49.1.1. ASSESSING THE RESULTS FROM PROGRAMME GRIN

Please note that the .kout file does not contain the water molecules originally reported in the pdb.

The goal of our tutorial is to predict the favourable hydrophobic interactions around the protein. Accordingly, the hydrophobic probe called DRY will be selected.

To do that select:

Probes->Choose probes from the menu (or press the Probes button) and the following window will appear.

Select the DRY probe clicking on its name so the "selected" column reads "IN" and press OK; note that "DRY" now is listed in the "probes" box in the upper right corner on the Greater window.

You are now studying a complete protein, and your GRID map will be not restricted to a part of the protein structure like in the previous tutorial. However, the computation over the whole protein will not take more than few minutes on a modern UNIX processor (few seconds in Linux operating system). Therefore it will not be necessary to define the grid cage that will be automatically selected by GRID.

Now you can define some more GRID options, modifying the keywords located in the bottom right corner of the main Greater window.

Choose, in the "box" section, NPLA. Select "2" as input value modifying the default value; press OK to confirm.

Now select Compute->Run (or press the Run button) and press, in the following window, the OK button to start the GRID run.

49.1.2. ASSESSING THE RESULTS FROM PROGRAMME GRID

The status bar of the Greater main window will report "running" and after some time will turn to "Completed". Once the GRID computation will be completed and the map is generated the Targets->View fields command becomes active along with a new tab in the Targets->View text files window containing the GRIDLONT output. Select:

Targets->View text files option and click on GRIDLONT button.

If you go at the end of the GRIDLONT output you can read the most negative interaction energy for the probe that is about -3.0 Kcal/mol. This shows that the DRY probe would be strongly attracted to this place on the protein. Such a big value for hydrophobic scale is typical for protein, while it is normally smaller for ligand molecules.

Now we are ready to visualise the GRID map graphically;select the Targets->View fields command (or press the View button) and the GVIEW programme will start. More information about the Gview programme is found in the Gview page.

The Molecular Interaction Field produced by the DRY probe will be reported on the screen together with the protein structure. This contour level has been automatically selected by the Gview program. To change the energy levels, select:

Edit->Field style

Move the cursor of the interaction energy levels up to -0.5 and then press Exit on the dialogue box. Move the image pressing the left button of the mouse and moving the mouse at the same time.

Now import the water molecules in the original structure PDB_out.pdb by using:

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

Graphic visualisation shows there are 143 water molecules around the PDB structure, and, as expected, the hydrophobic contours of the DRY probe almost always define regions which are NOT occupied by those waters.

Please make active the mouse toggle mode and then click on the atom of the structure. Gview will report the position of the row atom in the original PDB file, the atom type and the residual name. Click the right mouse button to clean the label from the graphic screen.

Only one water (O1135 close to Tyr63) is within the DRY region. Four reasons can be proposed to explain this result: the DRY probe can be incorrect at that position; the experimental results could be incorrect for that water molecule; the water molecule observed by the crystallographer could be something else, like NH4+ ion that has exactly the same number of electrons to diffract X-rays like a water molecule; finally another possibility is that the water is really at that place, but because the position is favoured by a polar side-chain from the next protein molecule in the crystal. Of course without the mentioned structure GRID cannot take into account this effect.

The most negative energy occurs at grid point number 46,47,53. These are the indices defining the grid point, and not the actual xyz coordinates which are 14.500, 10.000 and 33.000 Angstrom. You can find this information inspecting the GRIDLONT file as in the previous tutorial.

The strong hydrophobic interaction in this region is mainly due to the interaction of the hydrophobic probe with three residues, such as Trp64, Trp109 and Val99.

It is interesting to note that very close to this highly hydrophobic region there are four water molecules strongly linked to each other by hydrogen bonds. These water molecules are the O1073, O1074, O1075 and O1173 located close to the triptophan residue 109. To find more information about this region, edit the file GRIDLONT and look for Z=51.

The figure below reports the GRID map produced around these supposed water molecules, using a ON probe in grid.in command file. This probe refers to the oxygen's atom of a nitro group. When the energy is contoured at -5.0 kcal, then two of the three oxygens are inside the grid contour. Try to reproduce yourself the view of the figure below.

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

Please, continue with Tutorial03.