C687 Tutorial:

Surface Area Measurements, Electrostatic Field Calculations, and Computer-Assisted Ligand Design


You only need to complete 2 of the 3 sections:

Part 1A: Measuring Solvent-Accessible Surface Areas

Duration: ~20 minutes
  1. The Solvation module is currently licensed only on stereo3 and splatter. Therefore, when working on other machines it is necessary to run the program remotely on stereo3 or splatter, then display on your local monitor. Half of the class should choose each machine.
    To run remotely on stereo3:
    1. Type xhost stereo3
    2. Type telnet stereo3, then login
    3. cd to the appropriate directory
    4. Type insightII
    You can also log onto splatter using the same procedure.
  2. Restore your zinc-finger folder (saved during the Atomic & Molecular Properties tutorial). Or download the zinc finger PDB file into your home directory, read this structure into InsightII, and Fix/Fix/Fix potentials.
  3. Choose the Solvation module and browse through the menu options.
  4. To set up your system, click on Setup/System, and select your protein.
  5. Set up your parameters: Select Surface Area Only.
  6. Select Low Accuracy_Level (just to limit the time required for the calculation for this tutorial) If you want to calculate surface areas for your research, choose a higher level of accuracy. Set Output level to atom.
  7. To run, click on solvation_run/run.
  8. Wait a few minutes for the calculation to proceed.
  9. When the calculation is finished, wait until the Solvation Results Table is finished being created and displayed. Then delete this box: click on the square in the upper left corner of this window, then select close.
  10. In a UNIX window, list the files that were created. Examine the contents of the file named your_molecule#.hydro_log.
  11. Read through this file and identify which residues are solvent exposed and which are buried in the protein structure. Which atoms of solvent-exposed residues are buried?
  12. Copy the file your_molecule#.hydro_log to a file named surf_area.hydro_log in your home directory.

Part 1B: Measuring Free Energies of Solvation

Duration: ~20 minutes

Drug designers often like to calculate the free energy of solvation from octanol to water. Octanol is assumed to approximate the environment inside the protein. Therefore, the free energy of solvation measures the non-specific "affinity" of the drug for the protein binding site vs. the aqueous solution around the protein.

Some chemists consider the hydrophobicity to be the free energy of solvation from a vacuum to water, while others consider the free energy of solvation from octanol to water to be a more accurate measurement. What do you think?

  1. If you are not running the solvation module from stereo3 or splatter, see the instructions in the Solvent Accessible Surface Areas section of this tutorial.
  2. Select the Builder/fragment/get menu, turn off the To_Modify_Bond option and get a leucine amino acid and a lysine amino acid as 2 moleules (do NOT connect them with a bond).
  3. Select the Atom/hybridization menu, and set the hybidization of the lysine's nitrogen side chain atom to sp3+ hybridization.
  4. Select the Modify/hydrogens menu, and add hydrogens with Un-Charged termini.
  5. Click on Forcefield/select and select the CFF91 forcefield. The Eisenberg_McLachlan Octanol_Water_Model is based upon the (electrostatic and van der Waals) CFF91 parameters---other forcefields will NOT work!
  6. Click on Forcefield/potential and fix the atom potentials, fix the partial charges, and fix the formal charges of the two amino acids. While you aren't using the zinc finger for this part of the tutorial, you should also fix/fix/fix the zinc finger (or you can change the forcefield back to CVFF for the electrostatics calculation).
  7. Perform the following measurement two times, once for each amino acid:
    1. Select the Solvation module, select the Setup/system menu, and select the amino acid to set up your system.
    2. Select the Setup/ parameters menu:
      ONLY select the CFF91 Parameter_Set and the Eisenberg_McLachlan Octanol_Water_Model. Select Low Accuracy_Level (just to limit the time required for the calculation for this tutorial). If you want to calculate surface areas for your research, choose a higher level of accuracy. Set Output level to atom.
    3. To run, select the Solvation_run menu and run.
    4. Wait about 10-30 seconds for the calculation to proceed.
    5. When the calculation is finished, wait until the Solvation Results Table is finished being created and displayed. Then delete this box: click on the square in the upper left corner of this window, then select Close.
  8. In a UNIX window, list the files that were created. (CAUTION: If you are remotely logged onto splatter or stereo3 and you start a UNIX shell from InsightII, the UNIX shell will NOT pop forward as normal---you must iconize the InsightII window to see your UNIX shell). Examine the contents of the file your_molecule#.hydro_log.
  9. Compare the results of the two calculations.
  10. Append the contents of the lysine hydro_log file to the end of the leucine hydro_log file. Copy this leucine/lsyine hydro_log file to a file named solv_energy.hydro_log in your home directory.

Part 2: Calculating Electrostatic Fields

Duration: ~30 minutes

This tutorial will generate a 3D grid of points centered on your molecule. The electostatic potential felt by each grid point from the partial charges of each atom is then calculated. Finally, the surface of the protein is created and colored based upon the electrostatic values.

How the electrostatic potential calculation is accomplished:
Consider two points in space that are fairly close together, both of which have a partial charge:

  1. One point "radiates" it's electrostatic potential, and the other point "feels" this potential. The second point "reacts" (via dipolar reorientation and electronic polarization) to this potential and the electrostatic potential of the second point changes in value.
  2. The second point, with it's new value, "radiates" it's potential, and the first point "feels" this potential. The first point "reacts" to this potential, and the electrostatic potential of the first point changes in value.
  3. Since the first point has changed it's electrostatic potential value, the calculation returns to step 1 with this new value of the first point. This calculation loop is repeated until the changes in the electrostatic potential values are very small.
The total electrostatic potential is equal to the electrostatic charge of the atom plus the electrostatic potential generated by this "reaction field" (caused by the presence of the other atoms), as described by the Poisson-Boltzman equation. More information about this method is available from the DelPhi manual in room A701.

Each grid point is calculated based upon it's "reaction" to the values of the 6 neighboring grid points. This causes two problems:

Unfortunately, if we try to avoid both problems, we need to create a very large grid with very many grid points. This can take a very long time to calculate. For the purposes of this tutorial, we will calculate one grid with a very large size and only average resolution. If you attempt this calculation and you want research-grade results, you can apply the following trick:
  1. Calculate a very large grid with only average resolution, as described below.
  2. Then repeat the calculation, select a smaller grid (e.g., a grid that is not much larger than the size of the protein), set the Boundary to focussing, and set the focussing grid to the grid that you calculated in step 1.
This will set the boundary points of the second (focused) grid to values interpolated from the nearest points found in the first (focussing) grid. Thus, the boundary points of the second grid will feel an approximation of the "reaction field" of the grid points of the first (focussing) grid that lie outside the second (focused) grid. This method has been shown to work very well.

The tutorial performs these steps:

  1. If you are not running InsightII from stereo3 or splatter, see the instructions in the Solvent Accessible Surface Areas section of this tutorial.
  2. Restore your zinc_finger folder or download the zinc finger PDB file into your home directory, read this structure into InsightII, and Fix/Fix/Fix potentials.
  3. Select the DelPhi module.
  4. Select the Setup/Boundary menu, select Full coulombic
  5. Select the Setup/Grid menu.
    Select display_grid.
    Set Grid Center to Molecule_Region.
    Set Molecule Region to your molecule.
    Set Grid Size to Border_Space.
    Set Border Space to 10.
    Set Grid Resolution to Point_Spacing.
    Set Angstroms/Grid Pt to 1.
  6. View the parameters in the Setup/Solute and Setup/Solvent menus. These default parameters are set up for a typical protein solute in a physiological solvent.
  7. Select the Run_DelPhi/Run menu. Turn on the Auto_Get_Grid option, and run your calculation in the background.
  8. The calculation can take several minutes. Once the calculation is finished, a box will appear to notify you or a message wil appear at the bottom of the InsightII window.
  9. If you forgot to turn on the Auto_Get_Grid option in the Run_DelPhi step, select the Grid/get menu and get the new grid.
  10. To generate a surface of your protein, click on Molecule/Surface. Create a Solid Connolly surface with an atom radius of 1.4 and an atom radius incr of 0.00. For this tutorial, select a low-resolution surface quality.
  11. Wait a few minutes for this surface to be calculated.
  12. Color the surface of the molecule via the Molecule/Color menu.
    Set Color Method to Grid.
    Set spectrum Name to CHARGE_SPECTRUM.
    Set Scalar Grid Name to the name of the grid you calculated.
  13. Save your results to a file named electro_surf.psv in your home directory.

Part 3: Computer-Assisted Ligand Design

Duration: ~30 minutes

  1. If you are not running InsightII from stereo3 or splatter, see the instructions in the Solvent Accessible Surface Areas section of this tutorial.
  2. Restore the cruzain.psv folder. You downloaded this folder during the Docking Tutorial, so you may already have this folder in your directory.
  3. Select the Ligand_Design Module. Notice that only the Ludi and Background Job menus are unitque to Ludi---the other menus are identical to menus in the Builder and Biopolymer Modules.
  4. Select the Ludi/Parameters menu. Turn on the Invert option at the bottom of the menu.
    Examine the other options:
    1. Preselect specifies a criteria for "preselecting" fragments that may fit well to the receptor. Range = 1.5 to 2.5.
    2. Min Separation specifies the minimum separation between carbon atoms in the fragment and in the receptor. Minimum separations between other atom types are scaled relative to this parameter. Recommended range = 3.0 to 3.5 angstroms.
    3. Dens L specifies the density of grid points to represent the receptor pocket for lipophilic (hydrophobic) interactions. Larger values slow the calculation, but may find more fragments. Range = 10 to 25.
    4. Dens P specifies the density of grid points to represent the receptor pocket for H-bond interactions. Larger values slow the calculation, but may find more fragments. Range = 10 to 25.
    5. Min Surf specifies the minimum percentage of the fragment's surface area that must be in contact with the receptor. Default = 0. Increase this value if you are generating too many fragments.
    6. Relative Weighting terms:
      • Link Weight specifies the relative weight for aligning the new fragment with link sites on the ligand. Default = 1.0. If you change the weighting, this term should be weighted twice as high as the others to give it substantially more influence during the calculation.
      • Lipo Weight specifies the relative weight for aligning the new fragment with lipophilic interaction sites of the receptor. Default = 1.0. If you change the weighting, this term should be weighted 10 times as high as the others to give it substantially more influence during the calculation.
      • H Bod Weight specifies the relative weight for aligning the new fragment with hydrogen bond sites of the receptor. Default = 1.0. If you change the weighting, this term should be weighted 10 times as high as the others to give it substantially more influence during the calculation.
    7. If Aliphatic_Aromatic is turned on, then lipophilic interactions between a fragment's aliphatic groups and a recptor's aromatic groups, and interactions between a fragment's aromatic groups and a recptor's aliphatic groups, will NOT be considered.
    8. If Reject_Bifurcated is turned on, then bifurcated hydrogen bonds will NOT be considered. Default is to turn off this option.
    9. If No_Unpaired_Polar is turned on, then polar groups that are not paired with another polar group can NOT be buried in a hydrophobic pocket. Default is to turn on this option.
    10. If Electrostatic_Check is turned on, then LUDI will check for electrostatic repulsion between polar atoms. Default is to turn on this option. Turn this option off if you fit fragments to metal ion coordination sites in the receptor.
    11. ES Dist specifies the minimum distance between hydrogens of polar groups. Distances between other atoms of polar groups are scaled relative to this parameter. Default = 2.5.
    12. Minimum Score specifies the smallest allowable score for a fragment hit. Default = 0. Increase this parameter if you generate too many fragments. Defalt = 940, the size of the fragment library.
    13. Maximum Hits specifies the maximum number of fragment hits.
    14. Max Unfilled Cavity specifies the maximum size for buried cavities produced by a fragment fit. If a fragment creates a cavity larger than this size, the fragment will be rejected. Default = 0. Increase this value if you are not generating enough fragments.
    15. Scoring Function can be set to
      • Hbond_Lipo, which scores fragment fits based on geometric criteria (orientation & length of H bond; total area of hydrophobic contact)
      • Energy_Estimate, which scores fragments based a free energy calculation of H-bond strength and hydrophobicity
      Default = Energy_Estimate
  5. Select the Ludi/Run menu.
  6. Wait for the Ludi calculation to finish. An information box will appear when the calculation is finished. You can continue using InsightII during the calculation.
  7. When the calculation is finished, select the Ludi/Load menu, and load the results from your Ludi run. Turn on Load Fragments and Show Statistics.
  8. Examine the Ludi Table. Note which types of fragments get the highest scores.
  9. To select specific fragments, use the options in the Ludi/Review menu.
  10. Display only the fragment with the highest score, the receptor, and the ligand. Change the receptor to green, the ligand to red, and the fragment to yellow, and save the results in a folder named ludi_cruzain.psv in your home directory.

Part 4: Verify that you have completed this portion of the assignment

See the Docking/Ligand Design/Electrostatics Assignment page for details.


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Last updated: 01/23/2001