WARNING BEFORE YOU START: TO FIX frag_pick script do:
wget -O ~/tools/pick_frag/pick_frag.pl https://gremlin2.bakerlab.org/rosninja2016/KTSC/pick_frag.pl
In this tutorial we will try to predict the structure of the following sequence:
MQRQPVSSSRILSIGYDPDNRMLEIQFREQGTYQYLGVPERAHQNFMSAVSKGRFFDGVIKGKFLCRKIG
- From PFAM (PF13619):
This short domain is named after Lysine tRNA synthetase C-terminal domain. It is found at the C-terminus of some Lysyl tRNA synthetases as well as a single domain in bacterial proteins. The domain is about 60 amino acids in length and contains a reasonably conserved YXY motif in the centre of the sequence. The function of this domain is unknown but it could be an RNA binding domain.
- Setup a working directory
mkdir KTSC
cd KTSC
- Submit the sequence to the GREMLIN (co-evolution) server: http://gremlin.bakerlab.org/submit.php
In this case, I've already submitted the sequence and got the results: 1475969195 - Download the restraints to use in rosetta by click on "Generate restraints for ROSETTA Structure Modeling"
- Pick fragments
- Create a "flags" file:
-abinitio::fastrelax
-abinitio::increase_cycles 10 # increase for larger proteins
-abinitio::rg_reweight 0.5 # radius of gyration; set to 0.0 for long extended proteins
-abinitio::rsd_wt_helix 0.5
-abinitio::rsd_wt_loop 0.5
-in:file:native 4RGIA.pdb
-in:file:fasta KTSC.fas.fasta
-in:file:frag3 KTSC.fas.200.3mers
-in:file:frag9 KTSC.fas.200.9mers
-nstruct 10
-constraints:cst_weight 3
-constraints:cst_file 1475969195_cb.cst
-constraints:cst_fa_weight 3
-constraints:cst_fa_file 1475969195_cb.cst
- Launch pymol (because we want to watch!) This script launches a "listening" script inside pymol that can take data from rosetta and display it.
run /home/work/Rosetta/source/src/python/bindings/PyMOLPyRosettaServer.py
- Launch abinitio relax!
AbinitioRelax.default.linuxgccrelease @flags -show_simulation_in_pymol 1
- Extract models
I've already generated lots of decoys for you guys to play with:

wget https://gremlin2.bakerlab.org/rosninja2016/KTSC/default.out.zip
unzip default.out.zip By default the output of the protocol saves the models to a file called "default.out". All your models are saved in this single file. To extract ALL models (not a good idea), you can run the following command:
extract_pdbs.default.linuxgccrelease -in:file:silent default.out Instead you would want to only extract the top 5 (or more models). grep for the SCORE line from the default.out, sort by the score column and get the top 5 names:
grep ^SCORE default.out | awk '{print $2,$NF}' | sort -k1n | head -5 give the extract app the top 5 tags:
extract_pdbs.default.linuxgccrelease \ -in:file:silent default.out \ -in::file::tags S_00000041_27 S_00000052_22 S_00000054_5 S_00000052_49 S_00000024_34
- Lets Hybridize!
mkdir hyb; cd hyb create "hyb.flags" file:
-frag_weight_aligned 0.1
-beta # this flag enables the latest rosetta score function
-in:file:fasta ../KTSC.fas.fasta
-in:file:native ../4RGIA.pdb
-parser:protocol hyb.xml # rosetta script (see below)
-relax:minimize_bond_angles
-relax:jump_move true
-relax::dualspace
-default_max_cycles 200
-relax:min_type lbfgs_armijo_nonmonotone
-hybridize:stage1_probability 1.0
-hybridize:stage1_4_cycles 400
-nstruct 1 create "hyb.xml" file:
<ROSETTASCRIPTS>
<SCOREFXNS>
<stage1 weights="stage1.wts" symmetric=0>
<Reweight scoretype=atom_pair_constraint weight=3/>
</stage1>
<stage2 weights="stage2.wts" symmetric=0>
<Reweight scoretype=atom_pair_constraint weight=3/>
</stage2>
<fullatom weights="beta_cart.wts" symmetric=0>
<Reweight scoretype=atom_pair_constraint weight=3/>
</fullatom>
</SCOREFXNS>
<MOVERS>
<Hybridize name=hybridize stage1_scorefxn=stage1 stage2_scorefxn=stage2 fa_cst_file="../1475969195_cb.cst" fa_scorefxn=fullatom batch=1 stage1_increase_cycles=2.0 stage2_increase_cycles=1.0 linmin_only=0 skip_long_min=1>
<Fragments 3mers="../KTSC.fas.200.3mers" 9mers="../KTSC.fas.200.9mers"/>
<Template pdb="../S_00000041_27.pdb" weight="1" cst_file="../1475969195_cb.cst"/>
<Template pdb="../S_00000052_22.pdb" weight="1" cst_file="../1475969195_cb.cst"/>
<Template pdb="../S_00000054_5.pdb" weight="1" cst_file="../1475969195_cb.cst"/>
<Template pdb="../S_00000052_49.pdb" weight="1" cst_file="../1475969195_cb.cst"/> <Template pdb="../S_00000024_34.pdb" weight="1" cst_file="../1475969195_cb.cst"/>
</Hybridize>
</MOVERS>
<PROTOCOLS>
<Add mover=hybridize/>
</PROTOCOLS>
<OUTPUT scorefxn=fullatom/>
</ROSETTASCRIPTS> Download stage weight files:
wget https://gremlin2.bakerlab.org/rosninja2016/rattata/stage1.wts
wget https://gremlin2.bakerlab.org/rosninja2016/rattata/stage2.wts
Lets hybridize:
rosetta_scripts.default.linuxgccrelease @hyb.flags
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