Aims

• To reinforce the use of a probe sphere in defining the molecular surface.
• To give practice in using macromolecular docking software.

Objectives

After this practical you will:

• have used a program that demonstrates the principles of molecular surface calculations;
• have used the Hex macromolecular docking program;
• will able to initiate a docking calculation using Hex, and view the predicted docked conformations.

Introduction

In this tutorial you will use the Hex macromolecular docking program. If you need help using this program during the tutorial, please ask. If you need help when working on your own, a useful starting point is the Hex User Manual.

To run Hex, type:

    hex

Exercises

1. Experiment with Michael Connolly's 2D demo (Java) of molecular surface calculations.

2. Use Hex to perform a local docking search with the antibody HyHel-5 and its antigen, lysozyme.

   Start the Hex program by typing 'hex' at the Unix prompt. Ensure that the main "Hex 4.2" window and the "Hex Messages" windows are both visible.

   Use the File menu to Open the following three data files:

   • the "Receptor": the variable fragment (Fv) of the antibody HyHel-5 (data file 3hfl_fv.pdb);
   • the "Ligand": lysozyme (data file 3hfl_ly.pdb);
   • the "Complex": the known complex the antigen binding fragment (Fab) of HyHel-5 and lysozyme as the "Complex" (data file 3hfl.pdb).

   When initially loaded, the Receptor and Ligand superpose exactly on the Complex. Atom colouring is used for the Receptor and Ligand, while the Complex is shown in grey. Only the two constant domains of the Complex (CL and CH1) and some water molecules can be seen in grey.

   To rotate the molecule, place the cursor in the graphics window, press the right mouse button, and move the mouse. To translate the molecule, place the cursor in the graphics window, press the left mouse button, and move the mouse. To zoom in, use the vertical slider to the left of the main graphics window. To identify atoms, click on the "ID" icon to the left of the main graphics window then click on an atom. The atom will be identified in the Hex message window, and by a label in the main graphics window. Clicking on an atom a second time will remove the label from the graphics window. Click on the arrow icon to the left of the main graphics window to return to the default mouse mode.

   Now, pretending that we do not know the true native conformation of this complex (in grey), we shall use Hex to perform a constrained docking search to dock the Ligand to the Receptor. Before doing this, I recommend that you look through the start of section 5 and section 5.5 of the Hex User Manual. From the "Controls" menu, select "Docking". Set the docking parameters as follows:

   Search Mode	Full Rotation
   Correlation Type	Shape Only
   Radial Filter	None
   Post Processing	None
   Receptor Range	30	Samples	642
   Ligand Range	30	Ligand Samples	642
   Twist Range	360	Twist Samples	128
   Distance Range	40	Step Size	1.0
   Grid Dimension	0.6
   Steric Scan	16
   Final Search	25

   Then "Activate" the docking search. As docking proceeds, look at the output in the Hex messages window (ignore any error messages; these are about non-essential cache files and do not affect the docking result).

   When docking is complete, step through the top predictions interactively, and compare these visually with the native docked conformation (in grey). The first solution found by Hex is close to the native conformation (0.62Å RMS). With the cursor in the main graphics window, use the "Page Up" and "Page Down" keys to step through the list of solutions found by Hex. Alternatively, you can use the "Soln" slider in the Docking Control window to jump to a particular solution (solution 0 is the native conformation).

3. Repeat the docking calculation from Question 2, but instead of loading the data files using the File menu, use the "Macros" menu, select "Run" and then select macro file "3hfl.mac". This will load the Receptor, Ligand and Complex as before, but the Receptor will be coloured to highlight the complementarity determining regions, and the Ligand will be shown in orange with some of the intreface residues highlighted.