Fragment Positioning
General remarks
The option 'p' of the menu 'cnf'
allows to position molecules (fragments) in the active
site of a enzyme or the cavity of a receptor. It may be (mis-)used
to dock larger and flexible structures, but other programs are
better optimized for that purpose. For a basic, though somewhat
outdated, general description see the first section of the
Lego Tutorial.
The algorithm positions a fragment at various places (determined
by the positions entry) in various initial orientations (and,
optionally, in various initial conformations). Every
initial position of the fragment is subjected to a MAB force field
minimization in which a background entry is also present, though
with fixed atomic positions. The lowest-energy results of these
minimizations are kept in a conformation library. Clearly, if one
tries to position larger molecules with this algoritm
(full docking), they may entangle with the environment enty.
This may in unfavorable cases lead to a hang-up of the minimizer!
We use the B-chain of dihydrofolatereductase as cavity and the
diaminopyrimidine fragment of trimethoprim as the fragment to be
positioned. The corresponding files, 4dfr.pdb, and
trimethoprim.mab can be found in the moloc/dat directory.
Preliminary Actions
- Start Moloc
- Read in the structure trimethomprim, '.../g/m',
and call it e.g. 'dapm' (diaminopyrimidine).
- Delete it's benzyl substituent 'dTp/d' to be
left with just diaminopyrimidine.
- Read in from 4dfr.pdb the B-chain and its methotrexate
ligand as two separate entries '.../g/p'.
- Generate an environment entry, a small fraction of the B-chain,
that surrounds the ligand:
- Make a duplicate of the B-chain, '.../:d', and
call it e.g. '4dfr_B_env'.
- Generate an distance set on this entry from the ligand
'4dfr_B_MTX':
- '4dfr_B_env' should be active, '4dfr_B_MTX' visible, the rest
favorably invisible '.../a'.
- In the set menu, 'set', make a set of
'4dfr_B_MTX', 'e' and pick.
- Click 'd' and select 'by distance, entire monomers'.
A 6.3-A distance-set, called 'u2', on the active '4dfr_B_env' is
produced which you can accept by clicking 'accept' on the slider,
and then 'y'.
- Exit and go to the delete menu 'dTp/d'.
- Choose the whole environment entry, '4dfr_B_env',
'e' and pick.
- Subtract the predefined set 'u2', '!'
and select 'u2'. The set now only contains the atoms to be deleted.
- Exit to be left with the immediate environment of '4dfr_B_MTX'.
This environment set is produced in order to accelerate force-field
calculations, by reducing the number of atoms. However, the full
protein is still used to generate the positions at which the
fragment is placed before the minimization takes place.
- Save the environment entry e.g. as .mab file
'.../s/m'.
An example file 4dfr_B_env.mab can be found in the moloc/dat directory.
Generating the Entry of Initial Positions
- Enter the position generator 'cnf/p'. These
menus are handled best by treating point by point (if needed) from
left to right.
- 'k' This allows to use either a single structure
or an already existing conformation library as starting structures.
The second option may be useful in cases where only a single feature
is subjected to variation, but in a whole set of already existing
conformations.
- 'e' Specify the full B-chain, in order to produce
the positions. Before starting the job this point has to be clicked
again in order to specify the reduced environment entry for the
actual calculations.
- 's' To specify the surface atoms the set menu
is again entered. This time we want atoms of the B-chain specified.
Hence, set it active, the ligand visible, the rest invisible.
- set 'u1' as the current set 'c', it should
still contain the full ligand.
- By clicking 'd' and repeating the procedure
described above a set of surface atoms, 'u3', is generated on the
B-chain entry.
- Exit the set menu to find yourself again in the positioning
menu.
- Now, create positions by clicking 'c'. A entry
of positions, by default named 'ps_4dfr_B', is produced. It usually
contains much too many positions.
- The options 'd', 'o', and
'r' serve to reduce the number of positions. They
can be repeatedly aplied. Read the help text (click them with the
Ctrl-key pressed) to find out what they do. Parameters for their
actions can be modified in option 't'.
After clicking 'd' and 'o' there
remain usually some unwanted positions that can be removed with
'r'. (The set menu is entered. Pick the unwanted
positions with 'a'!)
- It is advisable to save the positions entry now,
'./s/j'. The default file name is
ps_4dfr_B.jkl.
A previously generated positions entry can
be read back and be specified as the relevant one for the current
calculation with option 'p'. Thus, one can avoid
repeating the somewhat lengthy procedure of this section.
Generate Library of Positioned Fragments
- The coverage parameter in option 't' determines
in how many initial orientations the fragment(s) will be placed at
each position. However, the number chosen is not equal to the number
of orientations!
- Parameters for the run can be set (modified) in option
'p'. In this separate menu one can also store the
current set of parameters, or read back previously defined ones.
In Moloc's .dat directory some example settings can be found
(extension .Fpar).
- Before starting the job we have to replace the whole protein
by the environment entry. Thus click 'e' again
and alter the environment setting.
- Now, with option 'm', specify the fragment
entry(ies) (dapm) to be positioned.
- 'i' By choosing interactive mode you can watch
the program at work. It will just write a conformation (position)
library dapm.mcl which is also kept in memory.
- 'b' For batch mode the program writes several
files: dapm.s.mcl, dapm.Gntr, dapm.Fpar,
and dapm.b.cif (the background entry) which contain all
the information needed to run the job. Then the job is started.
It produces the same conformation library dapm.mcl.
- Under Windows the job has to be explicitly started by issuing
the command 'Mlbch -f -c dapm', where the
program name has to be proceeded by the path of the Moloc bin
directory!
Generate a Further Library
The preparations of the previous sections can be reused to produce
further libraries of positioned structures. We demonstrate this with
the example of the entire trimethoprim structure.
- Start Moloc.
- Read in the background structure dapm.b.cif,
'.../g/i'.
- Read in the positions entry ps_4dfr_B.jkl,
'.../g/j'.
- Read in the structure of trimethoprim trimethoprim.mab,
'.../g/m', and rename it, e.g. to 'trmtpr'.
- Enter the position menu 'cnf/p'.
- 'e' select the environment '4dfr_B_env'.
- 'p' select the positions 'ps_4dfr_B'.
- 'm' select the structure to be positioned
'trmtpr'.
- 'i,b' run the job to obtain the library
trmtpr.mcl.
- Watching the execution of this job will exemplify the dificulties
that may arise when one tries to position (dock) a large structure
with this direct algorithm! Nevertheless, the resulting (two)
low-energy structures are reasonably placed in the active site,
showing the characteristic salt bridge between the diaminopyrimidine
group and the Asp27 residue of dihydrofolate reductase.
Analyze Positions
The generated positions for a conformation library and can be
analyzed completely analogous to normal conformations (see section
'Analyze conformations' of the
Conformation Analysis Tutorial).
A few trivial tips:
- Of course, positioned fragments only make sense in context
with the background entry.
- For further treatment, the extraction option
'cnf/m/e' may be needed in order to have the
various positions of the fragment in the form of single entries.
- Do not use the superposition option 'cnf/m/r'
with the superposition parameter set to 'move'! ;-)