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

KEYWORDS: NONCANONICALS UTILITIES

This demo was written by P. Douglas Renfrew (renfrew at nyu dot edu).

This demo illustrates the make_rot_lib protocol. It was originally written as the 2nd (of 4) parts of a "protocol capture" acompanying the paper "Using Noncanonical Amino Acids in Computational Protein-Peptide Interface Design" by P. Douglas Renfrew, Eun Jung Choi, Brian Kuhlman (2011) PLoS One, from the RosettaCon2010 special PLoS ONE special issue. The four separate protocol captures that describe the four different ways Rosetta was used in the publication: creating Rosetta ResidueType parameter files for NCAAs, creating backbone dependent rotamer libraries for NCAAs, calculating explicit unfolded state reference energies for NCAAs, and the running of the DougsDockDesignMinimizeInterface protocol using NCAAs. The protocol caputres for creating ResidueType paramater files, rotamer libraries and explicite unfolded state energies describe the process used in the publication but are written from the standpoint of a researcher looking to add an additional NCAA to Rosetta.

Creating a Noncanonical Amino Acid (NCAA) rotamer library is the second of two steps toward being able to use a NCAA in Rosetta. To add a new NCAA or to better understand how the NCAAs in the related publication were added one should have already completed or understand the steps in HowToMakeResidueTypeParamFiles.

Rotamer libraries are sets of common side chain conformations that generally correspond to local minima on the side chain conformational energy landscape. Side chain conformations are usually represented as a set of mean angles and a standard deviation to indicate variability. Rotamer libraries are used in for two main purposes in Rosetta: to provide starting points for side chain optimization routines, and the relative frequency is used as a pseudo-energy. Traditionally rotamer libraries are created by collecting statistics from protein structures. Rosetta uses the backbone dependent Drunbrack rotamer libraries. Since there are not enough structures containing NCAAs they must be generated.

Running the MakeRotLib protocol consists of four steps:

1. Creating and input template and generating the MakeRotLib options files.
2. Running the MakeRotLib protocol on each option file.
3. Assembling the individual rotamer libraries in a single file.
4. Modifying the ResidueType parameter file to be aware of our new rotamer library.

Step 1: Making input files

Rosetta primarily uses backbone dependent rotamer libraries. Backbone-dependent rotamer libraries list provide side chain conformations sampled from residue positions whose backbone dihedral angles fall in particular bins. In the case of the Drunbrack rotamer libraries used by Rosetta the bins are in 10 degree intervals for for both phi and psi for a total of 1296 (36x36) phi/psi bins. To replicate this for the NCAAs we need to create a set of side chain rotamers for each member of a set of phi/psi bins.

The MakeRotLib protocol takes an option file as input. It requires an options file for each phi/psi bin. The first step in running it is creating these 1296 options files. Continuing from the HowToMakeResidueTypeParamFiles protocol capture we are again using ornithine as an example. Ornithine has 3 sidechain dihedral angles (chi). We want to sample each chi angle from 0 to 360 degrees in 30 degree intervals, and based on the chemistry of the side chain we predict that were will probably be three preferred angles for each chi angle at 60, 180, and 300 degrees for a total of 27 rotamers (3x3x3). We setup our MakeRotLib options file template as shown bellow.

<<<<< C40_rot_lib_options_XXX_YYY.in start >>>>>
AA_NAME C40
PHI_RANGE XXX XXX 0
PSI_RANGE YYY YYY 0
NUM_CHI 3
CHI_RANGE 1 0  330  30
CHI_RANGE 2 0  330  30
CHI_RANGE 3 0  330  30
CENTROID 300 1 300 1 300 1
CENTROID 300 1 300 1 180 2
CENTROID 300 1 300 1  60 3
CENTROID 300 1 180 2 300 1
CENTROID 300 1 180 2 180 2
CENTROID 300 1 180 2  60 3
CENTROID 300 1  60 3 300 1
CENTROID 300 1  60 3 180 2
CENTROID 300 1  60 3  60 3
CENTROID 180 2 300 1 300 1
CENTROID 180 2 300 1 180 2
CENTROID 180 2 300 1  60 3
CENTROID 180 2 180 2 300 1
CENTROID 180 2 180 2 180 2
CENTROID 180 2 180 2  60 3
CENTROID 180 2  60 3 300 1
CENTROID 180 2  60 3 180 2
CENTROID 180 2  60 3  60 3
CENTROID  60 3 300 1 300 1
CENTROID  60 3 300 1 180 2
CENTROID  60 3 300 1  60 3
CENTROID  60 3 180 2 300 1
CENTROID  60 3 180 2 180 2
CENTROID  60 3 180 2  60 3
CENTROID  60 3  60 3 300 1
CENTROID  60 3  60 3 180 2
CENTROID  60 3  60 3  60 3
<<<<< C40_rot_lib_options_XXX_YYY.in end >>>>>


Field definitions:

• AA_NAME <three letter code for the amno acid>

• PHI_RANGE <phi value for this bin> <phi value for this bin> 0 : The phi range functionality is not functional. Both values need to be the same and the interval set to 0

• PSI_RANGE <psi value for this bin> <psi value for this bin> 0 : The psi range functionality is not functional. Both values need to be the same and the interval set to 0

• NUM_CHI <number side chain dihedral angles> : This should be the same as in the parameter file.

• CHI_RANGE <chi number> <starting value> <ending value> <interval> : The number of CHI_RANGE fields needs to equal the values specified for NUM_CHI.

• CENTROID <Rotamer number for chi 1> <starting value> {<rotamer number for chi 2> <starting value>}{etc.} : CENTROIDS specify the starting points for the K-means clustering described in the related publication. A CENTROID field is needs for each potential rotamer. The number of CENTROID fields defines the number of rotamers listed in the resulting rotamer library.

To generate the 1296 input files we use a provided script that simply replace the XXX and YYY with the phi and psi values. The script is run as shown bellow.

$> cd inputs$> ../scripts/make_inputs.bash C40_rot_lib_options_XXX_YYY.in


The number of chi angles and the CHI_RANGE sampling interval are the primary determinants of the run time as they determine the number of rotamers that will be tested for each phi/psi bin. It is recommended to have at least 500 samples per chi. In the ornithine example we sample in 30 degree intervals for each of the 3 chi angles giving us a total of 1728 (12x12x12) conformations tested for each phi/psi bin. For a residue with a single chi 1 degree bins will suffice.

Step 2: Running the MakeRotLib protocol

The next step is to run the MakeRotLib protocol on each of the input files we created in step one. This is the most time consuming portion of the process and should probably be done on a cluster. As cluster setups vary, an example for a single MakeRotLib options file is provided. The other 1295 should be run identically.

$> cd outputs$> PATH/TO/ROSETTA/bin/MakeRotLib.macosgccrelease -rot_lib_options_file ../inputs/C40_rot_lib_options_-60_-40.in >& C40_rot_lib_options_-60_-40.log &


NOTE: The extension on your executable maybe different.

The only options passed to the executable are the path to the database and the MakeRotLib options file. After the run completes a file called C40_-60_-40_bbdep.rotlib should be in the output directory. This is the backbone dependent rotamer library for a phi of -60 and a psi of -40.

The log file from the rosetta run in includes quite a bit of useful output. There are three main sections to the log output, "ROTAMERS", "CENTROIDS" and "FINAL ROTAMERS" sections. Each one shows the following data: phi, psi, omega and epsilon backbone dihedral angles, probability, total energy, torsion energy, intra-residue repulsive, intra-residue attractive, the number of chi angles, the assigned cluster number, the set of input chi angles, the set of minimized chi angles, the standard deviation, and the distance from that point to each of the cluster centroids. The log file also displays the number of conformations per cluster, the average distance between the cluster center and the members of that cluster. Lack of conformations in a cluster and a large (>30) average cluster centroid distance suggests that that cluster is higher in energy.

Step 3: Assembling the individual rotamer libraries in to a single file

After the MakeRotLib protocol has been run on all of the MakeRotLib options files the individual rotamer libraries for each phi psi bin need to be assembled in to a single file. This is accomplished with a provided script as shown bellow.

$> cd outputs$> ../scripts/make_final_from_phi_psi.pl C40


The single file rotamer library should be called C40.rotlib. The file should be placed in the ncaa_rotlibs directory in the database.

> cp C40.rotlibs PATH/TO/DATABASE/rosetta_database/ncaa_rotlibs/


Step 4: Modifying the residue type PARAMS file

The last step is modifying the residue type parameter file to use the new rotamer library. To do this we need to add the name of the rotamer library, the number chi angles it describes, and how many bins there are for each chi angle to the Residue type parameter file. The ornithine rotamer library is called C40.rotlib and the rotamer library describes 3 chi angles and each of those 3 chi angle has 3 rotamer numbers. So we would use the following commands to add that information to the file we created in HowToMakeResidueTypeParamFiles.

> echo "NCAA_ROTLIB_PATH C40.rotlib" >> PATH/TO/rosetta_database/chemical/residue_type_sets/fa_standard/residue_types/l-ncaa/ornithine.params
> echo "NCAA_ROTLIB_NUM_ROTAMER_BINS 3 3 3 3" >> PATH/TO/rosetta_database/chemical/residue_type_sets/fa_standard/residue_types/l-ncaa/ornithine.params