned a rank. Usually high ranking fills are most likely to be ligand binding-pockets. The grid points shown in green are used by ADFR to seed the initial population of the genetic algorithm. This facilitates the docking procedure by placing the ligand into a reasonable initial position.

Note that the “generate maps …” button is now enabled. This is because we have a valid docking box and a valid set of fill-points defining the binding pocket (i.e. at least 1 pocket binding fill points is inside the docking box).

 

 

 

 

 

Step 5: Check the “for all atom types” button

When the ligand was loaded the list of AutoDock atom types found in it was used to initialize the list of atom types for which affinity maps would be computed, 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 zip file can be used for any ligand and is recommended for virtual screening of libraries of ligands. Alternatively, the “edit …” button will display an interface for manually selecting the atoms types for which affinity maps should be generated.

 

 

Step 6: Click on the “generate maps …” button to calculate affinity maps

A dialog comes up asking for a name for saving this set of maps. The name will be used to create a directory containing all the files (receptor, maps, fill points) and generate a corresponding Zip file. It is advisable to use a name that is descriptive. In this case we could use NativeCDK2BindingSite_rigid as we defined the docking box using the native ligand and we did not specify any flexible receptor side chains. The Zip file can be used by ADFR directly, or unzipped to extract the maps to be used with AutoDock.

Pressing the “OK” button will start the calculation in a separate thread, leaving the graphical user interface active. A progress bar displayed below the “generate maps …” button will indicate the level of completion of the calculation.

 

 

 

 

 

 

Use case 2: Docking ligands to an apo receptor in the proximity of particular receptor amino acids

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 associated 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 that the docking box grows to encompass the selected amino acids as they are selected.

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

 

The top portion of the residue selection widget interface supports selecting amino acids located within a distance cutoff of the 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 top left corner of the window.

Step 3: Click on the “compute pockets” button

For this docking box AutoSite identified 4 binding pockets with more than 50 points and created a fill containing all other high affinity points, which did not cluster. 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. Each binding pocket can be selected by checking its button in the ”fills” column.  The fill points of all selected (i.e. displayed) binding pockets will be used by ADFR as potential initial positions of the ligand during the search. In this example pick fill #1.

 

 

 

 

 

 

 

 

 

 

 

Step 4: Click on the “generate maps …” 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. These files will be saved in a zip file, directly usable as input for AutoDockFR. See Step 5 of Use case 1 for details

 

 

 

 

 

Use case 3: Blind docking into a 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 prepared receptor file (4EK3_rec.pdbqt) is available in the data associated 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 the entire receptor and identifies 19 binding pockets comprised of more than 50 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 maps …” button to calculate affinity maps

See Step 5 of Use case 1 for details. Save the maps as 4EK3_mainSite.zip.

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 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 maps…” button to calculate affinity maps

See Step 5 of Use case 1 for details. Save the maps as 4EK3_alternateSite.zip.

 

 

 

 

Use case 4: docking with flexible receptor side-chains (AutoDockFR only)

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 Cyclin-dependent kinase protein 2 (CDK2) (pdb: 4EK3) and one of its ligands (pdb: 1YKR). The files 4EK3_rec.pdbqt and 1YKR_lig.pdbqt are available in the data associated 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 click on  to focus the view on the docking box

This particular ligand overlaps with side chains of the receptor apo conformation. Label the receptor residues in the docking box () 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 modeled with flexible side chains during docking

The residue selection window displays all receptor amino acids currently in the docking box and having a flexible side chain (i.e. not alanine, glycine or proline). Selected 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 top 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 with the number such residues. Flexible residues can be specified manually by typing their identifiers into this widget. A residue can be specified by its residue number (e.g. “89”), or by its residue type and number (e.g. “LYS89”). Multiple residues must be separated by a comma ‘,’ or space characters, optionally preceded by a chain id (i.e. “A:120,LYS89”). If the chain id is omitted residues for all chains matching the selection string are selected. Residues from different chains can be specified by separating the selection string for each chain by a semi colon ‘;’ e.g. “A:10,24,35;B:12,34”

Step 5: Type “,88<return>” in type in widget for flexible residues

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 maps button is disabled and the buttons to its left appear as 2 red lights. These buttons indicate from left to right the validity of the set of binding pocket fill-points (i.e. at least one fill point is within the docking box) and validity of the docking box (i.e. the docking box covers all moving atoms from the receptor’s flexible side-chain).

 

 

 

 

 

 

 

Step 7: Click on the  button to adjust the docking box (i.e. to cover all flexible receptor atoms)

 

The docking box has been extended to the right to fully cover lysine 33 and lysine 89 and the button for flexible side chains validity is now green.

 

 

 

 

 

 

 

 

 

 

 

 

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

AutoSite identified a single pocket in the box and selected it. The binding pocket fill-points validity button is now green and the “generate maps …” button is now enabled.

Step 9: Click on the “generate maps …” button to calculate affinity maps

See Step 5 of Use case 1 for details. Save the maps as 4EK3_alternateSite.zip.

 

 

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.

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

Select receptor side chains to be made flexible during docking

Adjust docking box to encompass flexible receptor side chains