Protein X-Ray Model Building
The following two files for the 46-residue plant-seed protein crambin,
derived from the x-ray structure (Brookhaven code 1crn), can be found
in Moloc's data directory moloc/dat: crbn.pdb (structure),
crbn.mpf (calculated electron density map). In order to
save disk space the map resolution is rather low.
Prepare Incomplete Structure
- Start Moloc by typing 'Moloc -c crbn.pdb'
- Enter the 'dTp' (topology changes) menu.
- Select 'd' (delete set of atoms) to be thrown
into the set menu.
- Choose 'm' (monomers) ant pick any atom of each
of the three residues 'T28', 'Y29', and 'T30'.
- Upon leaving the set menu the three amino acids will be removed
from the structure.
- This incomplete structure will be the starting point of our
modeling example.
Rebuild Deleted Loop
- Read the map crbn.mpf 'map/g'
and generate a 2.0 contour 'map/k'
(see Maps).
- Enter the menu
'xry' (utility for x-ray model building).
- Choose 'k' (generate Calf structure)
and pick the incomplete Crambin structure to obtain a
new Moloc entry of type 'c' (Calf structure).
The all atom structure is now invisible, but still in Moloc's
memory.
- Enter the C-alpha builder by choosing
'c' (Calf building).
- Choose 'a' (build sequence, add, delete
residues) to obtain a new menu, displaying mainly
the list of natural amino acids in one letter code.
- Since the map suggests a alpha-helix conformation for the
residues following 'A27', keep the conformation suggested by
the menu header (which may, however, be changed with option
's' (secondary structure)) and pick
residue 'A27' to tell the program where to continue
building. It will confirm by 'C-terminal chosen'.
- Now add the three missing residues by clicking 'T',
'Y', and 'T'. Moloc adds the three residues in the
(pre-)specified conformation.
- Exit from the menu.
- Close the missing 'bond' between 'T30' and 'G31' by
choosing 'b' (make, break bonds) and
picking the two residues.
- Exit from the C-alpha builder.
The Calf structure is now complete, but the conformation may
not optimally fit into the electron density (although we have
arranged for a reasonable position of the new residues).
There are a couple of tools in Moloc to modify peptide
conformations. They can be found in the 'forge' and 'Calf
building' menus, but have been collected in a separate menu
'xry/m' (modifying (forging) Calf structure)
in order to have the frequently used options ready at hand.
Modify C-alpha Structure
In Moloc, protein structures can be modified interactively at
the C-alpha level. By this token, the view is kept simple,
while all degrees of freedom can still be varied.
- Go to 'xry/m' (modifying (forging) Calf structure)
- Option ',' (peptide representation) provides
the possibility to display a peptide representation of a Calf
structure by picking it. This representation indicates the orientation
of peptide plains and 'CA'-'CB' exit vectors. Picking the structure
with the middle mouse button removes this display.
- The simplest type of modification is to move around the amino acids
- Choose 'a' (move single residue).
- Pick a residue (e.g. 'Y29').
- Move it around by pressing the SHIFT-key and operating the
mouse with the middle button pressed for x,y-movements, or middle
and left-hand button pressed for z-movment.
- Bond length's and, if displayed, peptide-links and 'CA'-'CB' exit
vectors are monitored.
- Modify orientation of peptide links
- Choose 'l' (turn peptide links)
- Pick a bond, it will be highlighted.
- Press the SHIFT-key and move the mouse sideways while pressing
its left-hand button.
- Modify side chain 'chi'-angles
- Choose 'h' (drive chi angles)
- Pick a residue, e.g. 'Y29'. This will cause the display
of its side chain and backbone atoms.
- Keeping the 'Ctrl'-key pressed and hitting the middle mouse
button will toggle through Ponder-Richards rotamers (see also
help of the identical option in the forge menu).
- In order to update the original structure proceed as follows:
- Choose 'a' (generate full peptide)
and pick the Calf structure. This generates an all-atom structure
from the completed Calf. This will be the result-crbn.
- Choose 'u' (update full structure from Calf or from
all-atom fragment), pick the result-crbn structure first,
then the original (incomplete) crbn-structure. The coordinates of
all atoms of the original structure are transferred to the result.
The side chain of the added residues are added with standard chi
angles and, thus, will typically not fit well into the electron
density. This is particularly evident for 'Y29' (in case you have
not altered the chi-angles).
Search Database for loop conformations
The residues of the loop must already be present in the structure,
albeit in an arbitrary conformation.
- Go to 'xry/m' (modifying (forging) Calf structure)
- Choose 's' (search in database)
to enter a new menu.
- Pick 'b' (build database) to build up the
database to be searched. A new menu to read protein structures
appears.
- Select 'c' (mca-file (Calf structures)),
specify '.../moloc/dat/*', and select 'pdb.mca'. The program
will read the 30 entries found in that file and add them to the
database.
- You may want to use your own reference structures, which you
can read in here from any file containing protein information.
A multi-entry .mca format is well suited for that purpose.
- Exit from the database builder menu.
- Choose 's' (define search set) to be thrown
into the set menu.
- Specify a contiguous stretch around the three inserted residues:
'I25' to 'I33' (9 residues). Exit from the set menu.
- When choosing 'w' (set residue weights) a set
of 9 sliders appears, with which you can set a weight of each
specified residue, which determines how important its actual
conformation should be in the search.
- Set the weights of the three freshly inserted residues to zero
and accept the slider values.
- initiate the search with 'd' (do the search).
You will then be automatically be dropped into the examination menu,
'e' (examine hits).
- Select the conformation that pleases you most
(e.g. 3: DLARSYGIP; 0.75(0.37): 5p21). The help text gives the
sequence, the rmsd of the superimposition, and the name of the
database structure).
- Exit three times to the 'xry' menu.
Real Space Refinement of Newly Built Stretch
Moloc offers the possibility to adjust conformations automatically
by running a force field minimization in which the electron
density provides an additional force term.
- In the 'xry' menu choose 'r' (real space refinement of
protein structure) and pick the result-crbn structure.
This yields a new menu.
- Choose the relevant map 'm' (choose map)
(we have only one).
- Select 's' (choose set of atoms) to get into
the 'set' menu and specify the atoms of the three added residues
'T28', 'Y29', and 'T30' (e.g. with option 'm').
Then exit from the set menu.
- Set the environment mode,
'e' (set environment modus), to 'fixed environment'.
- To start the refinement hit 'r' (do refinement).
This generates a copy of the part of the structure needed for the
force field calculation and puts you into the optimizer menu.
- Hit 'o' to start optimization.
- The atoms are now pulled into the density, most evidently for
the side chain of 'Y29'.
- In cases where the refinement finds a local minimum that does
not optimally fit the density (e.g. orientation of the peptide
bond between 'T30' and 'G31', or for the side chain of 'T28'), one
may interrupt the optimization, make the necessary adjustments in
the forge menu 'f', and continue the optimization.
- When finished, exit from the optimizer.
- Select 'u' (update structure). This causes
the program to transfer the atomic coordinates from the copy to the
starting structure (result-crbn). The copy is then deleted.
Structure Update after Building
Structure building in Moloc makes use of its reduced C-alpha
representation. However, at the end, or for refinment a full
(all atom) structure is needed. From the C-alpha structure
coordinates of non-C-alpha atoms are reconstructed by
algorithm. Even if the C-alpha structure remained unchanged,
the result may slightly deviate from the start structure.
In order to keep the original coordinates for unchanged
residues, Moloc provides update procedures for various
cases:
- The C-alpha structure is of identical sequence but with
(some) changes of coorinates or dihedral angles (phi, psi,
chi). In this
case the original structure is unchanged for all residues
without a change. For the other residues, new atomic
coordinates are reconstructed from the corresponding data
of the C-alpha structure. (menus pca/u, xry/u)
- New residues have been added to the C-alpha structure.
In this case a fully reconstructed all-atom structure is
first produced (menus pca/a, xry/a). This will be the final structure after it
is updated from the original one, which is now considered
a fragment (see next point).
- Update from an all-atom fragment. The structure to be
updated must have a corresponding residue for each one
occurring in the fragment. When updating, the coordinates
of the fragment atoms are transferred to their
corresponding counterparts in the new structure.
Heterogens are not considered! (menux pca/u, xry/u)
Do not hesitate to save your work from time to time.
No program is totally crash-save!
Further X-ray Tools
Further available options are only listed here without detailed
demonstration:
- 'w' (add water molecules or isolated atoms).
In a separate menu isolated atoms can be added to a structure.
- 'p' (scan through residues),
'i' (scan through isolated atoms).
This option allows to scan through residues or isolated atoms.
Temperature factor and (optionally) real space correlation
coefficient and gradient with respect to the electron density
map are listed for quality judgment.
- 'q' (positioned comments). To monitor
peculiarities, e.g. in the electron density map, comments can
be written at required positions.
Modify a Ligand
A ligand is modified by the standard building tools in Moloc.
However, when the structure originates from a .pdb File it is
specified as monomer of a polymer and carries corresponding
information which may interfere with the building process.
How to deal with this is subject of a
separate topic.
Data Generation for Third-Party Software
While force-field parameters or geometric reference values
are commonly available in other programs for proteins, nucleic
acids, and a few common biomolecules, it is not always
straightforward to obtain such values for general organic molecules
as usually encountered e.g. in medicinal chemistry. Since Moloc's
force-field MAB is based on a generic parametrization it is able
to provide parameters for these cases. In order to facilitate
the combined use of third-party software, files containing the
necessary data for running the following programs can be
generated:
- XPLOR, X-ray refinement program.
Topology and parameter files.
- TNT, cristalographics package.
File defining geometrical constraints.
- REFMAC, cristalographics package.
File defining geometrical constraints.
- AMBER, molecular dynamics package.
Three AMBER-specific files (.lib, .dat, and .so).
- GROMOS, molecular dynamics package.
Topology file.
To generate such files, choose option
'dyn/g' (generate geometry-, topology-, and
parameter files).