The scripts and input files that accompany this demo can be found in the demos/public directory of the Rosetta weekly releases.

KEYWORDS: DESIGN NONCANONICALS

In the first part of this tutorial we show how to make a point mutation in a PDB file (1l2y) including two non-canonical amino acids (NVL and HLU). In the second part of this tutorial we show how to incorporate novel non-canonical amino acid side-chains into rosetta for design.

Updated in June 2016 during documentation XRW to enable automated testing (parisah@uw.edu).

# Designing with NCAA

Below are two command-lines for making point-mutations including the possibility of incorporating non-canonical amino acids. In this procedure the backbone is kept fixed and the Trp6 residue is allowed to be altered. Note: this example makes use of PyRosetta scripts, and requires that PyRosetta is installed.

• Trp6 → Canonical amino acids plus NVL and HLU
$>$ROSETTA3/bin/fixbb.default.linuxgccrelease -s starting_files/1l2y.pdb -use_input_sc -nstruct 1 -ex1 -ex2 -overwrite -minimize_sidechains -resfile rosetta_inputs/ncaa_resfile_pluscanon

where $ROSETTA3=path-to-Rosetta/main/source • Trp6 → NVL or HLU $> $ROSETTA3/bin/fixbb.default.linuxgccrelease -s starting_files/1l2y.pdb -use_input_sc -nstruct 1 -ex1 -ex2 -overwrite -minimize_sidechains -resfile rosetta_inputs/ncaa_resfile # Adding new sidechains to Rosetta ## Generating an initial sidechain model I usually do this in PyMOL but any molecular editor will work. PyMOL has rudimentary but useful editing functionality. The goal is to get the structure close to the final structure. Don't stress too much because the QM minimization will clean up most things. Make sure you double check chirality. To make sure the geometry for the atoms near the ends of the molecule is correct you will need to put capping groups on the molecule. I would recommend an acetyl (ACE) on the nitro-terminus of the betapeptide(BP) and a n-methyl (NME) on the carboxy terminus. This structure (ACE-X-NME) is usally called a dipeptide despite the fact that it only has one complete residue. Pro-tips: • Within PyMOL go to the "Mouse" drop down and select "3 button editing". • Within PyMOL type "help edit_keys" in the command box will bring up the instructions for the editor. • Within PyMOL type "set valence, 1" in the command box to show single, double, triple bonds. • Be consistent with atom order. It make future steps easier. I like to put all the atoms for the capping groups first, followed by backbone heavy atoms, side chain heavy atoms, and then then hydrogens. See the example pdb files in the folder stage_01_initial_structures and shown below: <<<<< ornithine.pdb start >>>>> ATOM 1 C ACE 1 0.984 0.045 -0.578 1.00 0.00 C ATOM 2 O ACE 1 1.815 -0.721 -1.083 1.00 0.00 O ATOM 3 CH3 ACE 1 -0.445 -0.396 -0.349 1.00 0.00 C ATOM 4 1HH3 ACE 1 -0.468 -1.251 0.313 1.00 0.00 H ATOM 5 2HH3 ACE 1 -1.013 0.407 0.098 1.00 0.00 H ATOM 6 3HH3 ACE 1 -0.904 -0.668 -1.288 1.00 0.00 H ATOM 26 N NME 3 4.937 2.266 0.684 1.00 0.00 N ATOM 27 CH3 NME 3 5.341 3.324 1.592 1.00 0.00 C ATOM 28 H NME 3 5.534 1.511 0.316 1.00 0.00 H ATOM 29 1HH3 NME 3 4.689 4.177 1.478 1.00 0.00 H ATOM 30 2HH3 NME 3 5.286 2.977 2.614 1.00 0.00 H ATOM 31 3HH3 NME 3 6.355 3.627 1.379 1.00 0.00 H ATOM 7 N ALA 2 1.392 1.405 -0.195 1.00 0.00 N ATOM 8 CA ALA 2 2.806 1.518 -0.539 1.00 0.00 C ATOM 9 C ALA 2 3.512 2.478 0.390 1.00 0.00 C ATOM 10 O ALA 2 2.929 3.435 0.912 1.00 0.00 O ATOM 11 CB ALA 2 2.895 1.945 -2.014 1.00 0.00 C ATOM 12 C01 ALA 2 2.214 0.926 -2.946 1.00 0.00 C ATOM 13 N01 ALA 2 1.680 0.434 -5.295 1.00 0.00 N ATOM 14 C02 ALA 2 2.334 1.404 -4.405 1.00 0.00 C ATOM 15 H ALA 2 0.770 2.167 0.251 1.00 0.00 H ATOM 16 HA ALA 2 3.283 0.529 -0.413 1.00 0.00 H ATOM 17 2HB ALA 2 2.409 2.923 -2.195 1.00 0.00 H ATOM 18 3HB ALA 2 3.943 2.035 -2.355 1.00 0.00 H ATOM 19 H01 ALA 2 2.698 -0.045 -2.840 1.00 0.00 H ATOM 20 H02 ALA 2 1.461 -0.405 -4.777 1.00 0.00 H ATOM 21 H03 ALA 2 1.161 0.837 -2.679 1.00 0.00 H ATOM 22 H04 ALA 2 2.300 0.206 -6.059 1.00 0.00 H ATOM 23 H05 ALA 2 3.387 1.492 -4.674 1.00 0.00 H ATOM 24 H06 ALA 2 1.850 2.375 -4.509 1.00 0.00 H ATOM 25 H07 ALA 2 0.828 0.835 -5.661 1.00 0.00 H END <<<<< ornithine.pdb end >>>>>  ## Making GAUSSIAN input We need to minimize the initial structure we made in PyMOL to get a good set of ideal bond lengths and angles. To do this we will take the coordinates from the pdb file and put them into a gaussian input file. Like the one shown below and in stage_02_gaussian_input. A discussion of the complete gaussian input structure is beyond the scope of this document. Documentation for gaussian can be found at http://www.gaussian.com/. In short the input is as follows: • line 1 sets the path for the checkpoint file • line 2 describes the level of theory, options, and convergence criteria. You may need to change the basis set to something smaller if you are using stuff bellow the 4th line of the periodic table. • lines 3-5 are comments • line 6 is the charge and multiplicity (this is usually 0 1 but ornithine is charged) • line 7-37 are the elemental type and xyz coordinates of the atoms from the pdb file in the same order as the pdb file • line 38 is blank • line 39-40 is the modredundant input that says that we want to keep the torsion formed by atoms 1 13 14 and 15 fixed at 150.00 degrees • line 41 is blank. Running Gaussian is simple but the minimizations take a long time (a few hours per structure). The command bellow will run gaussian on the input file in the stage_02 folder and put the output in the stage_03 folder. $ g03 stage_02_gaussian_input/ornithine.com stage_03_gaussian_output/ornithine.log


There is a new version of Gaussian called Gaussian09. If you are using this version, the commands and options might be slightly different. The input below will probably work with Gaussian09 but was written for and tested with Gaussian03.

The Gaussian output is very verbose. It's not included in this document but it is in the demo folder.

<<<<< ornithine.com start >>>>>
%Chk=stage02_ace_nme_res_ordered_pdbs/ornithine.chk
# HF/6-31G(d) Opt=ModRedundant SCF=Tight Test

scan rotamers

1  1
C        0.984   0.045  -0.578
O        1.815  -0.721  -1.083
C       -0.445  -0.396  -0.349
H       -0.468  -1.251   0.313
H       -1.013   0.407   0.098
H       -0.904  -0.668  -1.288
N        4.937   2.266   0.684
C        5.341   3.324   1.592
H        5.534   1.511   0.316
H        4.689   4.177   1.478
H        5.286   2.977   2.614
H        6.355   3.627   1.379
N        1.392   1.405  -0.195
C        2.806   1.518  -0.539
C        3.512   2.478   0.390
O        2.929   3.435   0.912
C        2.895   1.945  -2.014
C        2.214   0.926  -2.946
N        1.680   0.434  -5.295
C        2.334   1.404  -4.405
H        0.770   2.167   0.251
H        3.283   0.529  -0.413
H        2.409   2.923  -2.195
H        3.943   2.035  -2.355
H        2.698  -0.045  -2.840
H        1.461  -0.405  -4.777
H        1.161   0.837  -2.679
H        2.300   0.206  -6.059
H        3.387   1.492  -4.674
H        1.850   2.375  -4.509
H        0.828   0.835  -5.661

1 13 14 15  -150.00 F
13 14 15 7  150.00 F
<<<<< ornithine.com end >>>>>


## Converting GAUSSIAN output to molfile

The program bable we built in the first step can convert the gaussian output to a molfile.

Bable says it can handle output form Gaussian09. I am not sure when it was added, the current version is 2.3.0 and the modified version I have included is based on version 2.2.0. The changes to 2.2.0 were not extensive and could probably be ported to 2.3.0.

The command bellow will convert the gaussian output to molfile format for the peptide and peptoid examples:

$openbabel/install/bin/babel -i g03 stage_03_gaussian_output/ornithine.log -o mol stage_04_molfile/ornithine.mol$ openbabel/install/bin/babel -i g03 stage_03_gaussian_output/amino2.log -o mol stage_04_molfile/amino2.mol

<<<<< ornithine.mol start >>>>>
OpenBabel01101117083D

31 30  0  0  0  0  0  0  0  0999 V2000
0.4866    2.2296   -0.2354 C   0  0  0  0  0
1.2081    1.9315   -1.1549 O   0  0  0  0  0
0.4369    3.6268    0.3352 C   0  0  0  0  0
-0.3187    4.1933   -0.1996 H   0  0  0  0  0
0.1852    3.6362    1.3888 H   0  0  0  0  0
1.3920    4.1075    0.1799 H   0  0  0  0  0
-2.6920   -1.1572   -0.6650 N   0  0  0  0  0
-4.0550   -1.5847   -0.3922 C   0  0  0  0  0
-2.3500   -1.2554   -1.5934 H   0  0  0  0  0
-4.1238   -1.9440    0.6231 H   0  0  0  0  0
-4.7601   -0.7727   -0.5220 H   0  0  0  0  0
-4.3066   -2.3874   -1.0709 H   0  0  0  0  0
-0.3415    1.3293    0.3431 N   0  0  0  0  0
-0.6023    0.0286   -0.2270 C   0  0  0  0  0
-2.0235   -0.3660    0.1877 C   0  0  0  0  0
-2.4547   -0.0133    1.2526 O   0  0  0  0  0
0.3558   -1.0672    0.2829 C   0  0  0  0  0
1.8184   -0.8036   -0.0848 C   0  0  0  0  0
4.1559   -1.6146    0.0004 N   0  0  0  0  0
2.7190   -1.9434    0.3678 C   0  0  0  0  0
-0.9580    1.6065    1.0765 H   0  0  0  0  0
-0.5197    0.1005   -1.3044 H   0  0  0  0  0
0.2486   -1.1369    1.3608 H   0  0  0  0  0
0.0400   -2.0197   -0.1352 H   0  0  0  0  0
1.9103   -0.6656   -1.1566 H   0  0  0  0  0
4.2542   -1.4797   -0.9970 H   0  0  0  0  0
2.1487    0.1188    0.3755 H   0  0  0  0  0
4.7999   -2.3436    0.2758 H   0  0  0  0  0
2.4931   -2.8811   -0.1191 H   0  0  0  0  0
2.7087   -2.0883    1.4385 H   0  0  0  0  0
4.4568   -0.7571    0.4437 H   0  0  0  0  0
2  1  2  0  0  0
1  3  1  0  0  0
1 13  1  0  0  0
4  3  1  0  0  0
6  3  1  0  0  0
3  5  1  0  0  0
9  7  1  0  0  0
7  8  1  0  0  0
7 15  1  0  0  0
12  8  1  0  0  0
11  8  1  0  0  0
8 10  1  0  0  0
14 13  1  0  0  0
13 21  1  0  0  0
22 14  1  0  0  0
14 15  1  0  0  0
14 17  1  0  0  0
15 16  2  0  0  0
24 17  1  0  0  0
18 17  1  0  0  0
17 23  1  0  0  0
25 18  1  0  0  0
18 20  1  0  0  0
18 27  1  0  0  0
26 19  1  0  0  0
19 28  1  0  0  0
19 20  1  0  0  0
19 31  1  0  0  0
29 20  1  0  0  0
20 30  1  0  0  0
M  END
<<<<< ornithine.mol end >>>>>


## Modifying the molfiles

The molfile2params_polymer.py script requires some additional data to be added to the end of the molfile. This data is specified at the end of the file after the bond information. It is a list of variable names and then a list of values. The variable are described bellow.

• ROOT: Single numerical value. Atom number (acording to the order above). Where the atom tree is rooted for this residue type. Should probably be the nitrogen of the central residue.

• POLY_N_BB, POLY_CA_BB, POLY_C_BB, POLY_CO_BB: Single numerical value. The backbone nitrogen, alpha-carbon, carbonyl-carbon and carbonyl-oxygen. These get special rosetta atom types and so are listed here special. Note: You will need to add some additional code to work with your additional backbone atom.

• POLY_IGNORE: List of numerical values. Atom number (acording to the order in the molfile). These are the atoms for the capping groups with the exception of the upper and lower connect atoms. They will not be listed in the atoms and bonds in the params file but are used in determining the atom types.

• POLY_UPPER, POLY_LOWER: Single numerical value. Atom number (according to the order in the molfile). These are the atoms in the capping groups that connect to the residue. They will not be listed in the atoms and bonds in the params file but are used in determining the atom types and they are listed in the internal coordinate section.

• POLY_CHG: Single numerical value. Overall charge on the residue.

• POLY_PROPERTIES: List of alpha-numerical values. These get used by rosetta at various places in the program. You can say something like:

if (pose.residue(10).type().is_protein() ) { /* do someting */ }
You will want to create a new property called "BETAPEPTIDE" and corresponding functions to the residue type class.
• END: The end of the file.

Note that there are 2 spaces between the "M" and the variable name. If you have to make multiple residue types (which it sounds like you will), keeping the atoms in the same order makes assigning all these numbers easier.

Additional info for peptide ornithine:

M  ROOT 13
M  POLY_N_BB 13
M  POLY_CA_BB 14
M  POLY_C_BB 15
M  POLY_O_BB 16
M  POLY_IGNORE 2 3 4 5 6 8 9 10 11 12
M  POLY_UPPER 7
M  POLY_LOWER 1
M  POLY_CHG 1
M  POLY_PROPERTIES PROTEIN POLAR CHARGED
M  END


Additional info for amino2:

M  ROOT 1
M  POLY_N_BB 1
M  POLY_CA_BB 2
M  POLY_C_BB 3
M  POLY_O_BB 4
M  POLY_IGNORE 9 10 22 23 24 27 28 29 30 31 32 33 34
M  POLY_UPPER 26
M  POLY_LOWER 7
M  POLY_CHG 1
M  POLY_PROPERTIES PEPTOID POLAR CHARGED
M  END


## Running molfile2params.py

Finally, the molfile2params_polymer.py script will convert the modified molfile to a params file. The commands bellow work for the example files:

> ./molfile_to_params_polymer.py --clobber --polymer --no-pdb --name C40 -k ornithine.kin stage_05_modified_molfile/ornithine.mol
> ./molfile_to_params_polymer.py --clobber --polymer --peptoid --no-pdb --name P01 -k amino2.kin stage_05_modified_molfile/amino2.mol

There is additional tweaking that needs to happen to the params files to make them work. Compare these to the ones in the database for reference.

The molfile2params.py script can produce a kinemage file. This is super handy as it lets you check how Rosetta will build the atom tree, and the atom type assignments, and other stuff. You can open kinemage files using the KiNG program from the Richardson lab at Duke (http://kinemage.biochem.duke.edu/software/king.php).

# Using your shiny new params file

Rosetta params files live in the database at database/chemical/residue_type_sets/(fa_standard)/..., but you can just pass your new parameters in on command line via -extra_res_fa or -extra_res (the latter for centroid).

# Rotamer library design

For questions on backbone-dependent rotamer library design please contact either Doug Renfrew (dougrenfrew at gmail dot com) or Tim Craven (twc254 at nyu dot edu).