The scripts and input files that accompany this demo can be found in the
demos/protocol_captures directory of the Rosetta weekly releases.
This is a protocol capture, and represents the protocol at a fixed point in time. It may not work with the current version of Rosetta.
KEYWORDS: EXPERIMENTAL_DATA STRUCTURE_PREDICTION This protocol capture has been written by Tatjana Braun in January 2015 and demonstrates on an exemplary structure how the results presented in the PLos ONE RosettaCon collection paper "Combining evolutionary information and an iterative sampling strategy for accurate protein structure prediction" by Tatjana Braun, Julia Koehler Leman and Oliver Lange (2015) have been generated.
The following files and folders are provided in this protocol capture
scripts/: Python scripts for generating distance restraints,
analyzing the final results and setting up a RASREC runrosetta_flags/: All necessary flagfiles for setting up the initial and
refinement RASREC runinputs/: All necessary input files, including EVFold prediction, fasta
file, fragment files and native structureoutputs/: Exemplary outputs for all steps carried out in this tutorialThe following section is written in style of a tutorial and reflects exactly what has been done for each target in the manuscript. This tutorial will predict the structure of 1wvn with all necessary input files provided . All commands listed in this tutorial are executed from the protocol_capture folder.
For being able to directly copy all commands from this tutorial store the path of this tutorial in in the following variable:
PROTOCOL=~/path/to/tutorial
cd $PROTOCOL
The contact predictions used in the manuscript have been generated with the
EVFold webserver (available at
http://evfold.org/evfold-web/newprediction.do) using standard parameters.
The results can be downloaded in form of a compressed folder, which is
subdivided into several subdirectories. The all-by-all residue pairing
scores are stored in {jobname}_{scoringmethod}.txt in the ev_couplings
folder. In case of the standard scoring method (PLM), the file is named
{jobname}_PLM.txt.
An exemplary prediction for 1wvn is provided in inputs/ev_couplings/1wvn_PLM.txt.
From this score file, the N top-ranked residue pairing scores having a minimum distance of 5 residues are extracted and translated into Rosetta specific distance restraints. This can be done with the following two steps:
Generate NxN contact map
$PROTOCOL/scripts/create_evfold_contactmap.py -i inputs/ev_couplings/1wvn_PLM.txt -f inputs/1wvn.fasta -o 1wvn_PLM.cmp
This command generates a file called 1wvn_PLM.cmp in you protocol folder. It should be identical to the one provided in $PROTOCOL/outputs/1_contactmap
Translate top scoring pairs into Rosetta specific distance restraints
$PROTOCOL/scripts/extract_top_cm_restraints.py 1wvn_PLM.cmp -r_fasta inputs/1wvn.fasta -r_num_perc 1.0
This command generates the restraint file called "1wvn_PLM_ub8_lb1.5_w1_m5_CB_70_SIGMOID.cst". An already precomputed restraints file is stored in $PROTOCOL/outputs/2_restraints
The Rosetta software package version 3.6 or higher has to be obtained from www.rosettacommons.org. Rosetta applications are denoted with the extension <.ext>, which should be replaced with the system and compiler dependent extension. For instance, for gcc compiled Rosetta on a Linux system use .linuxgccrelease.
Note: The RASREC protocol requires MPI with a minimum of 4 computes cores (higher numbers are highly recommended). Rosetta can be compiled in MPI mode with the following commands:
cd /path/to/Rosetta/main/source
./scons.py -j <number of processors> bin mode=release extras=mpi
RASREC requires substantial computer resources. The required time depends on several factors including size, fold complexity, number and information content of restraints. For instance, target 1r9h requires 26 hours on 96 compute cores (2.6 GHz AMD Opteron Processors). The run time can be reduced by decreasing the standard pool size of 500, however this is not recommended as this will directly affect the final prediction accuracy.
We have run the fragment picker for all our targets with the following command:
make_fragments.pl -nohoms
The flag -nohoms leads to exclusion of fragments from homologous proteins. This flag should be omitted when not used for benchmarking.
Alternatively, fragments can be generated using the webserver Robetta (available at http://www.robetta.org/).
For the tutorial, this step can be omitted as fragments are already provided in $PROTOCOL/inputs/fragments.
All runs in our manuscript have been set up with the CSRosetta Toolbox (available at http://www.csrosetta.org/). In case, the Toolbox is not available, a folder containing all necessary flag files is provided. Both ways (with and without CSRosetta toolbox) to set up a RASREC run will be described below.
Using CSRosetta Toolbox
The following path assembles target related input files and stores the setup in ~/cs_targetlib.
setup_target -method rasrec -fasta inputs/1wvn.fasta -frags inputs/fragments/* -native inputs/1wvnA_ref.pdb -native_restrict inputs/1wvnA_core.rigid -target 1wvn_standard -restraints 1wvn_PLM_ub8_lb1.5_w1_m5_CB_70_SIGMOID.cst.
A run ready directory as specified with -dir containing all input files and flags is created with the following command:
setup_run -method rasrec -target 1wvn_standard -pool_size 500 -dir RASREC_runs -extras mpi
After using this command, the final run ready directory will be RASREC_runs/1wvn/. Along with all necessary input files, run-scripts for different cluster settings have been generated.
In case a local machine is used, RASREC can be started with the following commands:
cd $PROTOCOL/RASREC_runs/1wvn_standard/run
mkdir logs
mpirun -np <CORES> minirosetta.mpi.<ext> -out:level 300 -mute all_high_mpi_rank_filebuf -out:mpi_tracer_to_file logs/log -out:file:silent decoys.out @flags_denovo @flags_rasrec @flags_iterative -run:archive -out:nstruct <CORES-1>
Without the use of the CSRosetta Toolbox
In case no CSRosetta toolbox is installed on your system, you can setup the RASREC run manually. All flag files are provided in the rosetta_flags. These flags are ready-to-run for this tutorial. All necessary modifications for different targets will be explained below.
The following commands will create a run folder containing all necessary flags and files for a RASREC run
cd $PROTOCOL
mkdir -p RASREC_runs/1wvn_standard/run/logs
cp rosetta_flags/standard/* RASREC_runs/1wvn_standard/run
cd RASREC_runs/1wvn_standard/run
The final RASREC run can either be started with one of the run-scripts (for different cluster settings) or by executing the following commands:
mpirun -np <CORES> /path/to/Rosetta/main/source/bin/minirosetta.mpi.<ext> -out:level 300 -mute all_high_mpi_rank_filebuf -out:mpi_tracer_to_file logs/log -out:file:silent decoys.out @flags_denovo @flags_rasrec @flags_iterative -run:archive -out:nstruct <CORES-1>
For running RASREC with user specific files, the following flags have to be adapted:
flags_denovo:
-frag3 <3mer fragment file>
-frag9 <9mer fragment file>
-in:file:fasta <input.fasta>
flags_rasrec:
-broker:setup <broker setup file>
-in:file:native <native.pdb>
#specifies residues used for RMSD calculation/ can be omitted if all resdidues should be used
-evaluation:rmsd NATIVE _full <resiudes.rigid>
setup_init.tpb:
file <restraints.cst>
A folder containing template files and a README showing which flags need to be changed is provided in $PROTOCOL/rosetta_flags/template
A RASREC run is finished, once the fullatom_pool_ has been generated. The following Error message occurs after a successful RASREC run and can be ignored:
ERROR: quick exit from job-distributor due to flag jd2::mpi_nowait_for_remaining_jobs --- this is not an error
The final RASREC models are stored in fullatom_pool/decoys.out
Using the CSRosetta Toolbox, the top scoring ensemble can be extracted from the pool of final models with the following commands:
extract_decoys decoys.out -score 30 > low_30.out
pack_pdbs -silent low_30.out > low_30.pdb
Without the toolbox, the PDB ensemble can be extracted as follows:
$PROTOCOL/scripts/silent_data.py decoys.out score description | sort -nk 1 | head -n 30 | awk '{print $2}' > pdb_list.txt
score_jd2.default.<ext> -in:file:silent decoys.out -in:file:tags $(cat pdb_list.txt) -rescore:skip -out:file:silent low_30.out
extract_pdbs.default.<ext> -in:file:silent decoys.out -in:file:tags $(cat pdb_list.txt)
for i in $(echo batch*.pdb); do echo "MODEL $i"; cat "$i"; echo "ENDMDL"; done >> low_30.pdb
The average energy and rmsd of 30 lowest-energy models can be analyzed with the following command
$PROTOCOL/scripts/silent_data.py low_30.out score rms_full | sort -nk 1 | $PROTOCOL/scripts/median.py
which yields
median Q1 Q3 hi lo
-280.880 -281.639 -278.708 -277.760 -284.937 #score
3.091 2.810 3.261 3.621 2.795 #rms_full
The following command shows the residues being converged within 2A
ensemble_analysis.default.<ext> -in:file:silent low_30.out -wRMSD 2 -rigid:out core_2.rigid -rigid:cutoff 2 -calc:rmsd
The output is as follows:
main: computed RMSD on #Converged residues listed below
main: RIGID 4 38 0 0 0 # 4-38
main: RIGID 42 64 0 0 0 # 42-64
main: RIGID 66 98 0 0 0 # 66 -98
main: RIGID 107 142 0 0 0 # 107-142
main: RIGID 164 166 0 0 0 # 164-166
main:
main: number of atoms from 189 for mean RMSD: 130
main: fraction of residues converged: 0.69
main: mean RMSD to average structure: 1.94
main: mean pairwise RMSD: 2.88
main: mean pairwise RMSD * superposed_fraction_of_atoms^-1: 4.19
If the convergence of the initial RASREC run is not sufficient enough (< 90%), a second RASREC run can be carried out. This run will reuse restraints from both predicted contact map and the previous result
The following command generates two restraint files given a model ensemble and a contact map.
cd $PROTOCOL
$PROTOCOL/scripts/repick_restraints_final.py -c 1wvn_PLM.cmp -s RASREC_runs/1wvn_standard/run/fullatom_pool/low_30.pdb -p 1 -o restraints
As output, the following files are generated:
restraints_converged_distances.cst: converged distances in low_30.pdb
translated to strict bounded potentials around the average distance
restraints_filtered_contactmaps.cst: additional restraints from the
contactmap that do not completely disagree with the previous results. Here a
more widely bounded potential is used.
The flags and patches used for the refinement RASREC run are identical to the ones listed in Section 2.3). The two RASREC runs only differ in the restraints used for structural guiding. The restraint files are added to a RASREC run in the broker file.
Using the CSRosetta Toolbox
Setup prediction
setup_target -method rasrec -fasta inputs/1wvnA.fasta -frags inputs/fragments/* -native inputs/*ref.pdb -native_restrict inputs/*_core.rigid -target 1wvn_rerun -restraints *_converged_distances.cst *filtered_contactmaps.cst
Generate run folder
setup_run -method rasrec -target 1wvn_rerun -pool_size 500 -dir RASREC_runs -extras mpi
The final run folder for the refinement run can be found RASREC_runs/1wvn_rerun/
Please COMBINE_RATIO for the filtered restraints set in the broker file (setup_init) as follows:
CLAIMER ConstraintClaimer
file restraints_filtered_contactmaps.cst
COMBINE_RATIO 2 # ADD THIS LINE!
FULLATOM
CENTROID
SKIP_REDUNDANT 0
FILTER_WEIGHT 1.00
FILTER_NAME restraints_filtered_contactmaps.cst
END_CLAIMER
This is done so that potentially wrong restriants do not affect the structure prediction in a wrong way.
RASREC can either be executed with one of the run-scripts or with the following commands:
cd RASREC_runs/1wvn_rerun/run
mkdir logs
mpirun -np <CORES> minirosetta.mpi.<ext> -out:level 300 -mute all_high_mpi_rank_filebuf -out:mpi_tracer_to_file logs/log -out:file:silent decoys.out @flags_denovo @flags_rasrec @flags_iterative -run:archive -out:nstruct <CORES-1>
Without the CSRosetta Toolbox
The folder with input files for the refinement run can be set up as follows:
mkdir -p RASREC_runs/1wvn_rerun/run/logs
cp rosetta_flags/rerun/* RASREC_runs/1wvn_rerun/run
cd RASREC_runs/1wvn_rerun/run
mpirun -np <CORES> minirosetta.mpi.<ext> -out:level 300 -mute all_high_mpi_rank_filebuf -out:mpi_tracer_to_file logs/log -out:file:silent decoys.out @flags_denovo @flags_rasrec @flags_iterative -run:archive -out:nstruct <CORES-1>
In case the protocol is applied to different targets, the parameters have to be changed as described in 2.3.2)
SVN Revision 57296