LESSON 3: Partitioning and Mapping a MacroMolecule

1. Read in the HIV1 molecule

   Go to the Molecule pulldown on the top menu bar and select the Get command. From Get File Type select PDB, and enter ./hiv1.pdb in the File Name parameter box . Press Execute.

2. Clean up HIV1

   The HIV1 molecule needs to have the proper end-groups, and InsightII needs to be informed that the lack of proper potential types is acceptable. From the Module (Biosym Logo) menu pulldown the Biopolymer item. First we must split HIV-1 into its two component subunits so that they can be properly capped. From the Modify pulldown select the Unmerge command; enter HIV1:101-199 as Piece to Unmerge and HIV1B as the New Molecule Name; press Execute. Now, from the Protein pulldown on the lower menu bar select the Cap command. Set the Molecule Name to HIV1. Then set the C_Terminus to on and the C Cap Groups to COO-, and N_Terminus to on and N Cap Groups to NH3. Press Execute. Next set Molecule Name to HIV1B for the second chain in HIV1. Now we can recombine the two chains of HIV-1. Pulldown the Merge command from the Modify menu. Select HIV1 as Molecule Name and HIV1B as Molecule to Merge. From the Forcefield pulldown, choose the Potentials command. Use the Potential Action of Accept and Execute this command. This action tells InsightII that we know that the potentials are not properly set. Hint does not need them for a macromolecule.

3. Partition HIV1 using the Dictionary method

   Go to the Partition pulldown on the Hint menu bar and select the Molecule command. Note that the Molecule is defined as HIV1. For proteins or other macromolecules consisting of well-defined monomers, more reproducible Hint results are obtained when the Partition Method is set to Dictionary. The Partition_File option gives you an opportunity to write an ASCII file containing the output from the Molecule Partition calculation. Leave this parameter off. Hydrogen_Treatment dictates how Hint will calculate parameters for the defined hydrogens in the current molecule. Choose Polar_Only now.

   In the Solvent Condition parameter box there are four options for partitioning the macromolecule. Acidic, Neutral, and Basic represent the most benign and generalized definition of these solvent conditions. This gives you an opportunity to rapidly explore the effect of pH on the hydropathy of a macromolecule. The fourth option, Inferred, calculates the protonation state for each monomer based on the number and connectivity of the hydrogens actually present in the macromolecule. (Note that this is not compatible with Hydrogen_Treatment set to United.) For this lesson select the Neutral Solvent Condition. Finally, select the Execute button to perform the Molecule Partition calculation for HIV1. The reported LogP for HIV1 (neutral solvent condition) is -4.136.

4. Calculate the Molecule HintMap for HIV

   From the Setup pulldown on the Hint menu bar, select the Grid command. In the Grid Setup parameter block, leave the View Grid Box parameter set to on so that the border of the grid is displayed when the command is executed. The Grid Center parameter should be set to Molecule_Region. Select the Molecule Region parameter box and select HIV1 from the Molecules Value Aid. Next, select the Border_Space option as the Grid_Size, and type 5.0 as the Border Space. Finally, select Point_Spacing as the Grid Resolution and type 1.0 in the Angstroms/Grid Pt parameter box. This specifies that there will be 1.0 Angstroms between grid points in all (x, y, z) dimensions. Select Execute to set the Grid parameters.

   Once again from the Setup pulldown select the Grid_Molecule command. This parameter block guides you through the setup of a HintGrid Molecule calculation. From Grid_Types select Hydrophobic/Polar to request a map where positive grid field values represent hydrophobic regions of space, and negative grid field values represent polar (hydrophilic) regions of space. Enter a value of 6.0 for the CutOff Radius. Set the Volume_Averaging parameter to off and set the Inside Mol Cutoff parameter to off. Press Execute to set the Grid_Molecule parameters.

   Next, from the Setup pulldown, set the Hint_DistFunct. There are two parts: the Hydropathic Term and the Steric_Term. For a HintGrid Molecule calculation select exp(-nr) for the Hydropathic Term; set the Steric_Term to off; and set Direction Vectors to None (Spherical). Press Execute to set the Hint_DistFunct parameters.

   Now, from the HintGrid pulldown on the Hint menu bar, select the Molecule command. Parameters in this block initiate the Molecule HintGrid calculation. First make sure that HIV1 is the parameter in the Molecule parameter box. Next, choose Background as the Execute_Mode parameter. The Job Name and Grid Filename parameters will be chosen for you automatically if you touch these boxes and press return. Enter a textual Comment of your choice. Select Auto_Get_Grid to be on. Finally, press Execute to begin the map calculation. This map calculation should take 1-3 hours.

   Repeat the map calculation to create a parallel Acid/Base map for HIV1. Return to Grid_Molecule Setup to select Acid/Base from Grid_Types. Choose Lewis as the Acid/Base Definition to use the Lewis model for acids and bases. Make sure all other parameters are the same as before. Press Execute. Return to Molecule HintGrid and repeat the calculation; new default values for Job Name and Grid Filename will be chosen.

   When this second map calculation is complete (about 1/2 hour) you may contour the HintGrid maps for display. Go to the Grid pulldown on the Hint menu bar and select the Contour command. First select the Grid Object box and pick HIV1_HP from the associated Value Aid. Next select the Contour Name Root box and enter HIVH. This will be the root portion of all Contour Objects created from HIV_HP. For the Contour Select parameter pick the Single Value option. Contour the Hydrophobic portion of this map. Enter a value of 10.0 for the Contour Level and pick a green for the Contour Color parameter. Now select the Grid Object box and pick HIV1_AB from the associated Value Aid. Next select the Contour Name Root box and enter HIVP. Contour this map with -20.0 (red) and 20.0 (blue). This display shows hydrophobic regions and differentiates between two types of Polar regions (Acid-like and Base-like) based on Lewis definitions of acids and bases.

   HINT hydropathic map of HIV-1 Protease