Frequently asked questions (FAQ) and examples

I want to build a model of a chimeric protein based on two known structures. Alternatively, I want to build a multi-domain protein model using templates corresponding only to the individual domains.
I don't want to use one region of a template for construction of my model.
I want to explicitly force certain Pro residues to the cis $\omega$ conformation.
How can I select/remove/add a set of restraints?
I want to change the default optimization or refinement protocol.
I want to build an all hydrogen atom model with water molecules and other non-protein atoms (atoms in the HETATM records in the PDB file).
How do I build a model with water molecules or residues that do not have an entry in the topology and/or parameter files?
How do I define my own residue types, such as D-amino acids, special ligands, and unnatural amino-acids?
How do I define my own patching residue types?
Is it possible to restrain secondary structure in the target sequence?
I want to patch the N-terminal or (C-terminal) residue (e.g., to model acetylation properly), but the model.patch() command does not work.
Is it possible to use templates with the coordinates for ${C}_\alpha$ atoms only?
How do I analyze the output log file?
How do I prevent ``knots'' in the final models?
What is considered to be the minimum length of a sequence motif necessary to derive meaningful constraints from the alignment to use in modeling.. one, two, three, or more?
Does Modeller have a graphical interface (GUI) ?
What do the `Alignment sequence not found in PDB file' or `Number of residues in the alignment and pdb files are different' errors mean?
Can I make a web interface or GUI for Modeller?

I want to build a model of a chimeric protein based on two known structures. Alternatively, I want to build a multi-domain protein model using templates corresponding only to the individual domains.
This can be accomplished using the standard modeling routine. The alignment should be as follows when the chimera is a combination of proteins A and B:
```
proteinA  aaaaaaaaaaaaaaaaaaaaaaaaaaaa----------------------------------
proteinB  ----------------------------bbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbb 
chimera   aaaaaaaaaaaaaaaaaaaaaaaaaaaabbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbb
```
In the PIR format the alignment file is:
```
>P1;proteinA
structureX:proteinA
aaaaaaaaaaaaaaaaaaaaaaaaaaaa----------------------------------*
>P1;proteinB
structureX:proteinB
----------------------------bbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbb*
>P1;chimera
sequence:chimera
aaaaaaaaaaaaaaaaaaaaaaaaaaaabbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbb*
```
If no additional information is available about the relative orientation of the two domains the resulting model will probably have an incorrect relative orientation of the two domains when the overlap between A and B is non-existing or short. To obtain satisfactory relative orientation of modeled domains in such cases, orient the two template structures appropriately before the modeling.

I don't want to use one region of a template for construction of my model.
The easiest way to achieve this is to not align that region of the template with the target sequence. If region 'bbbbbbbb' of the template should not be used as a template for region 'eeeee' of the target sequence the alignment should be like this:
```
template  aaaaaaaaaaaaaaaaaaaaaaaa-----bbbbbbbbcccccccccccccccccccccccccccccc
target    ddddddddddddddddddddddddeeeee--------ffffffffffffffffffffffffffffff
```
The effect of this alignment is that no homology-derived restraints will be produced for region 'eeeee'.

I want to explicitly force certain Pro residues to the cis $\omega$ conformation.

MODELLER should usually be allowed to handle this automatically via the omega dihedral angle restraints, which are calculated by default.

from modeller import *
from modeller.automodel import *
from modeller.scripts import cispeptide

# Redefine the special_restraints routine to force Pro to cis conformation:
# (this routine is empty by default):
class MyModel(automodel):
    def special_restraints(self, aln):
        a = self.atoms
        cispeptide(self.restraints,
                   atom_ids1=(a['O:4'], a['C:4'], a['N:5'], a['CA:5']),
                   atom_ids2=(a['CA:4'], a['C:4'], a['N:5'], a['CA:5']))

# This is as usual:
log.verbose()
env = environ()

a = MyModel(env, alnfile='align1.ali', knowns='templ1', sequence='targ1')
a.make()

How can I select/remove/add a set of restraints?
Restraints can be read from a file by Restraints.append(), calculated by Restraints.make() or Restraints.make_distance(), or added ``manually'' by Restraints.add(). Restraints.pick() picks those restraints for objective function calculation that restrain the selected atoms only. The 'automodel.homcsr()' routine contains examples of selecting atoms when generating restraints by Restraints.make_distance(). There are also commands for adding and unselecting single restraints, Restraints.add() and Restraints.unpick(), respectively. If you do Restraints.condense(), the unselected restraints will be deleted. This is useful for getting rid of the unwanted restraints completely.

I want to change the default optimization or refinement protocol.
See Section 2.2.2.

I want to build an all hydrogen atom model with water molecules and other non-protein atoms (atoms in the HETATM records in the PDB file).

See Sections 2.2.1 and 2.2.5 for some examples.

from modeller import *
from modeller.automodel import *

log.verbose()
env = environ()
env.io.hydrogen = env.io.hetatm = env.io.water = True

a = allhmodel(env, alnfile='align1.ali', knowns='templ1', sequence='targ1')
a.make()

How do I build a model with water molecules or residues that do not have an entry in the topology and/or parameter files?
See Section 2.2.1 for an example.

How do I define my own residue types, such as D-amino acids, special ligands, and unnatural amino-acids?

This is a painful area in all molecular modeling programs. However, CHARMM and X-PLOR provide a reasonably straightforward solution via the residue topology and parameter libraries. MODELLER uses CHARMM topology and parameter library format and also extends the options by allowing for a generic ``BLK'' residue type (Section 5.2.1). This BLK residue type circumvents the need for editing any library files, but it is not always possible to use it. Due to its conformational rigidity, it is also not as accurate as a normal residue type. In order to define a new residue type in the MODELLER libraries, you have to follow the series of steps described below. As an example, we will define the ALA residue without any hydrogen atoms. You can add an entry to the MODELLER topology or parameter file; you can also use your own topology or parameter files. For more information, please see the CHARMM manual.

Define the new residue entry in the residue topology file (RTF), say 'top_heav.lib'.
```
RESI ALA      0.00000
ATOM N    NH1    -0.29792
ATOM CA   CT1     0.09563
ATOM CB   CT3    -0.17115
ATOM C    C       0.69672
ATOM O    O      -0.32328
BOND CB CA    N CA    O C    C CA    C +N
IMPR C CA +N O     CA N C CB
IC -C    N     CA    C         1.3551  126.4900  180.0000  114.4400    1.5390
IC N     CA    C     +N        1.4592  114.4400  180.0000  116.8400    1.3558
IC +N    CA    *C    O         1.3558  116.8400  180.0000  122.5200    1.2297
IC CA    C     +N    +CA       1.5390  116.8400  180.0000  126.7700    1.4613
IC N     C     *CA   CB        1.4592  114.4400  123.2300  111.0900    1.5461
IC N     CA    C     O         1.4300  107.0000    0.0000  122.5200    1.2297
PATC FIRS NTER LAST CTER
```
You can obtain an initial approximation to this entry by defining the new residue type using the residue type editor in QUANTA and then writing it to a file.
The RESI record specifies the CHARMM residue name, which can be up to four characters long and is usually the same as the PDB residue name (exceptions are the potentially charged residues where the different charge states correspond to different CHARMM residue types). The number gives the total residue charge.
The ATOM records specify the IUPAC (i.e., PDB) atom names and the CHARMM atom types for all the atoms in the residue. The number at the end of each ATOM record gives the partial atomic charge.
The BOND records specify all the covalent bonds between the atoms in the residue (e.g., there are bonds CB-CA, N-CA, O-C, etc.). In addition, symbol '+' is used to indicate the bonds to the subsequent residue in the chain (e.g., C - +N). The covalent angles and dihedral angles are calculated automatically from the list of chemical bonds.
The IMPR records specify the improper dihedral angles, generally used to restrain the planarity of various groups (e.g., peptide bonds and sidechain rings). See also below.
The IC (internal coordinate) records are used for constructing the initial Cartesian coordinates of a residue. An entry

$\displaystyle IC \quad a \quad b \quad c \quad d \quad d_{ab} \quad \alpha_{abc} \quad \Theta_{abcd} \quad \alpha_{bcd} \quad d_{cd}$

specifies distances , angles $\alpha$ , and either dihedral angles or improper dihedral angles $\Theta$ between atoms , , and , given by their IUPAC names. The improper dihedral angle is specified when the third atom, , is preceded by a star, '*'. As before, the '-' and '+' prefixes for the atom names select the corresponding atom from the preceding and subsequent residues, respectively. The distances are in angstroms, angles in degrees. The distinction between the dihedral angles and improper dihedral angles is unfortunate since they are the same mathematically, except that by convention when using the equations, the order of the atoms for a dihedral angle is and for an improper dihedral angle it is .
The PATC record specifies the default patching residue type when the current residue type is the first or the last residue in a chain.
You have to make sure that all the CHARMM atom types of the new residue type occur in the MASS records at the beginning of the topology library: Add your entry at the end of the MASS list if necessary. If you added any new CHARMM atom types, you also have to add them to the radii libraries, 'modlib/radii.lib' and 'modlib/radii14.lib'. These libraries list the atomic radii for the different topology models, for the long range and 1-4 non-bonded soft-sphere terms, respectively. The full names of the files that are used during calculation are given by the environment variables $RADII_LIB and $RADII14_LIB.

Optionally, you can add the residue entry to the library of MODELLER topology models, 'modlib/models.lib'. The runtime version of this library is specified by the environment variable $MODELS_LIB. This library specifies which subsets of atoms in the residue are used for each of the possible topologies. Currently, there are 10 topologies selected by Topology.submodel (3 is default):

1	ALLH	all atoms
2	POL	polar hydrogens only
3	HEAV	non-hydrogen atoms only
4	MCCB	non-hydrogen mainchain (N, C, CA, O) and CB atoms
5	MNCH	non-hydrogen mainchain atoms only
6	MCWO	non-hydrogen mainchain atoms without carbonyl O
7	CA	CA atoms only
8	MNSS	non-hydrogen mainchain atoms and disulfide bonds
9	CA3H	reduced model with a small number of sidechain interaction centers
10	CACB	CA and CB atoms only

The Ala entry is:

#
         ALLH POLH HEAV MCCB MNCH MCWO CA   MNSS CA3H CACB
*
RESI ALA
ATOM     NH1  NH1  NH1  NH1  NH1  NH1  #### NH1  #### ####
ATOM     H    HN   #### #### #### #### #### #### #### ####
ATOM     CT1  CT1  CT1  CT1  CT1  CT1  CT1  CT1  CAH  CT1
ATOM     HB   #### #### #### #### #### #### #### CH3E ####
ATOM     CT3  CT3  CT3  CT3  #### #### #### #### #### CT2
ATOM     HA   #### #### #### #### #### #### #### #### ####
ATOM     HA   #### #### #### #### #### #### #### #### ####
ATOM     HA   #### #### #### #### #### #### #### #### ####
ATOM     C    C    C    C    C    C    #### C    #### ####
ATOM     O    O    O    O    O    #### #### O    #### ####

The residue entries in this library are separated by stars. The '####' string indicates a missing atom. The atom names for the present atoms are arbitrary. The order of the atoms must be the same as in the CHARMM residue topology library. If a residue type does not have an entry in this library, all atoms are used for all topologies.

You have to add the new residue type to the residue type library, 'modlib/restyp.lib'. The execution version of this file is specified by the environment variable $RESTYP_LIB. See the comments in the file for further information.
Every residue in the CHARMM topology file has to have an entry in the $RESTYP_LIB library, but not every residue entry in the $RESTYP_LIB library needs an entry in the residue topology file. If you need to edit the $RESTYP_LIB file, it is recommended that you change a copy of it, and provide that file to the environ() constructor.
In general, when you add a new residue type, you also add new chemical bonds, angles, dihedral angles, improper dihedral angles, and non-bonded interactions, new in the sense that a unique combination of CHARMM atoms types is involved whose interaction parameters are not yet specified in the parameter library (see also Section 5.2.1). In such a case, you will get a number of warning and/or error messages when you generate the stereochemical restraints by the Restraints.make() command. These messages can sometimes be ignored because MODELLER will guess the values for the missing parameters from the current Cartesian coordinates of the model. When this is not accurate enough or if the necessary coordinates are undefined you have to specify the parameters explicitly in the parameter library. Search for BOND, ANGL, DIHE, and IMPR sections in the parameters library file and use the existing entries to guess your new entries. Note that you can use dummy atom types 'X' to create general dihedral (i.e., X A A X) and improper dihedral angle (i.e., A X X A) entries, where A stands for any of the real CHARMM atom types. For the dihedral angle cosine terms, the CHARMM convention for the phase is different for 180 ${}^{o}$ from MODELLER's (Eq. A.83). If you use non-bonded Lennard-Jones terms, you also have to add a NONB entry for each new atom type. If you use the default soft-sphere non-bonded restraints, you have already taken care of it by adding the new atom types to the $RADII_LIB and $RADII_LIB libraries.

How do I define my own patching residue types?
This is even messier than defining a new residue type. As an example, we will define the patching residue for establishing a disulfide bond between two CYS residues.
```
PRES DISU         -0.36 ! Patch for disulfides. Patch must be 1-CYS and 2-CYS.
ATOM 1:CB  CT2    -0.10 !
ATOM 1:SG  SM     -0.08 !           2:SG--2:CB--
ATOM 2:SG  SM     -0.08 !          /
ATOM 2:CB  CT2    -0.10 ! -1:CB--1:SG
DELETE ATOM 1:HG
DELETE ATOM 2:HG
BOND 1:SG 2:SG
IC 1:CA  1:CB  1:SG  2:SG      0.0000    0.0000  180.0000    0.0000    0.0000
IC 1:CB  1:SG  2:SG  2:CB      0.0000    0.0000   90.0000    0.0000    0.0000
IC 1:SG  2:SG  2:CB  2:CA      0.0000    0.0000  180.0000    0.0000    0.0000
```
The PRES record specifies the CHARMM patching residue name (up to four characters).
The ATOM records have the same meaning as for the RESI residue types described above. The extension is that the IUPAC atom names (listed first) must be prefixed by the index of the residue that is patched, if the patch affects multiple residues. In this example, there are two CYS residues that are patched, thus the prefixes 1 and 2. When using the model.patch() command, the order of the patched residues specified by residues must correspond to these indices (this is only important when the patch is not symmetric, unlike the 'DISU' patch in this example).
DELETE records specify the atoms to be deleted, the two hydrogens bonded to the two sulfurs in this case.
The BOND and IC (internal coordinate) records are the same as those for the RESI residues, except that the atom names are prefixed with the patched residue indices.

Is it possible to restrain secondary structure in the target sequence?
Yes -- see Section 2.2.10 for an example.

I want to patch the N-terminal or (C-terminal) residue (e.g., to model acetylation properly), but the model.patch() command does not work.
This is probably because the N-terminus is patched by default with the NTER patching residue (corresponding to -NH3 ) and a patched residue must not be patched again. The solution is to turn the default patching off by env.patch_default = False before the model.generate_topology() command is called.

Is it possible to use templates with the coordinates for ${C}_\alpha$ atoms only?
Yes. You do not have to do anything special.

How do I analyze the output log file?
First, check for the error messages by searching for string '_E>''. These messages can only rarely be ignored. Next, check for the warning messages by searching for string '_W>''. These messages can almost always be ignored. If everything is OK so far, the most important part of the log file is the output of the selection.energy() command for each model. This is where the violations of restraints are listed. When there are too many too violated restraints, more optimization or a different alignment is needed. What is too many and too much? It depends on the restraint type and is best learned by doing selection.energy() on an X-ray structure or a good model to get a feel for it. You may also want to look at the output of command alignment.check(), which should be self-explanatory. I usually ignore the other parts of the log file.

How do I prevent ``knots'' in the final models?
The best way to prevent knots is to start with a starting structure that is as close to the desired final model as possible. Other than that, the only solution at this point is to calculate independently many models and hope that in some runs there won't be knots. Knots usually occur when one or more neighboring long insertions (i.e., longer than 15 residues) are modeled from scratch. The reason is that an insertion is build from a randomized distorted structure that is located approximately between the two anchoring regions. Under such conditions, it is easy for the optimizer to ``fall'' into a knot and then not be able to recover from it. Sometimes knots result from an incorrect alignment, especially when more than one template is used. When the alignment is correct, knots are a result of optimization not being good enough. However, making optimization more thorough by increasing the CPU time would not be worth it on the average as knots occur relatively infrequently. The excluded volume restraints are already included in the standard comparative modeling routine.

What is considered to be the minimum length of a sequence motif necessary to derive meaningful constraints from the alignment to use in modeling.. one, two, three, or more?
Usually more than that (dozens if you want just to detect reliable similarity, and even more if you want a real model). It is good to have at least 35-40% sequence identity to build a model. Sometimes even 30% is OK.

Does Modeller have a graphical interface (GUI) ?
No; Modeller is run from the command line, and uses a Python script to direct it. Graphical interfaces to Modeller are commercially available from Accelrys, at http://www.accelrys.com/products/modeler/index.html.

What do the `Alignment sequence not found in PDB file' or `Number of residues in the alignment and pdb files are different' errors mean?
When you give MODELLER an alignment, it also needs to read the structure of the known proteins (templates) from PDB files. In order to correctly match coordinates to the residues specified in the alignment, the sequences in the PDB file and the alignment file must be the same (although obviously you can add gap or chain break characters to your alignment). If they are not, you see this error. (Note that MODELLER takes the PDB sequence from the ATOM and HETATM PDB records, not the SEQRES records.) You should also check the header of your alignment file, to make sure that you are reading the correct chain and residue numbers from your PDB.
To see the sequence that MODELLER reads from the PDB file '1BY8.pdb', use this short script to produce a '1BY8.seq' sequence file:
```
from modeller import *
env = environ()
code = '1BY8'
mdl = model(env, file=code)
aln = alignment(env)
aln.append_model(mdl, align_codes=code)
aln.write(file=code+'.seq')
```

Can I make a web interface or GUI for Modeller?
Certainly, although you should bear in mind that the Modeller license is non-transferable, and permits free usage only for academic purposes.
For web interfaces, users must obtain their own Modeller license key directly from us; your web interface should provide a text box into which users should put their license key, and then use that input to set the KEY_MODELLER9v4 environment variable, as is done by our own MODWEB and MODLOOP interfaces.
For GUIs or other interfaces (e.g. frameworks), users should obtain and license Modeller directly from us, rather than it being bundled with your software.
In all cases, please update the links page in the Modeller wiki, to advertise your software to Modeller users.