Creating a molecule requires the following steps:

1. Specifiying all the atoms and bonds
2. Modifying the potentials, e.g. defining for the program what type of C atom a particular C is (e.g. amide vs ester vs aromatic) This is necessary so that, when the program comes to do any calculations, it treats all of the atoms in a realistic way.
3. Modifying the charges on particular atoms
4. Putting 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: Starting up Biopolymer or Builder

1. In Insight, click on the Biosym icon-Builder.
   Note the second row of menu options that appears. Briefly browse through each menu to see what options there are. Use the command guide and index to help identify certain options. At this stage don't actually try using the commands - we'll do that later. Now choose Biopolymer and browse again.

Part 2: Building Some Short Regular Peptides

1. In Biopolymer, choose Residue-Append.
   Specify a molecule name and click on the Alpha_R_Helix motif. This will allow us to build a right-handed alpha helix. Click in the residue box, then click in sequence on several residue types; about 15 will give ~4 turns of helix. Turn the helix around and look at its properties. In particular, examine the groups at the termini - what sort of functional groups are they?
2. Measure-HBond
   Check out the positions and orientations of H-bonds in the helix.
3. Delete this helix. Now try building a new helix which is amphipathic.
4. Delete your amphipathic helix. Now we will build a beta hairpin.
   Residue-Append: Build a 15 residue beta strand
   Modify-Geometry: Try adjusting the conformations of the central residues (7 and 8) so that the overall conformation is a beta hairpin. This may take some time. It will probably be easier if you turn off the sidechain display. Pay particular attention to the relative positions of H-bond donors and acceptors.
   ---OR--- Transform-Torsion: Define a torsion angle or angles, then use the boxes in the lower left corner to rotate about the torsion angle:
   • 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.
   • 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).
   • The fourth atom selected will be the atom that moves.
   • You can select a fifth atom to define a second adjacent torsion angle (i.e., an angle with atoms #2, 3, 4, and 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 Next Torsion.
5. Now display the sidechains again. Choose a sidechain with an OH group. We will now methylate this group.
   Modify-Bond
   Create, fragment window, single.
   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 5 mins.
   
6. delete *

Part 3: 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 and by so doing to become familiar with the methods of modifying atoms, bonds, and potentials, and doing a very rough energy minimization.

Before you start, take a good hard look at 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. Forcefield-Select
   Choose Clear_Potentials, Clear_Charges, cvff.frc
   This selects the CVFF forcefield. I have chosen this because the Amber forcefield cannot account for the chlorine atoms. It is good to do this at the beginning to avoid confusion later.
2. Residue-Append
   Molecule name - Vanco
   Beta Strand
   Construct a linear heptapeptide with the sequence: Leu-Tyr-Asn-Gly-Gly-Tyr-Gly
   We will construct vancomycin by modifying this sequence
3. Modify-Hydrogens to put the correct functional groups at the termini
   lone pairs off
   Capping mode- charged
   Observe the changes in 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

   Modify-Bond: add a methyl group to the N-terminus
   Molecule-Label: Label property - potential, execute
   Check that the potential of each atom is correct according to the list of CVFF potentials in the manual If the potential of any atom needs to be changed go to Atom-Potential and make the appropriate assignment
   Molecule-Label: Clear
   Molecule-Label: Label property - formal charge
   Check the formal charges. The N-terminus should be +1. If not, modify using atom-charge.

   Residue 2: A derivative of Tyr

   Add the OH group at the beta position (try and do it with the correct stereochemistry). Under modify-bond you will have to chose the functional group fragment library. Replace one of the H atoms on the aromatic ring by a chlorine using Atom-Replace. If you want to make the Cl easier to see, try Modify-Element to change its color. Again check the potentials and formal charges for this residue.

   Residue 3: Asn

   There should not be any changes necessary. Check potentials and formal charges.

   Residues 4-7

   Continue for residues 4-7. Note stereochemistry when you add sidechains to residues 4, 5, and 7. Also, for residue 4 just put an OH group on the para position, since we are making the aglycone, and do not put any substituents in the meta positions since we will use the oxygens from residues 2 and 6.

5. Modify the sterochemistry of all backbone alpha carbons and the chiral sidechain carbons on residues 2 and 6 as necessary. To modify steroechemistry go to Modify-Invert. A atom and B atom are the two that you want to be swapped. Chiral atom is the one to which they are both bonded and about which the stereochemistry will be switched.
6. Forcefield-Potentials
   Potential action - fix
   Partial charge action - fix
   Formal charge action - fix
   Check for messages in the UNIX window from which you opened Insight. If there are problems, try fixing again but this time choosing the options to print the various results. This long list of results will hopefully contain some clues to which atoms are problematic, then you might have to make changes to the charges or potentials of these atoms. You should try to make all necessary corrections before you move on. However, note that we have encountered some situations when the program
   • consistently assigns incorrect potentials
   • consistently cannot get the formal charges and partial charges to balance
   So far we have not found any way around this - so don't waste too much time worrying about it at this stage.
7. We will now try to form the residue 4-6 cross-link. First we will manually try to adjust the backbone so that the groups to be cross-linked are reasonably close in space. Modify-Geometry, choose dihedral then pick the atoms that define the dihedral angle you want to modify. A good one to start with might be the HA-CA-CO-C angle of residue 4. You need to type in the dihedral angle manually Keep making 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 we will now energy minimize).

   Before forming the new bond, rotate the aromatic ring so that the chlorine is pointing in roughly the right direction.

   Now form a bond between the desired atoms and then check and fix all the potentials and charges, ready for the energy minimization. To make the new bond, it is probably easiest to just delete the 2 H atoms the go to Modify-Bond-create and specify the O and C atoms.

   To energy minimize, go to Builder-Optimize-Optimize Choose 200-500 iterations, derivative 0.1, and wait a few minutes while the molecule undergoes a crude minimization.

   Look at 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.

8. Repeat step 7 for the residue 5-7 crosslink
9. Repeat step 7 for the residue 2-4 crosslink
10. Take a good look at the binding 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 "LastnameA1.psv" (e.g. StoneA1.psv) Then place that file in the directory /ruser/instruct1/stone/C687/assignments