To prepare for this module, read Builder/Biopolymer notes.

This tutorial uses the Biopolymer Module. Almost all of the commands within Builder are also in Biopolymer; Biopolymer has extra menus for building biopolymers. Builder has an Optimize menu that Biopolymer doesn't have. Optimize minimizes the energy of molecules with pre-set parameters. Instead of using Builder/Optimize, you will use the Discover Module, which allows you to define your own energy minimization parameters.

If you ever use the Builder Module, the program will alert you about a licensing problem. Just ignore this mesage.

Creating a molecule requires the following steps:

1. Specifiy all of the atoms and bonds.
2. Specify the potentials, e.g. defining what type of C atom a particular C is (e.g. amide vs ester vs aromatic).
3. Specify atomic charges.
4. Put the molecule in a reasonable conformation. This can be done either manually or by automated energy minimization.

Throughout this tutorial, you should save your current work to a folder frequently!! This will allow you to retrieve your work if anything goes wrong.

Part 1: Building Short Regular Peptides

1. Start InsightII. Select the Biopolymer Module.
   A second row of menu options will appear. Briefly browse through each menu to see the options.
2. Select the Residue/Append menu.
   Specify a molecule name and click on the Alpha_R_Helix motif. This will allow you to build a right-handed alpha helix. Click in the residue box, then click in sequence on several residue types (except proline); selecting about 15 residues will generate ~4 turns of helix. Rotate the helix and look at its properties. In particular, examine the groups at the termini - what sort of functional groups are they?
3. Select the Measure/HBond menu.
   Examine the positions and orientations of H-bonds in the helix.
4. Delete this helix. Build a new helix that is amphipathic. Color your residues based on hydrophobicity. Save this model in a file named amphipathic_helix.psv in your home directory.
5. Delete your amphipathic helix. Build a beta hairpin.
   Select the Residue/Append menu. Build a 15-residue beta strand. At least 1 residue must be a serine or threonine.
   
6. Then:
   • Select the Modify/Geometry menu. Adjust the conformations of the central residues (7 and 8) so that the overall conformation is a beta hairpin. Measure dihedral angles and distances between hydrogen bond acceptors & hydrogens to help you make these adjustments. Compare your dihedral angles with the angle values listed in the hairpin diagram on this web page. This may take some time. It will be easier if you turn off the sidechain display. Pay particular attention to the relative positions of H-bond donors and acceptors.
     
   • OR select the Transform/Torsion menu. Define a torsion angle or angles, then use the Dial Slider Boxes in the lower left corner to rotate about the torsion angle:
     1. Select 4-atom torsion angles instead of 2-atom torsion angles. It is usually better to select the 4 atoms of the torsion angle, rather than selecting the middle two atoms and letting the program choose the first and fourth atoms.
     2. All four atoms must be contiguous (e.g., the first must be bonded to the second, the second must be bonded to the third, the third must be bonded to the fourth).
     3. The fourth atom selected will be the atom that moves.
     4. You can select a fifth atom to define a second adjacent torsion angle (i.e., an angle with atoms #2, 3, 4, and 5).
     5. You can define several torsion angles, but only one torsion angle (the one in yellow) is active. To select another torsion angle, click on the Next Torsion button.
7. Display the sidechains again. Choose a sidechain with an OH group. You will now methylate this group.
   Select the Modify/Bond menu.
   Select Create, Fragment Window, and single bond.
   For A Atom, click on the H of the OH group you want to modify.
   For B Atom, click on one of the H atoms of the methyl group in the fragment window. Your OH group is now methylated.
   Try experimenting with some other modifications for a few minutes.
   
8. Delete everything (delete *)

Part 2: A Crude Model of the Antibiotic Vancomycin

The aim of this section is to build a crude model of the aglycone portion of the antibiotic vancomycin. This will help you to become familiar with the methods of modifying atoms, bonds, and potentials, and doing a very rough energy minimization.

Before you start, examine the structure. Note in particular:

• the peptide backbone
• the sterochemistry of the backbone from the N-terminus R,R,S,R,R,S,S (S=L-amino acid; R=D-amino acid)
• the nature of the termini
• the modifications of various amino acid sidechains and unnatural amino acids
• the three sidechain-sidechain crosslinks

You will also need a list of the CVFF potential types to complete this exercise.

1. Select the Forcefield/Select menu.
   Choose Clear Potentials, Clear Charges, and cvff.frc
   This selects the CVFF forcefield. Only the CVFF and ESFF forcefields have appropriate chlorine atom types.
2. Select the Residue/Append menu.
   Set Molecule Name to Vanco
   Select Beta Strand
   Construct a linear heptapeptide with the sequence:
   Leu-Tyr-Asn-Gly-Gly-Tyr-Gly
   You will construct vancomycin by modifying this sequence.
3. Select Modify/Hydrogens to put the correct functional groups at the termini
   Turn lone pairs off.
   Set capping mode to charged.
   Observe the changes on the terminal functional groups.
4. Modify each residue as required to give the correct covalent connectivities and also the correct potentials and charges. At this stage do not worry about stereochemistry and do not form the crosslinks between residues. Examples for residues 1-3 follow:

   Residue 1: N-Methyl leucine

   Select the Modify/Bond menu. Add a methyl group to the N-terminus.
   Select the Molecule/Label menu. Set property to potential, and execute.
   Check that the potential of each atom is correct according to the list of CVFF potentials. If the potential of any atom needs to be changed, use the Atom/Potential menu to make the appropriate assignment.
   In the Molecule/Label menu select clear, then set property to formal charge
   Check the formal charges. The N-terminus should be +1. If not, modify charges using atom/charge menu.

   Residue 2: A derivative of Tyr

   Add the OH group at the beta position (be sure to add this group to create the correct stereochemistry). Using the Modify/bond menu, chose the functional group fragment library. Replace one of the H atoms on the aromatic ring with a chlorine using the Atom/Replace menu. Again, check the potentials and formal charges for this residue.

   Residues 3 & 4: Asn & Gly

   Delete the nitrogen and associated hydrogens on the end of the side chain of residue 3. Break the peptide bond between residues 3 and 4. Create a bond between the carbonyl carbon of residue 3's side chain and the backbone nitrogen of residue 4. Check the potentials and charges for these residues.

   Residue 4: Gly

   Replace one of the hydrogens attached to the alpha-carbon with a phenol ring. The hydroxyl group on the phenol ring should be in the para position. Check the potentials and charges for this residue.

   Residue 5: Gly

   Replace one of the hydrogens attached to the alpha-carbon with a phenol ring. The hydroxyl group on the phenol ring should be in the para position. Check the potentials and charges for this residue.

   Residue 6: Tyr

   Add a hydroxyl group to the beta-carbon and a chloride atom to the phenyl ring. Check the potentials and charges for this residue.

   Residue 7: Gly

   Replace one of the hydrogens attached to the alpha-carbon with a di-phenol ring. The hydroxyl groups on the di-phenol ring should be in the meta position. Check the potentials and charges for this residue.

5. Modify the sterochemistry of all backbone alpha carbons and the chiral sidechain carbons on residues 2 and 6 as necessary. To modify steroechemistry, use the Modify/Invert menu. A atom and B atom are the two atoms that you want to swap. Chiral atom is the atom to which they are both bonded and about which the stereochemistry will be switched.
6. Select the Forcefield/Potentials menu.
   Set Potential action to fix
   Set Partial charge action to fix
   Set Formal charge action to fix
   Check for messages in the textport window. If there are problems, try fixing again, but choose the options to print the various results. This long list of results will hopefully contain some clues to which atoms are problematic. Also see Preparing a Molecule for Calculations
7. Form the residue 4-6 cross-link. Manually adjust the backbone so that the groups to be cross-linked are reasonably close in space. Use the Modify/Geometry menu, choose dihedral and pick the atoms that define the dihedral angle that you want to modify. For example, select the HA-CA-CO-C angle of residue 4. You need to type in the dihedral angle manually.
   Make similar adjustments until the sidechain atoms to be crosslinked are close enough to form a moderately realistic bond (it does not have to be too precise because the structure will be energy minimized).
   Before forming the new bond, rotate the aromatic ring so that the chlorine is pointing in roughly the correct direction.
   Form a bond between the desired atoms and then check and fix all the potentials and charges. To make the new bond, delete the 2 H atoms, then use the Modify/Bond menu to create a bond between the O and C atoms.
8. To energy minimize, select the Discover Module, then select the Parameter/Minimize menu. Choose 500 iterations, Steepest Decents minimizer, a derivative of 0.1, and cross, morse, and charges should be set to off. Execute this menu. Then select the Run/Run menu, make sure that your molecule name is listed and minimize is selected, and execute this menu. Wait while the molecule undergoes a crude minimization.
9. Examine the positions of the H-bonding groups. If some of the peptide groups are not oriented correctly (see figure) change their geometry manually then minimize again. You may have to break and then reform a bond to do this effectively.
10. Repeat for the residue 5-7 crosslink.
11. Repeat for the residue 2-4 crosslink.
12. Examine the pocket formed by vancomycin. Especially look at the positions of the H-bonding groups. Once you are satisfied that this is a reasonable model, make a representation that you think shows the binding pocket most clearly, and save it in a folder with the name vancomycin.psv in your home directory.

Part 3: Verify that you have completed this portion of the assignment