C687 Tutorial: Docking Module

The purpose of this tutorial is to become familiar with how to create a complex of two molecules via DOCKING the two molecules together. There are at least three methods used to dock two molecules. In this tutorial, we will attempt the first two methods. The third method is too computationally expensive to attempt on our Indy workstations.

Manual Docking with a Grid

Ideally, we would like to move the interacting molecules in real time on the screen of a workstation while computing the interaction energy. While the energy expression is straghtforward to compute, the computation time increases as the square of the number of interacting atoms, making the process too slow for many molecular systems. An energy grid approximating the larger of the two molecules can be precomputed. Since the interaction energy can then be approximated by calculating the energy between atoms of the moving molecule and the appropriate grid points, the docking can be accomplished much faster.

Docking with a grid was very popular during the 1980s when computers were not very powerful. Even though computing power has improved during the last decade, the grid method is still fairly popular, unless both molecules are relatively small.

Traction Beam Docking

If you know that two specific atoms of each molecule must interact (i.e., there is a hydrogen bond, a salt bridge, or an important van der Waals contact that must be present in the final complex), you can apply the "traction beam method" of docking. This method is just your basic energy minimization with one molecule held fixed and a low-force-constant distance restraint applied between the two atoms. When the non-fixed molecule is minimized, it's final conformation must also satisfy the distance restraint, so this molecule slowly "swims" towards the fixed molecule until the distance restraint is satisfied.

Variations of this method apply a step-wise approach to the distance restraint's force constant:

Another variation applies a high-force-constant (~100 kcal/mol) distance restraint so that the distance is only 2-3 angstroms shorter than the current distance. This minimization is repeated again and again; the distance between the atoms is restrained to a value 2-3 angstroms shorter than in the previous minimization for each round of minimization.

Other variations apply simulated annealing to allow the non-fixed molecule to avoid high-energy minima, which (presumably) culminate in "poor" molecular complexes.

AutoDock

A Monte Carlo simulation can be performed where a small ligand is allowed to "randomly walk" about the surface of a protein or nucleic acid. This method is very computationaly intensive. Since there is some randomness to the generation of the final molecular complex, and since there are usually many protein-ligand conformations with similar energy, an ensemble of ~10 structures is usually calcualted with the hope that most of the ~10 structures will show the same binding mode. This means that this computationally intensive program must be repeated ~10 times. To perform the Monte Carlo simulation:
  1. The temperature of the system is set to a high value (~500K)
    1. The coordinates of the ligand are changed randomly
      1. the energy of interaction between the two molecules is calculated
      2. if the energy of interaction is lower than the previous step, this new conformation is ACCEPTED
      3. if the energy of interaction is higher than the previous step, the difference in energy is calculated; the probability (between 0 and 1) that the energy could increase by this amount is then calculated based upon the Boltzman distribution at the system's temperature. A random number (between 0 and 1) is chosen. If the random number is larger than the probability, then this new conformation is REJECTED; if the random number is smaller than the probability, then this new conformation is ACCEPTED.
    2. Step 1 is repeated until the number of ACCEPTED or REJECTED steps reaches a pre-set limit (e.g., 100,000 steps)
  2. The temperature is reduced by a few percent, and step 1 is repeated at this new temperature.
  3. Once the final temperature reaches a very low value (~1K), the Monte Carlo simulation is finished.

Part 1: Preparing the System

Duration: ~10 min

1. Restore the folder /ruser/nmrsg1/mpagel/C687/cruzain_ligand.psv
2. fIx/fix/fix your potentials/partial charges/formal charges
3. Measure bumps and hydrogen bonds between the ligand and the protein with MONITOR ON

This complex consists of the protein cruzain and a ligand (an inhibitor); this system was kindly provided by Karl Scheidt, a member of the Roush research group.

There are two hydrogen bonds formed between residues 66 & 158 (in white) and the ligand:
Atom 1 Atom 2 Distance Angle
CRUZAIN:66:HN LIGAND:I2:O 2.32 153.51
LIGAND:I3H:HN CRUZAIN:158:O 2.13 141.77

There are many van der Waals bumps; most of these involve residue 25 (residue 25 is colored by atom type):
Atom 1 Atom 2 Distance
CRUZAIN:25:SG LIGAND:I3H:CA 2.72
CRUZAIN:25:CB LIGAND:1C:H12 2.08
CRUZAIN:25:HB2 LIGAND:1C:H22 1.89
CRUZAIN:25:SG LIGAND:1C:C1 1.89
CRUZAIN:25:SG LIGAND:1C:H12 0.85
CRUZAIN:25:SG LIGAND:1C:H11 2.44
CRUZAIN:25:SG LIGAND:1C:C2 2.96
CRUZAIN:25:HG LIGAND:1C:C1 2.05
CRUZAIN:25:HG LIGAND:1C:H12 1.55
CRUZAIN:25:HG LIGAND:1C:H11 1.91
CRUZAIN:67:HD21 LIGAND:I2:HN 1.78
CRUZAIN:158:O LIGAND:I3H:HN 2.13

This complex is actually a modification of the crystal structure: Origionally, the crystal structure had a covelent bond between the sulfur of Cysteine-25 and the C1 carbon of the ligand (to prove this, measure the distance between these two atoms; keep "monitor" ON. Is the a typical distance of a C-S bond? Remember this value---you will need it later.) I have removed this bond and added hydrogens to the sulfur and carbon, causing several large VDW overlaps.

In this assignment, we will dissociate the ligand and the protein, and then attempt to model how the ligand binds to the protein BEFORE the covalent bond is created. One of the biggest problems with this exercise is that the protein will NOT be allowed to move. Therefore, the protein is locked into it's conformation WITH the covelent bond, while we are assuming that the covalent bond is NOT yet made. Obviously, this allows Martin, Marty, and Brandt endless opportunities to ask you about this deficiency, SO BE PREPARED for their questions!

4. Connect to the ligand. Move the ligand until the distance between the C1 carbon atom of the ligand and the SG sulfur atom of cruzain-25 is 15 angstroms (this distance is already monitored and updated on the screen if you followed the instructions in the discussion above).
5. Turn off the monitored hydrogen bonds and bumps.

Part 2: Manually Docking with a Grid

Duration: 45-60 min
  1. Select the Docking Module
  2. Click on Docking_Grid/Create, and create a an enclosure-style grid about cruzain, with a border space of 5 angstroms and a 2 angstrom grid step.
  3. Click on Docking_Grid/Compute, and compute a docking grid of cruzain with Van_der_waals and Coulomb energies and a cutoff of 10 angstroms; make a visible grid by turning on make_vis_grid with a grid name of CRUZAIN_VGRD
  4. Click on Grid/Display, and display all points of CRUZAIN_VGRD
  5. Click on Grid/color and color all grid points using the charge_spectrum (turn "use_spectrum" ON)
  6. Click on Grid/Display, and turn off the display of the grid points
  7. Grid Slices
    Click on Grid/Slice
    Scalar grid name = CRUZAIN_VGRD
    Plane Number = 1
    Plane Direction = Z
    Plane Height = 59.5221
    H intvals = 20
    Plane Width = 59.5221
    W intvals = 20
    Plane Type = Mapplane
    Spectrum Name = SLICE_SPECTRUM
    Plane Style = Filled
    Click execute. Move the plane by moving the slide bar in the Parameters menu. Notice that when the plane bisects the molecule, the center of the plane is blue: the interaction energy inside the molecule is high (positive, or BAD). When the plane is moved towards the ligand and outside the protein, the plane turns red: the interaction energy is low (negative, or GOOD). Position the plane near the ligand; notice that the plane is read in the center, and blue near the edges: the interaction energy is GOOD in the center and BAD at the edges, so the ligand is attracted towards the center---the active site! This model may actually be useful!

    Try a Contour plane in the X direction with a SLICE_SPECTRUM, and Contour_Levels = -2 and Displacement = -3.17
    Add a Plane Number 2 at a contour level of -3.
    Add a Plane Number 3 at a contour level of -4.
    Add a Plane Number 4 at a contour level of -5.
    Add a Plane Number 5 at a contour level of -6.
    Add a Plane Number 6 at a contour level of -7.
    Notice how the interaction energy becomes lower (more negative) nearer the active site.

    Try other planes and parameters. When you are done, set Plane Type to OFF fdor all planes.

  8. Grid Contours
    Click on Grid/Contour
    Contour_Name_Root = contour1
    Level Specification = Single
    Contour Level = -15
    Display Style = Solid
    Color = yellow
    Note where the best interaction energies lie. Try other parameters (e.g., Contour Level = -10, -7, -5). Then delete all "contour*" objects.
  9. Evaluate the Intermolecular Energies
    Click on Evaluate/Intermolecular, and ADD a MONITORed energy between CRUZAIN and LIGAND via the GRID.
    The VdW, Elect, and Total energies are reported on the screen. Connect to the ligand and move it towards the active site. Note that the energies are updated fairly rapidly. Try to place the ligand in the active site in teh best conformation possible. This may take ~5-10 minutes. Be sure to turn OFF any contours or slices so that the graphics don't make this slow.
  10. Save a folder of your "best docked ligand-protein complex using a docking grid".
  11. Click on Evaluate/Intermolecular, and ADD a MONITORed energy between CRUZAIN and LIGAND. DO NOT use the grid; Use a cut-off of 10 angstroms, with both Van_der_waals and Coulomb energies. Move the ligand around. Notice that the calculation takes a LOT longer! Don't spend more than a minute or two in this mode.
  12. Delete all objects.

Part 3: Traction Beam Docking

Duration: ~20 min

  1. Restore the folder /ruser/nmrsg1/mpagel/C687/cruzain_ligand.psv.
  2. fIx/fix/fix your potentials/partial charges/formal charges.
  3. Connect to the ligand. Move the ligand until the distance between the C1 carbon atom of the ligand and the SG sulfur atom of cruzain-25 is 15 angstroms (monitor this distance).
  4. Click on Assembly/Associate and associate cruzain and the ligand using a New Assembly Name of "complex".
  5. Select the Discover module.
  6. Fix the entire cruzain molecule.
  7. set a generic distance constraint between CRUZAIN:25:SG and LIGAND:1C:C1
    GenericDis_Upr_Bnd = 2.73
    GenericDis_Lwr_Bnd = 2.73
    GenericDis_Upr_K = 5.00
    GenericDis_Lwr_K = 5.00
    GenericDis_Max_Frc = 50
  8. Minimize using Conjugate Gradients (the structures are already at a fairly low minimum energy, so we don't have to use steepest decents) to a derivative of 0.5 or 1500 iterations, whichever comes first. Use charges, but don't use cross or morse terms.
  9. Set a dielectric of 1.0, no distance dependence
  10. Set a cutoff of 20 angstroms.
  11. Click on Run/run and INTERACTIVEly RUN_MINIMIZATION on the COMPLEX.
  12. Save a folder of your "best docked ligand-protein complex using a traction beam".

Part 4: Submit your work

Duration = ~5-10 min
  1. Rename the cruzain to "cruzain_tb". Merge your ligand into "cruzain_tb".
  2. Color cruzain_tb with a light blue color.
  3. Recall your "best docked ligand-protein complex using a docking grid"
  4. Rename the cruzain to "cruzain_grid". Merge your ligand into "cruzain_grid".
  5. Color cruzain_grid with a green color.
  6. Superimpose all cruzain heavy atoms in cruzain_tb with all cruzain heavy atoms in cruzain_grid. DO NOT superimpose the atoms of the ligands.
  7. Delete everything except cruzain_tb and cruzain_grid.
  8. Save the superimposed structures in a folder.
  9. Compare the final conformations of your ligands. Which method worked better?
  10. In a UNIX shell, copy your folder to /ruser/instruct1/stone/C687/assignments/your_name.dock.psv
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Last updated: 01/23/2001