Difference between revisions of "Team:TU Darmstadt/Model/Rosetta Guide"

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Now you successfully installed Rosetta and can continue with the tutorials either on our Wiki page, the Meiler Lab page or the Rosetta Documentation itself.
 
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            Rosetta Docking
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Revision as of 17:29, 21 October 2020

image/svg+xml - O O



Introduction Installation Rosetta Docking Rosetta CM

Rosetta Guide

Rosetta is a console-based software suite capable of solving multiple computational macromolecular problem such as de novo protein design, enzyme design, ligand docking and structure prediction of biological macromolecules or macromolecular complexes. Since its very hard to start with for beginners we want to provide a guide for everyone wanting to take their first steps with Rosetta. In this guide we closely orientated on the Rosetta Documentation, which can be pretty confusing for beginners so we added our own experiences and some extra steps that may not be obvious to starters.

Installation

Unfortunately, Rosetta does not provide you an installer with a graphical user interface to easily install the program with a few clicks. Instead you need to download the source code, unpack it in the location you want to install Rosetta and compile it with a C++ compiler. You can only install Rosetta on Linux operating systems. Luckily, Windows already has a built in Linux subsystem and you can easily an OS like Ubuntu in the Microsoft App Store. Alternatively, you can install a virtual machine software such as VirtualBox and install Linux manually. After you downloaded the source code change the directory to the installation using the cd command. Simply add the path you want to change to after the cd. If you want to have a look at the directory you are currently in you can type ls to see the content in this directory. After you found your rosetta[releasenumber].tar.gz file you unpack it with the tar command and change directory to the rosetta[releasenumber]/main/source folder. We added an example of Rosetta unpacking below. 
#Assuming you downloaded the Rosetta Source Code to the directory “Software” on your c drive on a linux subsystem for windows this would be your way of unpacking:
>cd /mnt/c/Software
>tar -xvzf rosetta[releasenumber].tar.gz
>cd rosetta[releasenumber]/main/source 
#[releasenumber] can be simply replaced by the Rosetta version you downloaded
We recommend gcc as a compiler because it provides goof performance and can easily installed using apt-get. After you installed gcc you can continue to the main Rosetta installation. 
#sudo gives superuser allowance, that is needed to install the program. You also need to enter your password before the installation begins.
>sudo apt-get gcc
Rosetta provides you a python script that is able to locate your installed compiler and automatically compile the program afterwards. Use the following command to change your current directory to the source folder of Rosetta. 
#The -j flags defines the number of cores Rosetta can use to compile the source code. The more cores you pick the shorter the installation will take. Take in mind that a installation of Rosetta will take really long, if you are compiling on a single core it can take longer than 24h.
> ./scons.py -j  mode=release bin
After the installation is finished the bin directory should be added to your source folder. You can check if the installation was successful by running one of Rosetta’s multiple in the bin folder. This is done by simply writing the name of the program in the command prompt. If you want extras added to your installation like MPI, which can be used for big modelling runs because it allows the execution of multiple Rosetta tasks on multiple cores you can add an additional flag in your scons command. You can see all options in the Rosetta documentation. 
#Checking if the installation was successful: When the error thrown after running the application is thrown by Rosetta the installation succeeded. 
>$PATH_TO_ROSETTA$/main/source/bin/AbinitioRelax.default.linuxgccrelease
ERROR: Error: can't read sequence! Use -in::file::fasta sequence.fasta or -in::file::native native.pdb!
ERROR:: Exit from: src/protocols/abinitio/AbrelaxApplication.cc line: 372
[ ERROR ]: Caught exception:
File: src/protocols/abinitio/AbrelaxApplication.cc:372
[ ERROR ] UtilityExitException
ERROR: Error: can't read sequence! Use -in::file::fasta sequence.fasta or -in::file::native native.pdb! 
#The internal Rosetta error shows us that Rosetta is installed.

#Rosetta wih MPI support can be added by adding the mpi flag to your scons command:
>./scons.py extras=mpi
Now you successfully installed Rosetta and can continue with the tutorials either on our Wiki page, the Meiler Lab page or the Rosetta Documentation itself.

Rosetta Docking

Rosetta CM

An amazing tool for structure prediction is RosettaCM. To model a protein sequence’s structure, you need to find 3D-structures of proteins that possess a high homology to your target sequence. These structures can be found using blast applications such as NCBI blasptp by blasting your target sequence against the PDB database. The results can than be aligned to your target sequence using the Clustal Omega webserver. You then have to convert the alignments to the Rosetta .grishin datatype. We provide you an example of our target sequence EreB aligned with pdb entry 2RAD_A. 
## WP_063844486 2rad.pdb
#
scores_from_program: 0
0 ---------------------------------------MRFEEWVKDKHIPFKLNHPDDNYDDFKPLRKIIGDTRVVALGENSHFIKEFFLLRHTLLRFFIEDLGFTTFAFEFGFAEGQIINNWIHGQGTDDEIGRFLKHFYYPEELKTTFLWLREYNKAA--KEKITFLGIDIPRNGGSYLPNMEIVHDFFRTADKEALHIIDDAFNIAKKIDYFSTSQAALNLHELTDSEKCRLTSQLARVKVRLEAMAPIHIEKYGIDKYETILHYANGMIYLDYNIQAMSGFISG----GGMQG-DMGAKDKYMADSVLWHLKNPQSEQKVIVVAHNAHIQKTPILYDGFLSCLPMGQRLKNAIGDDYMSLGITSYSGHTAALYPEVDTKYGFRVDNFQLQEPNE-----GSVEK------AISGCGVTNSFVFFRNIPEDLQSIPNMIRFDSIYMKAELEKAFDGIFQIEKSSVSEVVYE-------
0 MKKKIIIAIVASAITMTHFVGNTYADSKTEVSVTAPYNTNQIAKWLEAHAKPLKTTNPTASLNDLKPLKNMVGSASIVGLGEATHGAHEVFTMKHRIVKYLVSEKGFTNLVLEEGWDRALELDRYVLTGK--GNPSQHLTPVFKTKEMLDLLDWIRQYNANPKHKSKVRVIGMDIQSVNENVYNN---IIEYIKANNSKLLPRVEEKIKG---LIPVTK--DMNTFESLTKEEKEKYVLDAKTISALLEENKS----------YLN--GKSKEFAWIKQNARIIEQFTTMLATPPDKPADFYLKHDIAMYENAKWTEEH--L-GKTIVWGHNGHVSKTNML--SFIYPKVAGQHLAEYYGKRYVSIGTSVYEGQYNVKNSDGEFG---PYGTLKSDDPNSYNYIFGQVKKDQFFIDLRKANGVTKTWLN—EQHPIFAGITTEGPDIPKTVDISLGKAFDILVQIQKVSPSQVHQLEHHHHHH
#The first sequence is the target sequence, the second sequence the template sequence. Note that the sequences should both be written in one line and both start with a 0 and a space. Further information on the Grishin format can be found in the Rosetta Documentation.
Now you can use the Rosetta partial thread applications to thread the target sequence on the structure of your template. 
#Threading of the template sequences. This step is performed for every structure used as a template. You need to provide the templates structure as a pdb file, the targets sequence as a fasta file, the structures’ alignment and the path to the Rosetta database.
> $PATH_TO_ROSETTA$/main/source/bin/partial_thread.default.linuxgccrelease -database $PATH_TO_ROSETTA$/main/database/ -in:file:fasta “target_structure.fasta” -in:file:alignment “alignment.grishin” -in:file:template_pdb “template.pdb” -ignore_unrecognized_res
To enhance the model’s precision structure fragments can be generated using the old Robetta webserver. The main script for Rosetta is a so-called XML File. You can add all steps Rosetta should perform by simply adding them to the Script. 
#This is the example XML script similar to the one provided on the Rosetta Documentation site. 
    
<ROSETTASCRIPTS>
    <TASKOPERATIONS>
    </TASKOPERATIONS>
    <SCOREFXNS>
        <ScoreFunction name="stage1" weights="score3" symmetric="0">
            <Reweight scoretype="atom_pair_constraint" weight="0.5"/>
        </ScoreFunction>
        <ScoreFunction name="stage2" weights="score4_smooth_cart" symmetric="0">
            <Reweight scoretype="atom_pair_constraint" weight="0.5"/>
        </ScoreFunction>
        <ScoreFunction name="fullatom" weights="talaris2013_cart" symmetric="0">
            <Reweight scoretype="atom_pair_constraint" weight="0.5"/>
        </ScoreFunction>
    </SCOREFXNS>
    <FILTERS>
    </FILTERS>
    <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" linmin_only="1">
            <Fragments three_mers="aat000_03_05.200_v1_3" nine_mers="aat000_09_05.200_v1_3"/>
            <Template pdb="2qgm.pdb.pdb" cst_file="AUTO" weight="1.000" />
            <Template pdb="2rad.pdb.pdb" cst_file="AUTO" weight="1.000"/>
            <Template pdb="3b55.pdb.pdb" cst_file="AUTO" weight="1.000"/>
        </Hybridize>
    </MOVERS>
    <APPLY_TO_POSE>
    </APPLY_TO_POSE>
    <PROTOCOLS>
        <Add mover="hybridize"/>
    </PROTOCOLS>
</ROSETTASCRIPTS>
Additionally, you have to give Rosetta the instructions on where to find the XML file, the target sequence and other options for example for the final structure relaxation. You can pick whatever options are suited for your simulation and simply add them to a text file that you add in the command prompt when starting the Rosetta application. 
#This is an example for an options file. You have to add the names of the structures behind the flags (start with a – and the description, e.g. -nstruct). You can also provide this information directly in the command prompt but it is easier to have a options file. Let’s assume we named our file “options”
# i/o
-in:file:fasta EreB.fasta
-parser:protocol ereb_hybridization.xml
-nstruct 1
-database /mnt/c/Software/rosetta_src_2020.08.61146_bundle/main/database
#-seed_offset  	# seed offset if necessary

# relax options
-relax:minimize_bond_angles
-relax:minimize_bond_lengths
-relax:jump_move true
-default_max_cycles 200
-relax:min_type lbfgs_armijo_nonmonotone
-relax:jump_move true
-hybridize:stage1_probability 1.0
-restore_talaris_behavior

# reduce memory footprint
-chemical:exclude_patches LowerDNA  UpperDNA Cterm_amidation SpecialRotamer VirtualBB ShoveBB VirtualDNAPhosphate VirtualNTerm CTermConnect sc_orbitals pro_hydroxylated_case1 pro_hydroxylated_case2 ser_phosphorylated thr_phosphorylated  tyr_phosphorylated tyr_sulfated lys_dimethylated lys_monomethylated  lys_trimethylated lys_acetylated glu_carboxylated cys_acetylated tyr_diiodinated N_acetylated C_methylamidated MethylatedProteinCterm

#Now the main simulation can be started using the Rosetta Scripts application, that is able to read the XML file and execute the commands:
>$PATH_TO_ROSETTA$/main/source/bin/rosetta_scripts.linuxgccrelease @options -database $PATH_TO_ROSETTA$/main/database

University

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Experts

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Former iGEMers

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Others

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