ROSETTA Scripts

Some Rosetta Scripts that allow for various tasks. For questions or updates, e-mail jose.manuel.pereira@ua.pt

1. RELAX (relax.py)

Performs a relaxation protocol for energy minimization of a PDB structure.

Spawns a defined number of decoys (100 by default) that perform the protocol in parallel, using positional restraints on the backbone atoms (Turned on by default). The restraints weight can also be set (1.0 by default). Simulation results are saved in a .fasc file that can be analyzed to find the best structures (See get_best.py script). Use relax.py -h for more detailed information regarding the optional arguments of this script.

2. DESIGN (design.py)

Performs a design protocol by randomly mutating the aminoacid sequence in a target region in order to minimize the energy of the structure.

The design effort will be localized in the surrounding aminoacids (with a cutoff of 9.0 angstrom) of a ligand peptide, defined as a single chain (B, by default) of the input PDB structure. Spawns a defined number of decoys (100 by default) that perform the protocol in parallel. A cycle of design is comprised of two steps: (1) Monte Carlo design using a rotamer packer; (2) Minimization step; The whole protocol is comprised of several of these cycles (4 by default). Simulation results are saved in a .fasc file that can be analyzed to find the best structures (See get_best.py script). Use relax.py -h for more detailed information regarding the optional arguments of this script.

3. MULTI DOCK (multi_dock.py)

Performs a Position And Sequence Simultaneous Optimization (PASSO) protocol from multiple starting positions.

The PASSO protocol intends to simultaneously optimize both the position of part of a protein (for example, a new grafted domain) and design the interface region between the movable and fixed parts. This protocol expects an "ABC Model", where the target protein is divided in 3 different chains on the input PDB file:

1. Chain A - Fixed part of the protein
2. Chain B - Movable part of the protein
3. Chain C - Target ligand (fixed). Will be used to define the designable region of the protein.

See the single_dock_decoy.py script docstring for more information on how to set up ABC models using the ze_util/molecule_manipulation.py tools.

PASSO protocol: A single step of the PASSO protocol is comprised of 3 steps:

1. Random placement of the movable block using a rigid body movement (translation of maximum 0.5 ansgtrom + rotation of maximum 3 degrees);
2. Evaluation of a pre filter (See the PreFilter class docstring on ze_utils/pyrosetta_classes.py for more detailed information);
3. If the newly proposed structure passes the pre filter on step 2 design the interface residues (Uses the design protocol from the design.py script by default, see hb_design.py script for alternatives);

Reporter files: A total of 3 files will be created when running this protocol, in addition to structural PDBs:

1. Run data file (.JSON): holds information regarding each of the steps performed by the protocol.
2. Energy file (.DAT): holds energy information regarding the accepted steps whose design stage produced mutants with energy values lower than the initial conformation.
3. Status file (.TXT): a single line file that gets overwrited every step. Simply holds information regarding the current step and the maximum number of steps of this simulation.

See the PASSO class docstring on ze_utils/pyrosetta_classes.py for more detailed information.

Docking grid: The multi_dock.py script attempts to initialize this conformational search process from multiple different starting positions for the movable part of the protein, each defined as a point in space on a grid. By default, the grid is centered on a virtual point on the vector between chain A and chain B centroids, at the same distance from chain B centroid as the chain C centroid. See the DockingGrid class docstring on ze_utils/pyrosetta_classes.py for more detailed information.

SLURM parallelization: This protocol is performed by calling the single_dock.py script, who is in turn responsible for spawning multiple (100 by default) decoys of single_dock_decoys.py scripts. If the SLURM flag is set when launching the multi_dock.py script, individual slurm bash scripts are automatically created and added to the slurm queue. This release of new decoys is controlled by a check step that verifies that there is enough space on the user queue (limited to a set number of queued jobs - 500 by default).

4. LOOP (loop.py)

Performs loop closure with automatic design of the loop aminoacids.

The loop to add can be obtained from an existing loop (from another pose), or generated from a string. Furthermore, existing loops can be extended with additional residues at the C/N terminal. The initial conformation for the loop closure can be set as the original, helix, beta sheet or straight conformation.

5. ANALYZE (analyze.py)

Analizes a single PDB file and outputs revelant intra-pose metrics.

1. Total energy
2. Individual residues energy per selection
3. Interaction energy metric (If a ligand is present)
4. Hydrogen bonding network energy

6. ALIGN, ALIGN_PYROSETTA and MULTI_ALIGN (align.py, align_pyrosetta.py and multi_align.py)

align.py script allows the alignment and RMSD calculation of two PDB files, while multi_align.py applies the same protocol to all the 1 on 1 combinations for all the PDB files in the current working directory. align_pyrosetta.py offers the same functionalities without leaving the pyrosetta framework.

Optinally, a subset of atoms can be dined by atom name (such as CA only, for example). Makes use of ze_util/molecule_manipulation.py tools.

7. GET BEST STRUCTURES FROM FASC FILES (get_best.py)

Reads a .fasc file and prints the best structures.

By default, uses 'total_score' parameter for structure comparison, and prints the n structures (5 by default) with the lowest values for the parameter. If the reverse flag is set to True, print the n structures with the highest values instead.

8. CONVERGENCE_FUNNEL (convergence_funnel.py)

Reads a .fasc file and plot the RMSD variance towards the lowest energy structure vs the energy variance.

9. STATUS (status.py)

Used to verify the current status of a multi_dock.py script running.

By default prints the completion percentage of each docking grid point and the n structures (5, by default) with the lowest 'total_score' parameter. If used in a SLURM environment, print the current percentage of rescources being used by the user.

10. BLOSUM62 and MULTI_BLOSUM62 (blosum62.py and multi_blosum62.py)

blosum62.py script performs the calculation of BLOSUM62 score and sequence conservation percentage between two sequences, while multi_blosum62.py performs the same task but between all .pdb or .gro files indentifies in the query folder.

Any two sequences analyzed by this script need to be of the exat same length, as it does not, on its current version, perform any sort of pre sequence alignment. In the case of multi_blossum62.py script, the scores printed to the user can be ordered based on either the blossum62 score or the sequence conservation percentage (using the -s flag, choose between blosum62 or conserved). The results can also be displayed is reverse order (lower values first) by using the -r flag.

11. STABILITY (stability.py)

stability.py script performs a relaxation protocol, without constraints, followed by RMSD calculation.

The objective is to measure the stability of the current conformation, if allowed to relax without constraints. Lower RMSD values infer higher stability.