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RosettaDesign
 
RosettaDesign
RosettaDesign is a program for finding low free energy sequences for a given target protein backbone.

Instructions for running RosettaDesign (rDesign)     (last modified 12/02/03)

rDesign can be run in several different modes.  They include:

1) repacking side chains on a fixed backbone
2) redesigning on a fixed backbone
3) redesigning with a flexible backbone

See below for a description of the files and commands you will need to run
in these different modes.

The RosettaDesign distribution comes with directories that contain 
sample runs for the different modes.

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files you will want in your running directory:

      1) starting pdb structure
      2) paths.txt (this file specifies the location of input/output 
                 for rDesign, a default paths.txt file is supplied with the 
                 rosetta source code)
      3) resfile (this file is optional, it is used to specifiy a subset 
               of residues to redesign.  The format for this file is 
               described below.)

      additional files for designing with a flexible backbone
          
           4) fragment files (see below) 		      
           5) a pdb file with a name of the form xxxx.pdb that agrees
               with the fragement names (see below)

other files/directories you will need

      rosetta_database directory (this directory is supplied with rosetta
                and contains a number of databases needed by rosetta.  This
                directory can be anywhere in your file system, it is just 
                neccessary to specify the location in the paths.txt file.)
      
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editing the paths.txt file

	most lines can be left unchanged
	make sure the path for 'data files' points to your rosetta_database
        if running with a flexible backbone, make sure that the names
           for the fragment files are correctly defined.

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obtaining fragment files for designing with a flexible backbone.s

       fragment files can be obtained at:
       http://robetta.bakerlab.org/fragmentsubmit.jsp

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command line flags:

  flags common to all modes of rosettadesign
     -design                       :tells rosetta to run in design mode,
     -s [filename]                 :starting structure
     -l [filename]                 :list of starting structures
     -resfile [filename]           :specifies which residues to redesign 
     -ex1                          :extra chi 1 rotamers
     -ex2                          :extra chi 2 rotamers
     -extrachi_cutoff [integer]    :residues with more neighbors than 
                                    the specified number get extra 
                                    chi 1 and chi 2 rotamers if 
                                    ex1 and ex2 are true
     -ala_ref [real]               :this energy will be subtracted 
                                    from all ala
     -cys_ref [real]               :ditto for cys


  flags for repacking sidechains
    -onlypack       :just repack sidechains


  flags for redesigning a fixed backbone
    -fixbb          :just redesign sequence
    -ndruns [int]   :number of design runs, used when fixbb set true
    -pdbout [char]  :specify prefix for pdb files that will be output,
                       only used when fixbb set true

  flags for designing with a flexible backbone
    -mvbb           :move the backbone


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example command lines:

redesign using a resfile, output 3 structures each with a name that 
begins with test1
  % pFOLD.absoft -s 1ubq.pdb -design -fixbb -resfile restest 
-ndruns 3 -pdbout test1 >& log

just repack a protein with extra chi1 rotamers
  % pFOLD.absoft -s 1ubq.pdb -design -onlypack -ex1 

move the backbone and design: 
  -in this case the 3 standard arguments to rosetta must be used. these are: 
      (1) a two letter id code, for example 'ab'
      (2) 4 character code name for the protein, this must agree with 
              the name of the fragement files and a pdb file named
              xxxx.pdb that has the same number of residues as the
              fragments
      (3) chain id
  -your paths.txt file must point to the appropriate fragment files 
  -the starting structure must be idealized, see below for a description
     on how to idealize a structure

  % pFOLD.absoft aa 1hz5 _ -s 1hz5_idl.pdb -design -mvbb  >& log

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how to create a resfile: an input file that specifies which residues you 
   would like to redesign and with which amino acids

to make initial file use the program makeresfile provided with rosetta

command line arguments:

-p [protein name]
-nchains [integer] : default is 1
-chain [protein chain] (default _)
-chain2 [protein chain2]
-chain3 [protein chain3]
-chain4 [protein chain4]
-resfile [outout file name]
-default [id]  (default setting for each residue, default is NATRO)

example:

%> makeresfile -p 1ubq.pdb -default NATAA -resfile restest

Once the intitial resfile is made, you can edit it by hand to select the
residues that will be redesigned.  Here is a sample resfile.  All lines 
before the start are just header explaining the format of the file.  

---- sample resfile ----------------------------------
                                                  
 Column   2:  Chain                               
 Column   4-7:  sequential residue number         
 Column   9-12:  pdb residue number                
 Column  14-18: id  (described below)             
 Column  20-40: amino acids to be used            
                                                  
 NATAA  => use native amino acid                  
 ALLAA  => all amino acids                        
 NATRO  => native amino acid and rotamer          
 PIKAA  => select inividual amino acids (the program is dumb, 
            leave two spaces before inserting one letter codes)          
 POLAR  => polar amino acids                      
 APOLA  => apolar amino acids                     
                                                  
 The following demo lines are in the proper format
                                                  
 A    1    3 NATAA                                
 A    2    4 ALLAA                                
 A    3    6 NATRO                                
 A    4    7 NATAA                                
 B    5    1 PIKAA  DFLM   (two spaces before D important)              
 B    6    2 PIKAA  HIL                           
 B    7    3 POLAR                                
 -------------------------------------------------
start                                                  
 _    1    1 NATAA
 _    2    2 NATAA
 _    3    3 NATAA
 _    4    4 PIKAA  LKDIG
 _    5    5 NATAA
 _    6    6 POLAR
 _    7    7 NATAA
 _    8    8 NATRO
 _    9    9 NATAA
 _   10   10 ALLAA
 _   11   11 NATAA
 _   12   12 NATAA
 _   13   13 NATAA
 _   14   14 NATAA
     .
     .
     .
 _   73   73 NATAA
 _   74   74 NATAA
 _   75   75 NATAA
 _   76   76 NATAA


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how to idealize a structure: creates a structure that resembles the starting
structure but has ideal bond lengths and angles.

command line:
pFOLD.gnu -s 1ubq.pdb -idealize -fa_input > & log_idealize &



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Troubleshooting

1) The program seg faults immediately.  Check if your box has enough memory.  You can use the command size -A [executable name] to see how much memory the program wants.

2) You get the error message that max_res is exceeded.  Increase max_res in param.h and recompile.  If the program now requires too much memory try lowering maxrot.

RosettaDesign runs on Linux machines; the executable is:
/joule2/programs/rosetta/RosettaDesign/rosetta/pFOLD.gnu or just type "rosetta_design"

The program is installed in /joule2/programs/rosetta/RosettaDesign/rosetta/pFOLD.gnu:

The documentation is available from /joule2/programs/rosetta/RosettaDesign/README_user_guide

Examples are available from /joule2/programs/rosetta/RosettaDesign/example_fixbb_design and also /joule2/programs/rosetta/RosettaDesign/example_mvbb_design

The program is available from http://depts.washington.edu/ventures/UW_Technology/Express_Licenses/Rosetta/ but UCLA's technology transfer office has indicated that they will not accept the terms of the RosettaDesign academic license for the source code. So, they sent us an executable version of RosettaDesign.



Reference:
Simons et al. J Mol Biol 1997;268:209-225
Assembly of protein tertiary structures from fragments with similar local sequences using simulated annealing and Bayesian scoring functions.