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 EXPERIMENTAL_DATA

Written by Lei Shi. Nikolas Sgourakis drafted the previous version

We will use the chemical shifts to improve the fragments from which Rosetta builds up structures, and the NOEs+RDCs to guide the Rosetta calculations towards the native structure.

Please see references at:

  • rosetta abinitio: Bradley, P et al Science 2005
  • chemical shift fragments: Shen Y et al. PNAS 2008;105:4685-4690
  • chemical shift+NOE+RDC: Raman S, et al Science 2010

These Rosetta calculation steps are also described separately:

  • Sgourakis NG et al JACS,2011,133(16):6288-98:

In this demo, we will use PDB 2JY7, which is a small protein (for demo purpose) and has experimental data deposited. Several scripts are provided in the scripts folder for formattting purposes:

bmrb2talos.com
cst_map_toCB.py
upl2mini.csh
scores.score.cfg

If you are from David Baker lab, there are scripts available to make setup easier without going through public servers. The following instructions should work just fine without having direct access to any Baker lab cluster.

Running the demo

  1. Create following folders: 

    mkdir starting_inputs
mkdir rosetta_inputs
mkdir rosetta_inputs/talos_output
mkdir rosetta_inputs/pick_cs_fragments

  2. Download protein fasta and experimental data. Download fasta from http://www.pdb.org/pdb/explore/explore.do?structureId=2JY7 

    wget http://www.pdb.org/pdb/files/fasta.txt?structureIdList=2JY7 -O starting_inputs/t000_.fasta
    Download chemical shift data from http://www.bmrb.wisc.edu/data_library/summary/index.php?bmrbId=15591 
    wget http://rest.bmrb.wisc.edu/bmrb/NMR-STAR2/15591 -O starting_inputs/raw.cs.bmrb
    Download NOE data from http://restraintsgrid.bmrb.wisc.edu/NRG/MRGridServlet?pdb_id=2JY7&show_blocks=true&min_items=0: 
    #http://restraintsgrid.bmrb.wisc.edu/NRG/MRGridServlet?db_username=wattos1&format=ambi&mrblock_id=434910&pdb_id=2jy7&program=DYANA%2FDIANA&request_type=block&subtype=general+distance&type=distance
echo "save file as starting_inputs/NOE_data.upl"
    Download RDC data (used for a different demo) from http://restraintsgrid.bmrb.wisc.edu/NRG/MRGridServlet?pdb_id=2JY7&show_blocks=true&min_items=0: 
    #http://restraintsgrid.bmrb.wisc.edu/NRG/MRGridServlet?db_username=wattos1&format=n%2Fa&mrblock_id=23753&pdb_id=2jy7&request_type=archive&subtype=n%2Fa&type=dipolar+coupling
echo "save file as starting_inputs/nh_xplor.rdc"

  3. Format data for Rosetta use: Formatting NOE: Note only residues separated by more than 3 are kept in constraint. This script scripts/upl2mini.csh only works with cyana format NOE. 

    $> scripts/upl2mini.csh starting_inputs/NOE_data.upl > rosetta_inputs/NOE.cst
$> scripts/cst_map_toCB.py rosetta_inputs/NOE.cst > rosetta_inputs/NOE.centroid.cst
    Formmatting chemical shift data for TALOS. The required bmrb2talos script is part of the Rosetta toolbox, downloadable from The CS Rosetta web site. The output file is provided here. 
    $ bmrb2talos starting_inputs/raw.cs.bmrb > rosetta_inputs/cs.talos
    Formmatting RDC data for Rosetta: Note that rename HN to H in the rdc file: 
    $ awk '{print $4,$7,$11,$14,$16}' starting_inputs/nh_xplor.rdc > rosetta_inputs/nh.rdc

  4. Generating talos predictions using http://spin.niddk.nih.gov/bax/nmrserver/talosn/ using rosetta_inputs/cs.talos Save/copy pred.tab and predSS.tab to rosetta_inputs/talos_output

  5. Generate fragment/profile from RobettaServer http://www.robetta.org/fragmentqueue.jsp using starting_inputs/t000_.fasta Save/copy t000_.checkpoint to rosetta_inputs/

  6. Pick fragments using secondary structure profile and chemical shift data (where $ROSETTA3=path-to-Rosetta/main/source and $ROSETTA3_DB=path-to-database) 

    $> $ROSETTA3/bin/fragment_picker.default.linuxgccrelease -in::file::vall $ROSETTA3_DB/sampling/small.vall.gz -frags::n_frags 200 -frags::frag_sizes 3 9 -frags::sigmoid_cs_A 2 -frags::sigmoid_cs_B 4 -out::file::frag_prefix rosetta_inputs/pick_cs_fragments/frags.score -frags::describe_fragments rosetta_inputs/pick_cs_fragments/frags.fsc.score -frags::scoring::config scripts/scores.score.cfg -in:file:fasta starting_inputs/t000_.fasta -in:file:checkpoint rosetta_inputs/t000_.checkpoint -in:file:talos_cs rosetta_inputs/cs.talos -frags::ss_pred rosetta_inputs/talos_output/predSS.tab talos -in::file::talos_phi_psi rosetta_inputs/talos_output/pred.tab
    IMPORTANT The small.vall.gz used here for fragment picking is only used to speed up the demo. You have to change this to the vall database on your system!

  7. Run Rosetta with the fragments made above and use NOEs to guide search in both centroid sampling and full-atom optimization 

    $> $ROSETTA3/bin/minirosetta.default.linuxgccrelease -in:file:rdc rosetta_inputs/nh.rdc -cst_fa_file rosetta_inputs/NOE.cst -cst_file rosetta_inputs/NOE.centroid.cst -abinitio:stage1_patch scripts/patch_atom_pair_constraint_rdc -abinitio:stage2_patch scripts/patch_atom_pair_constraint_rdc -abinitio:stage3a_patch scripts/patch_atom_pair_constraint_rdc -abinitio:stage3b_patch scripts/patch_atom_pair_constraint_rdc -abinitio:stage4_patch scripts/patch_atom_pair_constraint_rdc -score:patch scripts/patch_atom_pair_constraint_rdc -in:file:fasta starting_inputs/t000_.fasta -file:frag3 rosetta_inputs/pick_cs_fragments/frags.score.200.3mers -file:frag9 rosetta_inputs/pick_cs_fragments/frags.score.200.9mers -nstruct 1 -out:file:silent csrosetta_noe_rdc.out -run:protocol abrelax -abinitio::increase_cycles 0.1 -abinitio::relax -overwrite

IMPORTANT

  • Change nstruct to generate desired number of models. Larger is better depending on your available computer time, etc.
  • change -abinitio::increase_cycles 0.1 to 10! (0.1 is chosen for tesing purposes only)

If you want to use non-linear-square fitting with Da or R fixed, use: 

-rdc:fit_method nls -rdc:fixDa 12.6 -rdc:fixR 0.42
You can/should adjust the weights of NOE and rdc constraints in scripts/patch_atom_pair_constraint_rdc. You can provide either -rdc:fixDa, -rdc:fixR or both of them Change nstruct to generate desired number of models. Larger is better depending on your available computer time, etc. Note that the demo in abinitio_w_chemicalshift_only, you can add flags such as: 
  -abinitio::rg_reweight 0.5
  -abinitio::rsd_wt_helix 0.5
  -abinitio::rsd_wt_loop 0.5
  -disable_co_filter true
  -abinitio::increase_cycles 10
to help sampling in centroid stage. They are not used probably NOE constraint helps guided the search.

Processing the output

  1. Extract the low energy models: 

    grep SCORE csrosetta_noe_rdc.out | sort –nk2 | head
    The second column contains the energies of the lowest energy 10 models. Select as the cutoff the energy on the last line. You should also use NOE constraint energy and RDC energy as a criteria to select structures. Example is only provided for total score.

  2. This script: 

    cull_silent.pl csrosetta_noe_rdc.out “score < cutoff”
    will produce csrosetta.select.silent which contains the lowest total energy 10 models.

  3. extract pdbs from silent files for a given tag in the silent file 

    $> $ROSETTA3/bin/extract_pdbs.default.linuxgccrelease -in::file::silent csrosetta_noe_rdc.out -in::file:tags S_00000001
$> mv S_00000001.pdb lowscore_1.pdb

  4. Check convergence by superimposing the ten low energy models in pymol or your favorite molecular graphics

  5. Check convergence by clustering the lowest energy models (see clustering demo for instructions)

  6. To see how NOE constraints are satisfied by a model: 

        $> $ROSETTA3/bin/r_cst_tool.default.linuxgccrelease -in:file:s lowscore_1.pdb -cst_file rosetta_inputs/NOE.cst
   ```
r_cst_tool is a pilot program by Oliver Lange in Rosetta/main/source/src/apps/pilot/olli/m

7. To see how NOE constraints are satisfied by a model:
Rosetta/main/source/bin/r_cst_tool.linuxgccrelease -database Rosetta/main/database/ -in:file:s lowscore_1.pdb -cst_file rosetta_inputs/NOE.cst
    r_cst_tool is a pilot program by Oliver Lange in Rosetta/main/source/src/apps/pilot/olli/ 
    
8. To see how rdc constraints are satisfied by a model
    Rosetta/main/source/bin/score_jd2.default.linuxgccrelease -database Rosetta/main/database/ -in:file:s lowscore_1.pdb -in::file::rdc rosetta_inputs/nh.rdc -score::patch scripts/patch_atom_pair_constraint_rdc -rdc:fit_method nls 1 -out:file:scorefile rdc_score_for_lowscore_1.sc -out:level 999 -mute core.chemical core.conformation -rdc:print_rdc_values lowscore_1.rdc_corr