48.2.1. ASSESSING THE RESULTS FROM PROGRAMME GRIN

Looking at the GRINLOUT text file in the last opened window you can read details about the quality of the conversion. In this case the target has a positive charge, because the pdb file does not contain any counter-ions. (Programme GRIN tells you the charge of the Target, in case you want to add counter-ions or to protonate some aminoacids before doing your "Molecular Interactione Field" GRID runs). Then the GRINLOUT file ends with the words:

*** THANK YOU FOR USING PROGRAM GRIN ***

This shows that everything has worked correctly, because the message would have been diverse if you had made a mistake, or if there had been significant errors in PDB.pdb.Please note that some new files were created by Greater in the Greater01 directory. They are the PDB_in.kout and PDB_in.lout. The .kout contains all the parameters required by GRID force field. Please note that the .kout file does not contain the water molecules originally reported in the pdb. All these molecule were filtered out by the semi-automatic filtering procedure.

If the User wants to consider these water molecules like part of the protein as actual water in the computation, no filtering should have been selected and the directive NETA must be activated. (Please refer to the GRID manual for better explanations).

However, the goal of our tutorial is to predict the favourable location(s) of water molecules around the protein. Accordingly, it was correct to eliminate the water molecules.

To inspect the protein, select:

Targets->View structure

Please note that the protein appears without water molecules. Some new files are created by Greater. They are named after the original input file (PDB in this case) with _in.pdb and _out.pdb appended to it. That is because Greater splits the ATOM records of the PDB.pdb file (they are now in PDB_in.pdb) from the HETATMS ones (the water molecules in our case that are now in PDB_out.pdb). The GRID computation will be carried out only on the former one that is PDB_in.pdb.

Click on the status bar with the right mouse button. Inspect the text files just produced by pressing in "view text files". If you now have a look at the PDB and GRINKOUT tabs you may notice that:

• The sequence of some lines in PDB has been shuffled, so that the lines in GRINKOUT are in the sequence defined by the conventions of the Protein Data Bank. For instance the first ATOM record in PDB is a C-alpha atom CA, but protein chains really start with a nitrogen. This anomaly has therefore been automatically corrected in the GRINKOUT file.

• Some numbers in GRUB have been modified before transfer to grinkout.dat For example, the charge on the N-terminal nitrogen (first row and 14th column of GRINKOUT) is much more positive (about 0.7) than the charge specified by GRUB (about -0.1). This is because the GRUB charge is (by default) appropriate for a mid-chain amide-type nitrogen, but the N-terminal nitrogen of a protein has a positive charge since it is a cationic amine-type atom.

Type exit to return on the Greater main window.

48.2.2. RUNNING THE PROGRAMME GRID

By this time you should have completed the previous section so you have generated a PDB_in.kout file of about 1400 lines and more than 120 columns. That file will be the primary input for your first GRID run.

Before running GRID you now need to set some options from the interface: the first thing is to define the type of probe which is to be used by GRID. To do that select:

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

Select the OH2 (water) probe clicking on its name so the "selected" column reads "IN" and press OK; note that "OH2" now is listed in the "probes" box in the upper right corner on the Greater window.

Now you will restrict your GRID map to a relatively small part of the whole protein structure to speed up the computation, making use of the Method->Define box size command. Please uncheck "automatic" and then select the residue GLN117 from the residue's list on the right of the box size window. Click twice on the GLN117 residue. The box dimensions TOPX ... TOPZ will be automatically updated. Press the Interactive button and study the location of the grid cage. Please note that the position of the cage can be modified interactively by clicking the 'ESC' keyboard button and pointing the black arrow mouse on one of the vertex of the cage. Press the left mouse button and move the mouse. The cage will be modified. The roto-traslation operations can be reactivated by pressing again the keyboard 'ESC' button. When moved, the grid cage coordinates will be updated automatically in the box dimensions window. Although one can move the cage in all the xyz positions, we will suggest you to produce a cage with approximately these coordinates: BOTX=24 BOTY=5 BOTZ=17 TOPX=28 TOPY=10 TOPZ=21

These values can also be inserted manually in the input boxes. When finished press OK.

Now you can define some more GRID options, modifying the important keywords.

Choose, in Method, Express setup section.

NPLA represents the number of planes per Angstrom and it determines the resolution of the computation. Select "4" as input value modifying the default value; the computation will be performed at 0.25 Å resolutions.

LEAU determines the way in which the water probe is treated during the computation. Select the default "zero" value.

MOVE controls the flexibility of the Target. In this run we use the rigid structure, so MOVE must be set to "zero".

NETA directive defines the number of extra target atoms to be considered as part of the protein. Our structure contains only aminoacids, and accordingly, NETA will not have any effect on calculations.

ALMD directive controls the printing of some extra information in the output file. Set ALMD to "one".

Press OK to confirm.

Now select Compute->Run (or press the Run button) and choose from the following window:

• visualise fields

• Run interactive

then press the "OK" button to actually start the GRID run.

48.2.3. ASSESSING THE RESULTS FROM PROGRAMME GRID

The status bar of the Greater main window will report "running" and after few seconds 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 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 -17 Kcal/mole. This shows that water molecules would be very strongly attracted to this place on the protein. Such a big value is not typical, because this region of the Target was specially selected for a demonstration run with the programmes.

Now we are ready to visualise the GRID map graphically selecting:

Targets->View fields option (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 water probe will be reported on the screen together with the protein structure.The yellow region refers to interaction energies between water probe and the protein. This contour level has been automatically selected by Gview programme.

To change the energy level, select:

Edit->Field style

Move the cursor of the interaction energy levels up to -5.8, select cyan colour and then press Exit. Move the image pressing the left button of the mouse and moving the mouse at the same time. To import the experimental water molecules together with the protein and the water MIF, use the commands:

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

Please refer to the Gview manual page for more information about Gview settings.

Graphic visualisation and interpretation shows that a water molecule is trapped in a funnel-shape potential. The most probable position for water molecule is close to the narrow part at the bottom, but all the positions into the blue region can be considered potentially water-populated. Thus, the cyan region could also represent a dynamic image of a water-protein interaction in this particular location of the macromolecule.

The most negative energy occurs at grid point coordinates which are close to 25.750, 7.500 and 19.750 Angstrom.

Now have a look at the GRIDLONT file using the command Targets->View text files where you can find a detailed table of results (of course you can still use your page editor to search through the file PDB.lont in the Tutorial01 directory).

This file lists, plane by plane, the atoms of the Target which are nearest to the most favourable position for the Probe. Search the Z plane with coordinates closest to 19.75. The associated Table shows that the water probe at coordinates 25.75 7.5 and 19.75 makes strong interactions with ARG and GLN residues. The water probe is able to donate two Hbonds (one to ARG and one to GLN) and to accept one from ARG. The overall Hbond energy account for more than -13 Kcal/mol. The remaining energy is mainly due to wan der waals interaction and to electrostatic components. Press "exit" to exit from the GRIDLONT file.

The X-ray crystallographer observed a water molecule at x=25.454 y=7.770 z=19.767 but Programme Grid took no account of that particular X-ray observation. Quite independently, the Grid computation found that the grid point at x=25.750 y=7.500 z=19.750 would be the best position when the grid points were spaced at 0.25 Angstrom.

From Gview 3D graphic plot press on:

Edit->Field style

Move the values up to -16 Kcal and then press Exit.

The sphere representing the experimental water molecule will be superposed to the negative field. Please note how the attractive grid map is coincident with the experimental location for the water molecule.

With a closer Grid spacing of 0.1 Angstrom the best point is at x=25.700 y=7.600 z=19.800. Of course more computer time is required to compute the close-packed points, but the distance between the observed and the predicted positions is now only 0.30 Angstrom. This close agreement shows that GRID has made a satisfactory prediction. GRID predicts that there should be a water molecule very near the place where the crystallographers actually found one.

The extended results obtained by using ALMD directive can be inspected graphically. From the Edit menu of programme Gview select "Field style option" and then move the cursor of the energy levels up to -10. To display the contribution to the Molecular Interaction Field produced by individual protein atoms, click on the "Style" tab and select "atom contribution"(atom type color). You can move the image by pressing the left button of the mouse and moving the mouse at the same time.

The negative blue region of water-protein attractive interaction will be coloured in red. This is due to the oxygens O 920 of Gln and O 884 of Arg residues (red coloured) that contribute most to the energetic of the interaction with water molecule in the reported region. The nitrogens 941 of the Arg residue nearby is contributing less than the previous oxygen atoms. To prove that both the oxygens of residues Gln and Arg are contributing to the total energy of interaction in this region, open again the Rendering window and select "atom contribution"(contrast color).

The region of water-protein attractive interaction will be coloured in different colours now, showing that the two oxygens are giving a co-operative interaction with water molecule. However, N 941 is also contributing to the total energy of interaction in this region (see Figure below).

Change the MIF contour using symbol. Activate the toggle mode pressing ESC or selecting

View->Toggle Mode

The distances between atom-pairs, MIF field points and/or MIF points and protein atom can be now reported on the screen. Press SHIFT button and click on any atom of the protein, then press SHIFT and click again in another atom of the protein. Their distances will be reported on the screen. To show the MIF point - ATOM distance press on any of the MIF symbol and on the selected protein atom.

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

Please, continue with the Greater tutorial 02.