The guidelines given in this tutorial are intentionally very loose. To prepare for this tutorial, read the InsightII Simulated Annealing notes. The overall aim is to do a simulated annealing run on the zinc finger structure.

This tutorial will be performed in the style of popular cooking shows: You set up the run (but don't actually run it), then you analyze the results of a run that I did earlier (there is not enough time to do the run and analyze it in one tutorial session).
All files relevant to the run can be downloaded:

• bennett.car
• bennett.inp
• bennett.mdf
• bennett.out
• bennett.pdb
• bennett.arc
  To obtain the bennett.arc file,
  
  1. Click here to go to the Bennett download directory
  2. Select the bennett.arc.tar file from the directory listing
  3. Save the bennett.arc.tar file in your home directory
  4. In a UNIX shell, type tar -xvf bennett.arc.tar
  5. Type rm bennett.arc.tar to remove the original tar file.
  6. Type ls to verify that you have the bennett.arc file.

This tutorial focuses on Lobodomide A, a potentially new type of antitumor lead optimization compound. Lobodomide A is shown below, and also shows the differences between Lobodomide A and Lobodomide B-F. In general, the Lobodomides consist of a macrocyclic ring and a long "main chain".

Chad Bennett and I are currently modeling Lobodomide A. We ran a simulated annealing run to search for low-energy conformations, and we are currently analyzing the results to identify the pharmacophore. The pharmacophore is the region of the molecule that contains functional groups that are responsible for binding and binding specificity. We plan to compare the pharmacophore of Lobodomide A with the pharmacophore of Salicylihalamide A, a similar antitumor lead optimization compound.

It is likely that the pharmacophores of Lobodomide A and Salicylihalamide A have the same functional groups. Can you identify these functional groups?

Part 1: Forcefields, Potentials, and Restraints

1. Start InsightII and read in the bennett.pdb PDB file of lobodomide.
2. Choose a Forcefield. See the Forcefield notes.
   
3. Rename your molecule lobodomide.
4. Fix/Fix/Fix all potentials and charges.
5. Select the Discover module.

Part 2: Setting Up the Command File

1. Select the Run/Run menu, and select the local workstation, input the molecule name, select COMMAND FILE, auto-assign parameters, only minimize, and Reduce Output. Then click EXECUTE. This step creates a .car, .inp, and .mdf file.
2. Start a UNIX shell and read through the command file which has the suffix .inp. To understand each line in the file, follow through the bennett.inp example.
3. At this stage, if you were actually going to perform a dyamics run you would edit the command file so that it would resemble the bennett.inp example file. However, for now it will suffice just to read through this example file and make sure you understand each command. Spend ~10 minutes doing this.
   Pay particular attention to the following features of the command file:
   
   • Comment lines begin with an exclamation mark (!)
   • Parameters for changing the non-bond cutoff, the dielectric, and the decay time constant for heating and cooling.
   • Initial quick minimization to reduce very high energies
   • Second, longer minimization before starting the dynamics
   • The equilibration step
   • The beginning and end of the dynamics loop
   • The steps within the dynamics loop
     
     • Heating and high temperature dynamics
       
     • Cooling and lower temperature dynamics (annealing)
       
     • First, quick minimization
       
     • Second, longer minimization
       
     • Recording the results
       
   
   

Part 3: Running the Dynamics

Part 4: Analyzing the Results

• Read the notes about directions about how to evaluate simulated annealing calculations.
  
  
• Click on Molecule/Get and get frame #1 of the bennett.arc file.
• Select the Analyze Module.
• Select the Trajectory/Get menu, and get the trajectory stored in the bennett.arc file.
• Animate the trajectory. Look for regions of structure that do not appear to change conformation.
• Make a graph of frame number vs. the H-C-C-H dihedral angle of the trans-HCCH-double bond in the main chain. Does this double bond prefer to be cis or trans?
• Make a graph of frame number vs. the H-C-C-H dihedral angle of the cis-double bond in the main chain. Does this double bond prefer to be cis or trans?
• Make a graph of frame number vs. the H-C-N-O dihedral angle of the trans-HCNO-double bond in the main chain vs. the total energy. This will be a 3D graph.
• Connect Object to the 3D graph. Examine this graph from different perspectives. Which angle value is associated with the lowest energies? Save only the graphs in a folder named dyn_graphs.psv in your home directory.
• Make other graphs of distances, dihedrals, energies, frame numbers, etc. Answer these questions:
  • Does the ring like to make a "boat" or "chair" conformation? Is there a conformation for the ring that seems to be in the same relative position in most low-energy conformers?
  • Where does the main chain lie relative to the ring? Does the OH-C2-C3-C1 dihedral angle (the angle about the first bond of the main chain) prefer a gauche+, gauche-, or anti arrangement?
  • Where is the pharmacophore? Where are the functional groups that seem to be in the same relative position in most low-energy conformers?
• Make a cluster graph. Can you identify clusters of similar structures? You may have to make several cluster graphs with different parameters before you see these clusters.
• Save only the cluster graph in a folder named cluster_graph.psv in your home directory.

Part 5: Verify that you have completed this portion of the assignment.