52.1.1. HYDRATION

Go from your working directory in the Tutorial05 directory:

cd greater05

To begin with we have supplied a Protein Data Bank file called PDB.pdb on the Tutorial05 directory.

Begin by typing :

Greater

and hitting the RETURN key. You will see the normal Greater main window appear. To hydrate a protein, the first thing to do is to import the PDB.pdb structure like in Tutorial 2 selecting Automatic in filtering mode. Then in order to start with water-hydration, select:

Targets->Add ions or water

select 10 water molecules to add, OH2 in ions/water option, default as setting interpolate options and -8 Kcal/mol for energy cutoff, then press OK to start. After a minute (in UNIX) and few seconds (in Linux) the process ends.

The PDB.pdb structure has been now modified, and ten water molecules has been automatically added at the bottom of the structure. Use Gview to locate the water molecules around the structure. To do this: click on the status bar with the PDB structure with the right mouse button and select "View structure". The added water molecules are reported as red crosses. Now, import the experimental water molecules, that are in the file PDB_out.pdb using:

File->Open select the PDB_out.pdb from the dialogue and press OK.

The water molecules will be displayed on the 3Dplot with yellow crosses. To change the rendering of atoms and bonds, select:

Edit->Select click on the "PDB_out" from the dialogue and then click on edit style button. Change the Rendering style to "balls & Sticks" and color the atom to "cyan". Then press Exit

Check the reliability of the predicted positions for water molecules.

52.1.2. NEUTRALISATION

Macromolecular targets are normally uncharged at physiological pH, being their net charges counterbalanced by the counterions in the biological solution. Since globally uncharged system are preferable for any force-field analysis, GRID provided subroutines for automatic target neutralisation. From this release, the work is totally automatic. To perform the neutralisation task, the User must only select the counterions to be added to the macromolecular Target. Grid automatically computes the appropriate map for the selected counterion, finds the energy minima, uses a simulating annealing procedure to postprocess the minima in order to select the minima location subset that gives the most favourable overall interaction energy, and fills the target with the counterions in those minima. The counterions are also automatically added to the PDB file, so that the User may inspect their position using Gview. The software works in a different manner respect to previous situation (hydration). In this case the presence of a counterion at one site would tend to repel a second counterion from any nearby sites, and so Grid would tend to distribute the counterions well away from each other. This counterion behaviour would therefore be different to the behaviour with water, which tends to form hydrogen-bonding water clusters as the map is populated.

First, delete the PDB molecule, then start again with the original PDB file. When imported (be sure the filtering mode is in Automatic), press

Targets->Neutralise

select Cl- molecules to add, default as the setting, interpolate options and -8 Kcal/mol for energy cutoff, then press OK to start. After a minute (in UNIX) and few seconds (in Linux) the process ends. The PDB.pdb structure has been now modified, and eight counterions has been automatically added at the bottom of the structure. Use Gview to locate the counterions molecules around the structure. To do this: click on the status bar with the PDB structure with the right mouse button and select view structure. The added counterions are reported as grey crosses.

You have now completed your fifth Tutorial. Well done!! We look forwards to hearing from you if we can help in any way.

Please, continue with Tutorial06.