View source for Rosetta High Resolution Protein Structure Refinement Protocol

== '''Introduction'''  ==

[http://depts.washington.edu/ventures/UW_Technology/Express_Licenses/rosetta.php Rosetta] is a molecular modeling software package developed in the Baker Laboratory (U. of Washington) for the study and prediction of protein structures, and protein-DNA and protein-protein interactions.&nbsp; This section describes how the program has been used for the unrestrained refinement of X-ray and NMR protein structures in the NESG&nbsp;(Ref. 1).

== '''Protocol'''  ==

Before running Rosetta, please make directory with: 

*rosetta_database (directory) 
**paths.txt 
**1pdb.pdb (PDB file for single xray or nmr structure, with chain A reference removed) 
**1pdb_.fasta (containing full length sequence) 
**aa1pdb_03_05.200_v1_3 (3mer fragment files generated for 1pdb sequence) 
**aa1pdb_09_05.200_v1_3 (9mer fragment files generated for 1pdb sequence)

Note: fragment files can be obtained from the [http://robetta.bakerlab.org/fragmentsubmit.jsp Robetta fragment server]. 

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=== '''X-ray Structure Refinement'''  ===

#Input pdb files into Rosetta format 
#Add "missing" residues, such as N- and C-term, relax 
#Idealize geometry 
#Relax single input structure with backbone perturbations and sidechain repacking.



=== '''NMR Structure Refinement'''  ===

#Input pdb files into Rosetta format 
#Add "missing" residues, such as N- and C-term, relax (not needed If NMR structure is for full length sequence) 
#Idealize geometry 
#



=== '''Commands''' ===
<pre>1. rosetta -score -fa_input -s xxxx.pdb -nstruct n -use_pdb_numbering
2. rosetta aa 1pdb _ -relax -looprlx -s &lt;your input file &gt; -fa_input -loop_file &lt;loop_file_name&gt; -nstruct1
3. rosetta -pose_idealize -s &lt;input file name&gt; -fa_input -nstruct 1
4. rosetta aa 1pdb _ -relax -farlx -looprlx -loop_file &lt;loop_file_name&gt; -ex1 -ex2 -fa_input -s &lt;input pdb file&gt; -random_loop -loop_skip_rate 0.3 -farlx_cycle_ratio 1.0 -full_filename -nstruct &lt; number of structures you want&gt;
5. rosetta aa 1pdb _ -relax -farlx -s &lt;input pdb file&gt; -fa_input -ex1 -ex2 -short_range_hb_weight 0.50 -long_range_hb_weight 1.0 -farlx_cycle_ratio 1.0 -full_filename -nstruct &lt;as many structures as you want&gt;
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=== '''Files for Download'''  ===

*Click to download [[media:Rosetta Protocol.doc|Rosetta_Protocol.doc]]: Rosetta protocol in word format 
*Click to download [[media:example NMR rosetta refinement.tar.gz|example_NMR_rosetta_refinement.tar.gz]]: Example files for NMR Rosetta Refinement 
*Click to download [[media:example Xray rosetta refinement.tar.gz|example_Xray_rosetta_refinement.tar.gz]]: Example files for Xray Rosetta Refinement

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== '''References'''  ==

1.&nbsp; Ramelot, T.A., Acton, T.B., Ma, L-C., Xiao, R., Montelione, G.T. and Kennedy, M.A. (2008) Improving NMR protein structure quality by Rosetta refinement: a molecular replacement study.''PROTEINS 75'': 147 - 167. 

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-- TheresaRamelot - 26 Aug 2008 

-- Edited by JimAramini - Nov 2009