Rosetta High Resolution Protein Structure Refinement Protocol

Introduction

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.  This section describes how the program has been used for the unrestrained refinement of X-ray and NMR protein structures in the NESG [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 Robetta fragment server.

X-ray Structure Refinement

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

NMR Structure Refinement

1. Input pdb files into Rosetta format
2. Add "missing" residues, such as N- and C-term, relax (not needed If NMR structure is for full length sequence)
3. Idealize geometry
4. Rebuild loops, relax and minimize

Commands

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

Files for Download

• Click to download Rosetta_Protocol.doc: Rosetta protocol in word format
• Click to download example_NMR_rosetta_refinement.tar.gz: Example files for NMR Rosetta Refinement
• Click to download example_Xray_rosetta_refinement.tar.gz: Example files for Xray Rosetta Refinement

References

1.  Ramelot, T.A., Raman, S., Kuzin, A.P., Xiao, R., Ma, L-C., Acton, T.B., Hunt, J.F., Montelione, G.T., Baker, D., and Kennedy, M.A. (2008) Improving NMR protein structure quality by Rosetta refinement: a molecular replacement study.PROTEINS 75: 147 - 167.