The scripts and input files that accompany this demo can be found in the demos/public directory of the Rosetta weekly releases.


Method 1: RosettaRemodel


  1. Preparing the PDB

    Take the PDB 3k2m.pdb. Select chain B and C. Get rid of HETATM. Do fast relax. (where $ROSETTA3=path-to-Rosetta/main/source)

    $> $ROSETTA3/bin/relax.default.linuxgccrelease -s input_files/3k2m_bc.pdb -ignore_unrecognized_res -use_input_sc -constrain_relax_to_start_coords -relax:fast -out:file:renumber_pdb

    Let's say the PDB is 3k2m_bc_relax.pdb. Delete the Chain ID column.

  2. Generating BluePrint

    $> cp scripts/ .
    $> ./ -pdbfile input_files/3k2m_bc_relax.pdb > test.blueprint

    The blueprint file has the information to direct the protocol on which residue to design and remodel.

  3. Editing BluePrint

    The interface on the monobody is Residue 182-187. We decided to rebuild with 1 residue insertion starting at 183. The blueprint has to be modified in the following way

    178 G .
    179 E .
    180 D .
    181 S .
    182 A L
    183 G L
    0 x L
    184 Y E
    185 M E
    186 F E
    187 M E
    188 Y .
    189 S .
    190 P .
    191 I .

    In the above example, "0 x L" will mean eXtension and the secondary structure assined for the inserted region in Loop.

    • Column 1 is the residue postion
    • Column 2 is the residue identity
    • Column 3 is the backbone behavior
  4. Running the remodel application (you should replace input_files/* with your files)

    > $ROSETTA3/bin/remodel.default.linuxgccrelease -s input_files/3k2m_bc_relax.pdb -remodel:blueprint input_files/test.blueprint -extrachi_cutoff 1 -ex1 -ex2 -use_input_sc -num_trajectory 3 -save_top 1 -use_clusters false -find_neighbors

Method 2: Loopmodel and Fixbb

We can use loopmodel with fragment files and fixbb with a resfile to design the monobody part of interface.

  1. Preparing the starting PDB

    Same as Step 1 in METHOD 1. We do not need to get rid of the Chai ID column in this case. The relaxed PDB 3k2m_bc_relax.pdb will be the input PDB.

  2. Creating fragment libraries

    Take the fasta file of 3k2m_bc_relax.pdb. Create fragment libraries of sizes 9 and 3 locally or through Robetta Server.

  3. Other input files

    Loop file: 3k2m.loop_file

    The format is as follows:

    #LOOP  start end cutpoint skip-rate extend
    LOOP 85 89 0 0.0 0
    LOOP 179 185 0 0.0 0


    column1  "LOOP":     The loop file identify tag
    column2  "integer":  Loop start residue number
    column3  "integer":  Loop end residue number
    column4  "integer":  Cut point residue number, >=startRes, <=endRes. default - let LoopRebuild choose cutpoint
    column5  "float":    Skip rate. default - never skip
    column6  "boolean":  Extend loop. Default false.
  4. Running the loopmodel application. For this run, you need to have your fragments ready in the input directory).

    > $ROSETTA3/bin/loopmodel.default.linuxgccrelease  @input_files/flags

    where the flags file consist of following options (edit path to database!):

    -loops:input_pdb input_files/3k2m_bc_relax.pdb
    -loops:loop_file 3k2m.loop_file
    -loops:frag_sizes 9 3 1
    -loops:frag_files aat000_09_05.200_v1_3 aat000_03_05.200_v1_3 none
    -loops:remodel quick_ccd
    -out:prefix 3k2m_
    -nstruct 5

    The actual experiment should have 1000-10000 nstruct.

  5. Running the fixbb application with a resfile:

    Create a list of the output PDBs from loop modeling.

    > $ROSETTA3/bin/fixbb.default.linuxgccrelease -l list -resfile resfile -extrachi_cutoff 1 -ex1 -ex2 -nstruct 5

    Resfile format:

    179 B ALLAAxc
    180 B ALLAAxc
    181 B ALLAAxc
    182 B ALLAAxc
    183 B ALLAAxc
    184 B ALLAAxc
    185 B ALLAAxc
  6. Select PDBs based on total_score

  7. Optional run for binding energy.