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

KEYWORDS: STRUCTURE_PREDICTION LOOPS

Authors: Roland Pache, Michal Sperber, Steven Combs, George Rosenberger
Last updated: August 2011 (RosettaCon9)

This demo shows how missing electron densities of several consecutive residues can be modeled using the loop modeling application (loopmodel) and the KInematic Closure algorithm (KIC).

The starting structure (1tr2_missing_density.pdb) is based on vinculin (1TR2). 5 loop residues have been removed (32-36) and should be replaced by the sequence VDGKA for loop modeling (simulating missing electron density). Afterwards, this PDB structure can be used to model the loop. For this demo, the water molecules (HOH) have been removed and the structure was truncated to the first 132 residues.

## Running the demo

1. Insert the new residues into the structure file

Open the file 1TR2_missing_density.pdb in the text editor of your choice. Search the first gap line (residue 32). Search for the first residue Valine in the file and copy all atoms to the new line. Repeat this step for all other residues (DGKA) and insert the coordinates below Valine. The file should then look like 1TR2_manually_added_dummy_residues.pdb. Renumber the residues you copied from another place to 32-36 and remove all eventually inserted new lines. Save this file as 1TR2_manually_added_dummy_residues_renumbered.pdb.

2. Create the loop file.

Create a new file, called 1TR2.loop, and open it in your text editor. Insert the following line:

LOOP 31 37 37 0 1


This excerpt from the loopmodel documentation describes the meaning of the 6 columns in that line:

column1  "LOOP":     Literally the string LOOP, identifying this line as a loop
In the future loop specification files may take other data.
column2  "integer":  Loop start residue number
column3  "integer":  Loop end residue number
column4  "integer":  Cut point residue number, >=startRes, <=endRes.
column5  "float":    Skip rate. default - never skip (0)
column6  "boolean":  Extend loop. Set to 1


For this example, we select the one residue before and after the loop to have real coordinates that can be used as anchor points by the KIC loop modeling algorithm. The cut point residue number is set to the last loop residue, since it must be inside the loop. The skip rate is set to 0 for this short example (since we want to model this loop) and the extend loop setting is set to true to idealize all bond lengths, bond angles and torsion angles of the loop residues before modeling.

3. Execution of the algorithm and definition of the flags

Assuming that Rosetta 3.3 is installed and all paths are set correctly, open your shell and change the directory to the one where the demo files are stored.

$> cp rosetta_inputs/* .$> \$ROSETTA3/bin/loopmodel.linuxgccrelease -s 1TR2_manually_added_dummy_residues_renumbered.pdb -loops:loop_file 1TR2.loop -loops:remodel perturb_kic -loops:refine refine_kic -ex1 -ex2 -nstruct 1 -loops:max_kic_build_attempts 100 -in:file:fullatom


Brief descriptions for all the components of this command-line:

loopmodel.linuxgccrelease : loopmodel application (linuxgccrealease or macosgccrelease)
-database : path to your Rosetta 3.3 DB
-loops:loop_file 1TR2.loop : name of your loops file
-loops:remodel perturb_kic : kinematic closure based loop modeling low resultion stage (side chains: centroids)
-loops:refine refine_kic : kinematic closure based loop modeling high resultion stage (side chains: fullatom)
-ex1 : extra chi rotamers for chi-1 angle (+/- 1 stddev from the optimal rotamer for better loop reconstruction)
-ex2 : extra chi rotamers for chi-2 angle (+/- 1 stddev from the optimal rotamer for better loop reconstruction)
-nstruct 1 : number of structures to generate (set to at least 1000 for real application; computationally expensive)
-loops:max_kic_build_attempts 100 : the maximal number of trials the algorithm should do to find a closed confirmation for the loop (default: 100); can be increased for difficult problems.
-in:file:fullatom : keep native amino acid side chain confirmations of the non-loop residues (residues within 10 Angstrom of the loop will be remodeled by default)

4. Analysis of the results

If you set -nstruct > 1, look at the Rosetta energy score in the standard output and identify the model with the lowest energy. Compare them visually using your favorite molecular visualization application (pymol, etc). Sample output files (incl. visualization using pymol) can be found in the output directory.