55.2.1. ASSESSING THE RESULTS FROM PROGRAMME GRIN

Now use your page editor (e.g. vi, jot, nedit, gedit) in order to study the output files grinlout.lout and grinkout.kout which have been produced by your first run with Programme GRIN. There may be a Message in grinlout.lout telling you the total electrostatic charge of the Target. In this case it 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 before doing your GRID runs). Then grinlout.lout ends with the words:

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

This shows that everything has worked correctly, because the message would have been less polite if you had made a mistake, or if there had been significant errors in PDB.pdb (In fact another pdb file with errors has been supplied on the tape. It is call PDB.dud and you can try using it later in order to see how Programme GRIN deals with input errors. Do not use it now while you are doing the example computations).

Notice in particular that:

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

• That some numbers in GRUB have been modified before transfer to grinkout.kout For example, the charge on the N-terminal nitrogen (first row of grinkout.kout) is much more positive than the charge specified by GRUB. 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.

You have now completed you first GRIN run, and are ready to use Programme GRID. Well done!!

55.2.2. WORKED EXAMPLE WITH PROGRAMME GRID

It was necessary to use Programme GRIN before GRID, and by this time you should have completed the previous worked example. You should have generated a grinkout.kout file of about 1553 lines and more than 120 columns. That file will be the primary input for your first GRID run.

Begin by typing Great as before.

Great (or /your_grid_directory/great)

Then in menu A you should type 2 in order to use Programme GRID.

2 (press Return)

Give the answer 3 in the intermediate menu, in order to call up the filenames and parameter values which are saved in the latest copy of the directive file grid.in

3 (press Return)

Amongst other things it is necessary for the command file grid.in to specify the type of Probe which is to be used for your Grid run. The file is set-up for a water Probe (OH2), and you should now give the reply 'YES' in order to confirm that you do want to use water for your first Grid run.

Yes (press Return)

As soon as you have typed YES the Main Menu J will be displayed.

Make sure you understand items 1 to 6 in Menu J. Then start your first GRID run by typing SUBMIT.

submit (press Return)

The next thing is to give a title to your computation. You could give the answer: "First trial with GRID cage round a binding site for water" and hit RETURN.

First trial with GRID cage round a binding site for water (press Return).

Then give the final confirmation that you want the job to proceed by typing "YES".

yes (press Return)

You will be reminded that the original copy of grid.in may be overwritten and lost, and you should answer YES or NO. (If you just hit the RETURN key the original version of grid.in will be saved to a file called temp.saved).

As soon as you have answered the questions, grid.in will be submitted to the computer to run Programme GRID as a batch job in background mode. You will be asked to hit RETURN once or twice, and MENU A will be displayed again. You should then type the number 7 in Menu A in order to return to the operating system, and in a few seconds your GRID run will be completed.

55.2.3. ASSESSING THE RESULTS FROM PROGRAMME GRID

The supervising Programme GREAT will have created a new command file grid.in and you can use your page editor to inspect this command file while the GRID computation is running. These lines should appear near the end of the new grid.in file:

  OH2                                                            
   TOPX     28.000                                                
   TOPY      9.500                                                
   TOPZ     21.000                                                
   BOTX     24.000                                                
   BOTY      5.500                                                
   BOTZ     17.000                                                
   IEND                                                           
   First trial with GRID cage round a binding site for water      
         0    1                                                  

They are interpreted like this:

1. You have been studying a complete protein, but your GRID map will be restricted to a relatively small part of the whole protein structure. This part is defined by the six directives TOPX .....BOTZ. These define a Grid CAGE whose top right back corner is at the position x=28.0 y=9.5 z=21.0 and whose bottom left front corner is at x=24.0 y=5.5 z=17.0. The positions are measured in Angstrom on the same coordinate axes which were used to define the atom positions in the pdb file. For your first job the Grid map will be computed for this restricted region alone.

2. Notice that there is quite a long list of directives in grid.in, starting with INPT and continuing through TOPX and ending with IEND. Of these, IEND is particularly important because it tells the computer that the list of directives has completely finished. The directive list must always finish with IEND as shown above. All the other directives (with the exception of the Probe) are optional, and you can read about them later in the MD User Manual.

3. "First trial with GRID cage round a binding site for water" is the title you typed in.

4. The next line contains 0 and 1. These are values for variables NZ1 and NZ2 which are described in the MD User Manual. You can always use 0 and 1 unless you are studying a very large Target on a small or slow computer.

5. OH2 is the symbol specifying the choice of Probe, which is water in this first Grid run. This symbol OH2 defines the properties of a water Probe, which can alternatively be defined by a set of eight or nine Energy Parameters. The Parameters are described in more detail below. The list of Probes is reported in the MD User Manual.

So you have been studying the interaction of water with an interesting part of a protein.

You have generated a Grid Map in file gridkont.kont and we will look at the map later.

The map is supplemented by some detailed tables of results in file gridlont.lont and we will look at them with the page editor as soon as the GRID job has finished.

Go to the end of file gridlont.lont and notice the most negative interaction energy.

tail gridlont.lont (press Return)

It is about -17 Kcal/mole and shows that water 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.

55.2.4. OUTPUT FROM GRID TO GRAPHICS – Programme GVIEW

From GRID version 19 Molecular Discovery Limited provides a graphic interface to display the output from Grid. The programme is called Gview. More information on Gview programme is reported in the MD User Manual - Gview.

Begin to use Gview by typing gview with the name of the grid kont file:

Gview gridkont.kont (or /your_grid_directory/gview gridkont.kont)

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 macromolecule structure together with the water MIF, use the commands:

File->Open and select the PDB.pdb from the dialogue. Then press Open.

The water molecules will be displayed on the 3Dplot with red crosses. 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 yellow 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. Use your page editor to search through the file gridlont.lont and you will find a detailed table of results.

This file lists, plane by plane, the atoms of the Target which are nearest to the most favourable position for the Probe. Search the plane 12. The asociated Table shows that the nearest atom is water molecule number 1067* which is actually 0.401 Angstrom from the best probe position. The asterisk in 1067* shows that the water molecules was NOT in fact considered as part of the Target when the Grid run was done. 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 0.33 Angstrom.

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.

In this directory we have supplied a Grid kont file called gridkont_almd.kont. This has an extended format containing extra information on the energy contribution made by each individual protein atom. A new directive ALMD was used in order to produce this special output, and if you want to know more about ALMD, click here.

The extended results obtained by using ALMD can be inspected graphically. Typing Gview with the name of the grid kont file:

Gview gridkont_almd.kont

To change the energy levels, select:

Edit->Field style

Move the cursor of the energy levels up to -12 Kcal. 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 Tutorial 02.