- Tools for detecting homology (from simple to complex)
- Uses blosum62 matrix
- pBLAST - does alignment based on short "word" matches.
- phmmer - alignment using dynamic programming
- Uses sequence profile information (PSSM or HMM)
- HHsearch (aka HHpred) - HMM + secondary structure
- RaptorX - PSSM + secondary structure + solvent accessible surface area
- SPARKS-X - PSSM + secondary structure + backbone torsion angle + solvent accessible surface area
- ROBETTA uses HHsearch + RaptorX + SPARKS-X and recombines the results using the hybridize mover in RosettaScripts
- Hybridize mover does the following:
- Takes a list of input templates
- Randomly picks one template (you can bias toward certain templates being picked using an assigned weight).
- Uses TMalign to superimpose the remaining templates to the picked template. If TMalign score < 0.5 the template is discarded.
- Cuts each of the template at the loops creating "chunks"
- Stage 1: torsion space fragment insertion (in regions not covered by templates) and Cartesian space chunk recombination
- Stage 2: loop closure
- Stage 3: relax (full-atom sidechain and backbone refinement)
TUTORIAL
- Create a working directory
mkdir rattata
cd rattata
- Go to the UniProt website and find the sequence of "Ribosome association toxin RatA" from E. coli.
- Select "FORMAT" -> "FASTA" save the file to your working directory as "rata.fasta"
- Go to the HHpred website and submit the sequence. Enable the "Realign with MAC" option.
I've already submitted here: https://toolkit.tuebingen.mpg.de/hhpred/results/RATA_ECOLI
- Hit "save" on the HHpred and copy the *.hhr file to your working directory.
- Convert the top 5 HHpred hits to grishin format.
wget -O hhs2partial_thread.pl https://gremlin2.bakerlab.org/rosninja2016/rattata/hhs2partial_thread.pl
perl hhs2partial_thread.pl -hhs *.hhr -fas rata.fasta -name rata -limit 5
- Run the Rosetta partial_thread app to convert the alignments into "partial thread" pdb files. (Note: if you are not using VirtualBox, make sure the path to rosetta inside process_rata file is correct).
bash process_rata
- Generate fragments (since we learned how to do this yesterday, we'll skip this step and just download pre-made ones):
wget https://gremlin2.bakerlab.org/rosninja2016/rattata/P0AGL5.fas.200.3mers.gz
wget https://gremlin2.bakerlab.org/rosninja2016/rattata/P0AGL5.fas.200.9mers.gz
- Create "flags" file:
-frag_weight_aligned 0 # change to 0.1 to allow fragment insertion everywhere
-sog_upper_bound 10
-beta # this flag enables the latest rosetta score function
-in:file:fasta rata.fasta
-parser:protocol 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 "xml" file:
<ROSETTASCRIPTS>
<SCOREFXNS>
<stage1 weights="stage1.wts" symmetric=0>
<Reweight scoretype=atom_pair_constraint weight=1.0/>
</stage1>
<stage2 weights="stage2.wts" symmetric=0>
<Reweight scoretype=atom_pair_constraint weight=0.5/>
</stage2>
<fullatom weights="beta_cart.wts" symmetric=0>
<Reweight scoretype=atom_pair_constraint weight=0.5/>
</fullatom>
</SCOREFXNS>
<MOVERS>
<Hybridize name=hybridize stage1_scorefxn=stage1 stage2_scorefxn=stage2 fa_scorefxn=fullatom batch=1 stage1_increase_cycles=2.0 stage2_increase_cycles=1.0 linmin_only=0 skip_long_min=1>
<Fragments 3mers="P0AGL5.fas.200.3mers.gz" 9mers="P0AGL5.fas.200.9mers.gz"/>
<Template pdb="1t17A_0001_099_rata.pdb" weight="1" cst_file="AUTO"/>
<Template pdb="3tfzA_0002_099_rata.pdb" weight="1" cst_file="AUTO"/>
<Template pdb="2d4rA_0003_099_rata.pdb" weight="1" cst_file="AUTO"/>
<Template pdb="3tvqA_0004_099_rata.pdb" weight="1" cst_file="AUTO"/>
<Template pdb="4xrwA_0005_099_rata.pdb" weight="1" cst_file="AUTO"/>
</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
- HYBRIDIZE!
rosetta_scripts.default.linuxgccrelease @flags
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