Metadata

Documentation by Frank DiMaio (dimaio@u.washington.edu), Sebastian Rämisch (raemisch@scripps.edu), and Jared Adolf-Bryfogle (jadolfbr@gmail.com)

Purpose

This protocol functions to create a homology model given PDB files corresponding to one or more template structures. It is used for comparative modeling of proteins.

References

High-resolution comparative modeling with RosettaCM.
Song Y, DiMaio F, Wang RY, Kim D, Miles C, Brunette T, Thompson J, Baker D., Structure. 2013 Oct 8;21(10):1735-42. doi:10.1016/j.str.2013.08.005. Epub 2013 Sep 12.

Algorithm

At a high-level, the algorithm consists of a long Monte Carlo trajectory starting from a randomly-chosen template. The MC trajectory employs the following moves: a) fragment insertion in unaligned regions, b) replacement of a randomly-chosen segment with that from a different template structure, and c) Cartesian-space minimization using a smooth (differentiable) version of the Rosetta centroid energy function. Finally, this is followed by all-atom optimization.

Running the RosettaCM protocol

Step 1 - Created one or several threaded model(s)
Step 2 - Run the Hybridize mover

Step 1: Create a threaded model

Alternative 1 - Running the setup_CM.py script
Alternative 2 - Manually preparing threaded template model

Running the setup_CM.py script

Warning: This script is experimental and may be problematic. Please verify your input and output alignments after running!

The most straightforward way to run the protocol is through the rosetta_tools script, protein_tools/scripts/setup_rosettacm.py. As input, you need the following:

  • Sequence alignment file in one of the supported formats (see below)
  • Template PDB file(s)
  • Target sequence fasta file

It is run with the following:

Rosetta/tools/protein_tools/scripts/setup_RosettaCM.py [-h] --fasta FASTA
                      [--alignment ALIGNMENT]
                      [--alignment_format ALIGNMENT_FORMAT]
                      [--templates [TEMPLATES [TEMPLATES ...]]]
                      [--rosetta_bin ROSETTA_BIN] [--build BUILD]
                      [--platform PLATFORM] [--compiler COMPILER]
                      [--compiling_mode COMPILING_MODE]
                      [--setup_script SETUP_SCRIPT] [-j J] [--run]
                      [--keep_files] [--run_dir RUN_DIR] [--equal_weight]
                      [--use_dna] [--verbose]

With the following important options:

--alignment ALIGNMENT --alignment_format ALIGNMENT_FORMAT

The alignment file must be passed as input, and the format must be specified. The following formats are valid: grishin, modeller, vie, hhsearch, clustalw, fasta.

--templates [TEMPLATES [TEMPLATES ...]]]

A list of PDBs of template structures. The alignment file "tags" must match the names of the template PDB files.

--rosetta_bin ROSETTA_BIN

The path to the Rosetta bin directory.

After setting up a job, cd to the rosetta_cm directory, and run (pointing to installed Rosetta location):

Rosetta/main/source/bin/rosetta_scripts.linuxgccrelease @flags -database Rosetta/main/database -nstruct 10

Manually setting up a RosettaCM job

Alternately, it may be desirable to manually set up a RosettaCM job. If one wishes to use nonstandard features (e.g. electron density data or user-specified constraints), this is recommended.

Given a fasta file sequence of the protein to be modeled, a multiple sequence alignment, and PDBs of template structures, the first step of the protocol is to thread the same sequence onto one or multiple templates. These templates can be homologous proteins or the same proteins from multiple crystal structures. The second step is to use the HybridizeMover through RosettaScripts to create a single model from the template(s).

First we need to produce template pdb files for Rosetta by threading the target sequence onto one or several template protein structures. As input, you need the following:

  • Sequence alignment file in Grishin format
  • Template PDB file(s)
  • Target sequence fasta file

An overview of the Grishin file format

The threaded model is then created with the following command:

Rosetta/main/source/bin/partial_thread.linuxgccrelease \
     -database Rosetta/main/database \
     -in:file:fasta target.fasta \
     -in:file:alignment alignment.aln \
     -in:file:template_pdb 1k3d.pdb 1y12.pdb \
     -ignore_unrecognized_res

This application should only take a few seconds.

NOTE It might be necessary to run this for every individual pdb file using alignment files with only one alignment each. It seems like providing multiple pdb files with -in:file:template_pdb does not always work.

Step 2: Run the Hybridize mover

This is the step where modeling is performed.

As input, you need the following:

  • RosettaScripts xml file
  • target sequence fasta file
  • threaded template pdb files from Step 1

A simple XML for performing modelling is listed below (see RosettaScripts for a guide to the XML syntax in Rosetta). More advanced options are specified on the HybridizeMover page.

<ROSETTASCRIPTS>
<SCOREFXNS>
    <stage1 weights=score3 symmetric=0>
        <Reweight scoretype=atom_pair_constraint weight=0.5/>
    </stage1>
    <stage2 weights=score4_smooth_cart symmetric=0>
        <Reweight scoretype=atom_pair_constraint weight=0.5/>
    </stage2>
    <fullatom weights=talaris2013_cart 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=1.0 stage2_increase_cycles=1.0>
        <Template pdb="1k3d_templ.pdb" cst_file="AUTO" weight=1.000 />
        <Template pdb="1y12_templ.pdb" cst_file="AUTO" weight=1.000 />
    </Hybridize>
<FastRelax name="relax" scorefxn=talaris2013  />
</MOVERS>
<PROTOCOLS>
    <Add mover=hybridize/>
    <Add mover=relax/>
</PROTOCOLS>
<OUTPUT scorefxn=talaris2013 />
</ROSETTASCRIPTS>

Save the above XML file as hybridize.xml. Then, RosettaCM is run using the following command:

Rosetta/main/source/bin/rosetta_scripts.linuxclangrelease \
     -database Rosetta/main/database \
     -in:file:fasta target.fasta \
     -parser:protocol **hybridize.xml** \
     -default_max_cycles 200 \
     -dualspace

Post Processing

See Analyzing Results: Tips for analyzing results generated using Rosetta

Other tips

How can I model with multiple chains?

In the input fasta file, separate sequences of individual chains with a '/' character.

How can I model with ligands/nucleic acids?

Add the tag use_hetatm=1 to the Hybridize mover line. Then, manually add the aligned ligand(s), with residue numbers starting one past the length of the fasta file. For nucleic acids, this is all that is needed.

Note: Ligands must be added to all templates with a non-zero weight in the XML file!

For ligands, the params files must be generated using the molfile_to_params script, with a few non-standard options. Given a mol2 file of the ligand in question, XXX.mol2, run:

python Rosetta/main/source/src/python/apps/public/molfile_to_params.py \
   --keep-names \
   --clobber \
   --extra_torsion_output \
   --centroid XXX.mol2 \
   -p XXX -n XXX

And the following flags must be additionally provided:

--extra_res_cen XXX.cen.params
--extra_res_fa  XXX.fa.params
--extra_improper_file XXX.tors

How can I model with symmetry?

If input templates are symmetric, then CM may be run using the symmetry of the template. See symmetry for creating a symmetry definition file. Then, two simple modifications must be made to the XML file:

Change:

<stage1 weights=score3 symmetric=0>
<stage2 weights=score4_smooth_cart symmetric=0>
<fullatom weights=talaris2013_cart symmetric=0>

To:

<stage1 weights=score3 symmetric=1>
<stage2 weights=score4_smooth_cart symmetric=1>
<fullatom weights=talaris2013_cart symmetric=1>

And add the symmdef file for each template, changing:

<Template pdb="1k3d_templ.pdb" cst_file="AUTO" weight=1.000 />
<Template pdb="1y12_templ.pdb" cst_file="AUTO" weight=1.000 />

To:

<Template pdb="1k3d_templ.pdb" cst_file="AUTO" weight=1.000 symmdef="dimer_1k3d.symm"/>
<Template pdb="1y12_templ.pdb" cst_file="AUTO" weight=1.000 symmdef="dimer_1y12.symm"/>

Note: As with ligands, if symmetry is used, all templates with a non-zero weight must have a specified symmetry definition file.

See Also