Design with flexible loops

KEYWORDS: DESIGN LOOPS

Method 1: RosettaRemodel

Documentation: https://www.rosettacommons.org/docs/latest/application_documentation/design/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/getBluePrintFromCoords.pl .
   $> ./getBluePrintFromCoords.pl -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
   

   where

   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!):

   -in:file:fullatom
   -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
   -loops:ccd_closure
   -loops:random_loop
   -out:prefix 3k2m_
   -mut core.io.database
   -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:

   NATAA
   start
   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.