Building a Small Molecule (Peptide)
Although small molecules can be built from scratch, a much more
frequent case is that an existing structure is modified to anticipate
possible chemical modifications. We will start from methotrexate
and rebuild it to arrive at trimethoprim, a alternative dihydrofolate
reductase inhibitor.
- Startup Moloc with my_project
by issuing the shell command 'mx my_project.ils'. This will
load the protein DHFR and its inhibitor methotrexate into Moloc.
- Produce a copy of methotrexate '.../+/c' and pick the entry.
You will be requested to give the copy a name (e.g. 'tmpr').
- Return to the main menu (2 times 'x').
- Click 'dTp' (topology changes) to enter the
builder menu.
Delete Redundant Atoms:
- Click 'd' (delete set of atoms). This puts you into
the set menu to specify the set of atoms to be deleted.
- Specify all atoms except the diaminopyrimidine. This can be done
by clicking 'l' (loops) and picking the three
atoms C4A, N5, and C8A in this order. (atom labels in './e/l').
All atoms to be removed are now in the set (marked by rings).
- Exit from the set menu to have the atoms of the set deleted.
You are left with diaminopyrimidine.
Add New Atoms and Fragments:
- Click 'a' (add and delete atoms) to enter
a menu in which atoms can be added, removed or changed. The header
'-C' indicates that picking an atom ads a single-bonded carbon atom.
- Pick atom C4A to add C9.
- Exit to return to the builder menu (dTp).
- Click 's' (add selected substituents) and select
'phenyl' as substituent to be added (new menu).
- Pick atom C9 to attach a phenyl ring.
- Exit to return to the builder menu (dTp).
- Click 'a' (add and delete atoms) again.
- Click 'O' to select oxygen as the atom type
to be attached. The header changes to '-O'.
- Pick atoms C12, C13, and C14 to add the oxygen atoms O16, O17, and O18
for the three methoxy groups.
- Change atom type back to 'C' and add the three methyl groups.
- Exit to return to the builder menu (dTp).
Adjust Initial Conformation Manually:
- Click 'f' (forge structures) to adjust the
conformation which in this simple case can be done by bond rotation.
- Click 't' (drive torsions) to adjust dihedral
angles of the methoxy group at C12 (to have C19 point away from O17).
- Pick bond C12-O16 and modify the angle by pressing the shift
key and moving the mouse sideways with pressed LEFT-hand button.
- Make the protein visible './a'.
- Position the trimethoxy-phenyl group roughly into the cavity
by bond rotations about C4A-C9 and C9-C10.
- Exit to return to the builder menu (dTp).
Check Silent Hydrogens:
- Set the newly built trimethoprim active './a' and check the
'silent hydrogen count' by clicking
'h' (check modify silent H's). The number at each
atom must correspond to the number of hydrogens attached to that atom.
These counts can be increase or decreased by picking the atoms with
LEFT-hand or MIDDLE mouse button, respectively.
Correct H-count's are mandatory to obtain a meaningful
force field treatment!
- Exit the H-count menu to return to the builder menu (dTp).
Optimize the Conformation within the Fixed Protein:
- Now set the protein also in the active state './a', and take
that no further entry is active. (You may have methotrexate visible
for comparison purposes!)
- Click 'o' (optimize active structures) to enter
the force field optimizer.
- Click 's' (define stationary (fixed) atoms) which
puts you into the set menu to define the atoms that will not change
coordinates during the optimization, although they will take part in
the force field calculation.
- Select the whole protein ('e' and pick it).
- Exit to return to the optimizer menu.
- Start the optimizer with 'o' (optimize). After
an initial period used to set up all the force field parameters,
the inhibitor stars moving to adjust to the forces experienced by
the surrounding fixed protein. Minimization can be stopped by pressing
the escape key. The optimizer menu gives access to geometry
and forge menus to allow for checks and
possible adjustment of conformation without having to repeat the
preparatory phase.
- Click 'e' (energy examination) to check energy
terms in color coded displays, e.g.:
- h: H-bonds
- r: repulsive v.d.Waals
For these non-bonding terms the switch {*: all, 1: intra, 2: inter)
allows to display either all interactions, or just the ones within
single entries, or only the ones between different entries,
respectively.
Save the New Structure together with the Protein onto a .pdb File
You are strongly discouraged to store structures on .pdb-type files, just
to save your work for later continuation, because impotant information on
non-protein structures cannot be stored, most importantly, the atomic
H-counts which determine the molecular topology. However, there are programs
which require this file type.
The format of a .pdb file is somewhat tricky. Moloc will write a correct
file, if the entries are properly prepared. This requires the following
steps:
- Shuffle everything to be stored in the .pdb file into a single Moloc
entry with the following steps:
- Remove monomer information of entry 'tmpr' (if it exists) by choosing
'.../e' (change entry settings), selecting 'tmpr' and
clicking 'o' (remove monomer information).
- Go to 'dTp/l' (add local entries) and pick first
'tmpr' then the protein.
- The program asks for a monomer code (enter e.g. 'TMP') a chain
identifier (chain 'A' is meaningful), a sequence number (e.g. '1'
for the first heterogen), and a insertion code (omit). These are all
data fields provided in the .pdb format which you need or may want to
specify.
- Now the structure has been added to the protein.
- Upon storing this entry to disk in .pdb format the relevant
information will be properly written.
In case the ligand is covalently bound, the procedure has to be
analogous, i.e. the ligand has to be built beforehand as a separate
entry, than to be added to the protein entry as a new heterogen. Only
then can the covalent connection be made.
The (not working!) alternative would be to build the complex by
extending the covalently bound residue with the ligand atoms. Although
this leads to the same topology, the monomer specification is different
and not compatible with Brookhaven rules. This, because upon extending a
residue Moloc changes the whole residue to type 'UNK' (unknown but still
in the chain and, thus, not a heterogen). Removing atoms from a residue
is, of course, allowed. It simply yields a incomplete residue.