This entry list the main requirements to execute a simple FunFolDes run.

Problem setup

Let's assume that we have:

template.pdb: A PDB that contains our template.
motif.pdb: A PDB that contains our motif.

And we know:

We will use chain A from template.pdb as our actual template.
We will select as our insert_point into the template the segment: 50A-65A.
We will define our motif as 30B-45B from motif.pdb.
We will consider chain A from motif.pdb as a binder to our motif.

Finally, we'll need a vall database in order to be able to create the fragments.

Making the script

ResidueSelectors

To properly run FunFolDes, one needs to add the ResidueSelectors to pick each of the working labels and guide the non-fixed parts of the process. The following ones are the ones needed to generate the FLL-standard behaviour, and one can more or less consider that they should always be added:

<RESIDUE_SELECTORS>
  <ResiduePDBInfoHasLabel name="MOTIF"     property="MOTIF" />
  <Not                    name="!MOTIF"    selector="MOTIF" />
  <ResiduePDBInfoHasLabel name="TEMPLATE"  property="TEMPLATE" />
  <Not                    name="!TEMPLATE" selector="TEMPLATE" />
  <ResiduePDBInfoHasLabel name="CONTEXT"   property="CONTEXT" />
  <Not                    name="!CONTEXT"  selector="CONTEXT" />
  <ResiduePDBInfoHasLabel name="FLEXIBLE"  property="FLEXIBLE" />
  <Not                    name="!FLEXIBLE" selector="FLEXIBLE" />
  <ResiduePDBInfoHasLabel name="HOTSPOT"   property="HOTSPOT" />
  <Not                    name="!HOTSPOT"  selector="HOTSPOT" />
  <ResiduePDBInfoHasLabel name="COLDSPOT"  property="COLDSPOT" />
  <Not                    name="!COLDSPOT" selector="COLDSPOT" />
  
  # can design
  <Or name="COLDSPOT_OR_TEMPLATE"              selectors="COLDSPOT,TEMPLATE" />
  # bb movement
  <Or name="FLEXIBLE_OR_TEMPLATE"              selectors="FLEXIBLE,TEMPLATE" />
  # chi movement
  <Or name="COLDSPOT_OR_FLEXIBLE_OR_TEMPLATE"  selectors="COLDSPOT,FLEXIBLE,TEMPLATE" />
  # prevent repacking and design
  <Or name="HOTSPOT_OR_CONTEXT"                selectors="HOTSPOT,CONTEXT" /> 
  <And name="HOTSPOT_OR_CONTEXT_AND_!FLEXIBLE" selectors="HOTSPOT_OR_CONTEXT,!FLEXIBLE" />
  # no design
  <And name="FLEXIBLE_AND_!COLDSPOT"           selectors="FLEXIBLE,!COLDSPOT" />

And we also need to add the specific selectors for our exercise. Be aware that the design ChainResidueSelector is setup to B. This is due to the fact that designs pull their chainID from the chainID of the motif. This way, there can be no repetition of the identifier if the binder is added.

  <Chain name="template" chains="A" />
  <Index name="insert" resnums="50A-65A" />
  <Index name="motif" resnums="30B-45B" />
  <Chain name="binder" chains="A" />
  <Chain name="design" chains="B" />
</RESIDUE_SELECTORS>

TaskOperations

There are 3 main TaskOperations needed for FunFolDes; the one that defines static-non designable residues (FFLMOTIF_TASKOP); the one that defines packable-non-designable residues (FFLFLEX_TASKOP) and the one that defines bbflex-designable residues (FFLTEMPLATE_TASKOP)

<TASKOPERATIONS>
  <OperateOnResidueSubset name="FFLMOTIF_TASKOP" selector="HOTSPOT_OR_CONTEXT_AND_!FLEXIBLE" >
    <PreventRepackingRLT/>
  </OperateOnResidueSubset>
  <OperateOnResidueSubset name="FFLFLEX_TASKOP" selector="FLEXIBLE_AND_!COLDSPOT" >
    <RestrictToRepackingRLT/>
  </OperateOnResidueSubset>
  <OperateOnResidueSubset name="FFLTEMPLATE_TASKOP" selector="COLDSPOT_OR_TEMPLATE" >
    <DisallowIfNonnativeRLT disallow_aas="C" />
  </OperateOnResidueSubset>
</TASKOPERATIONS>

MoveMapFactory

MoveMapFactory allows to generate MoveMaps on apply time through residue selectors. By defining this FFLSTANDARD_MOVEMAP, we can guide the non-fixed parts of the protocol so that they still behave as expected.

<MOVE_MAP_FACTORIES>
  <MoveMapFactory name="FFLSTANDARD_MOVEMAP" bb="false" chi="false" nu="false" branches="false" jumps="false" >
    <Backbone enable="true" residue_selector="FLEXIBLE_OR_TEMPLATE" />
    <Chi      enable="true" residue_selector="COLDSPOT_OR_FLEXIBLE_OR_TEMPLATE" />
  </MoveMapFactory>
</MOVE_MAP_FACTORIES>

Movers

First of all, we will need to load/make the structural-based fragments:

<StructFragmentMover name="FragmentPicker" prefix="auto"
  vall_file="path/to/vall/database/vall.jul19.2011.gz" output_frag_files="1"
  small_frag_file="auto.200.3mers" large_frag_file="auto.200.9mers"
/>

This will create the fragments unless the fragment files exist, in which case they will just be loaded. Take notice of the prefix attribute. This sets up the identifier of these fragments, as other fragments for other processes might also be added.

Before folding our protein it is recommended to generate atom_pair_constraints to guide it. This can be very easily done:

<AddConstraints name="foldingCST" >
  <AtomPairConstraintGenerator name="atompairCST1" sd="1.5" ca_only="true"
    use_harmonic="true" unweighted="true" min_seq_sep="6" max_distance="40" residue_selector="template"
  />
</AddConstraints>

Now we can call NubInitioMover:

<NubInitioMover name="FunFolDes" fragments_id="auto" template_motif_selector="insert" >
  <Nub pose_file="motif.pdb" residue_selector="motif" binder_selector="binder" />
</NubInitioMover>

One could also first load the PDB file into a pose and then pass it as a reference_pose; something like:

<SavePoseMover name="readMotif" reference_name="motif_pose" pdb_file="motif.pdb" />
<NubInitioMover name="FunFolDes" fragments_id="auto" template_motif_selector="insert" >
  <Nub reference_name="motif_pose" residue_selector="motif" binder_selector="binder" />
</NubInitioMover>

There are some alternatives to the most basic call of NubInitioMover.
For example, let's say that one wants two residues on each side of the motif to be able to move, hopping for a better fit into the template, and one also considers that residues 2,4,6,8,13 (inside the motif count) are not in actual contact with the binder and, thus, they can be designed. One can target specific parameters of each segment in the motif:

<NubInitioMover name="FunFolDes" fragments_id="auto" template_motif_selector="insert" >
  <Nub pose_file="motif.pdb" residue_selector="motif" binder_selector="binder" >
    <Segment order="1" n_term_flex="2" c_term_flex="2" editable="2,4,6,8,13" />
  </Nub>
</NubInitioMover>

Once the folding is done, we can guide the design process with the previously defined MoveMap and TaskOperations:

<FastDesign name="DesignRelax" scorefxn="fullatom" clear_designable_residues="true"
  task_operations="FFLMOTIF_TASKOP,FFLFLEX_TASKOP,FFLTEMPLATE_TASKOP"
  repeats="3" delete_virtual_residues_after_FastRelax="true"
  movemap_factory="FFLSTANDARD_MOVEMAP" >
</FastDesign>

And last, we make sure that the structure is properly closed (this is unnecessary with only one inserted segment, but in those cases it will simply not do anything).

<NubInitioLoopClosureMover name="loopC" fragments_id="auto"
  break_side_ramp="true" design="true" fullatom_scorefxn="fullatom" />

All together

In summary, let's put all together with some extra boilerplate, and a regular FunFolDes script without any special evaluation/filter or added design conditions will look like:

<ROSETTASCRIPTS>

  <SCOREFXNS>
    # A weight is added to small-range hbonds to favor helix, and beta pairing
    # and to take residue propensity into account. This way, we can favor
    # helix-forming residues in our designs.
    <ScoreFunction name="fullatom" weights="ref2015">
      <Reweight scoretype="atom_pair_constraint" weight="1.6" />
    </ScoreFunction>
  </SCOREFXNS>

  <RESIDUE_SELECTORS>
    <ResiduePDBInfoHasLabel name="MOTIF"     property="MOTIF" />
    <Not                    name="!MOTIF"    selector="MOTIF" />
    <ResiduePDBInfoHasLabel name="TEMPLATE"  property="TEMPLATE" />
    <Not                    name="!TEMPLATE" selector="TEMPLATE" />
    <ResiduePDBInfoHasLabel name="CONTEXT"   property="CONTEXT" />
    <Not                    name="!CONTEXT"  selector="CONTEXT" />
    <ResiduePDBInfoHasLabel name="FLEXIBLE"  property="FLEXIBLE" />
    <Not                    name="!FLEXIBLE" selector="FLEXIBLE" />
    <ResiduePDBInfoHasLabel name="HOTSPOT"   property="HOTSPOT" />
    <Not                    name="!HOTSPOT"  selector="HOTSPOT" />
    <ResiduePDBInfoHasLabel name="COLDSPOT"  property="COLDSPOT" />
    <Not                    name="!COLDSPOT" selector="COLDSPOT" />
  
    # can design
    <Or name="COLDSPOT_OR_TEMPLATE"              selectors="COLDSPOT,TEMPLATE" />
    # bb movement
    <Or name="FLEXIBLE_OR_TEMPLATE"              selectors="FLEXIBLE,TEMPLATE" />
    # chi movement
    <Or name="COLDSPOT_OR_FLEXIBLE_OR_TEMPLATE"  selectors="COLDSPOT,FLEXIBLE,TEMPLATE" />
    # prevent repacking and design
    <Or name="HOTSPOT_OR_CONTEXT"                selectors="HOTSPOT,CONTEXT" /> 
    <And name="HOTSPOT_OR_CONTEXT_AND_!FLEXIBLE" selectors="HOTSPOT_OR_CONTEXT,!FLEXIBLE" />
    # no design
    <And name="FLEXIBLE_AND_!COLDSPOT"           selectors="FLEXIBLE,!COLDSPOT" />

    <Chain name="template" chains="A" />
    <Index name="insert" resnums="50A-65A" />
    <Index name="motif" resnums="30B-45B" />
    <Chain name="binder" chains="A" />
    <Chain name="design" chains="B" />
  </RESIDUE_SELECTORS>
  <TASKOPERATIONS>
    <OperateOnResidueSubset name="FFLMOTIF_TASKOP" selector="HOTSPOT_OR_CONTEXT_AND_!FLEXIBLE" >
      <PreventRepackingRLT/>
    </OperateOnResidueSubset>
    <OperateOnResidueSubset name="FFLFLEX_TASKOP" selector="FLEXIBLE_AND_!COLDSPOT" >
      <RestrictToRepackingRLT/>
    </OperateOnResidueSubset>
    <OperateOnResidueSubset name="FFLTEMPLATE_TASKOP" selector="COLDSPOT_OR_TEMPLATE" >
      <DisallowIfNonnativeRLT disallow_aas="C" />
    </OperateOnResidueSubset>
  </TASKOPERATIONS>
  <MOVE_MAP_FACTORIES>
    <MoveMapFactory name="FFLSTANDARD_MOVEMAP" bb="false" chi="false" nu="false" branches="false" jumps="false" >
      <Backbone enable="true" residue_selector="FLEXIBLE_OR_TEMPLATE" />
      <Chi      enable="true" residue_selector="COLDSPOT_OR_FLEXIBLE_OR_TEMPLATE" />
    </MoveMapFactory>
  </MOVE_MAP_FACTORIES>
  <MOVERS>
    # ** SavePoseMover used like this does not need to be called during PROTOCOL to work.
    <SavePoseMover name="readMotif" reference_name="motif_pose" pdb_file="motif.pdb" />

    ## PREPROCESSING: MAKING FRAGMENTS
    <StructFragmentMover name="makeFrags" prefix="T12"
      small_frag_file="%%pdb%%.%%frags%%.200.3mers" large_frag_file="%%pdb%%.%%frags%%.200.9mers"
    />

    ## PREPROCESSING: CONSTRAINTS
    <StructFragmentMover name="FragmentPicker" prefix="auto"
      vall_file="path/t/vall/database/vall.jul19.2011.gz" output_frag_files="1"
      small_frag_file="auto.200.3mers" large_frag_file="auto.200.9mers"
    />

    # MAIN: NUBINITIO FOLDING
    <NubInitioMover name="FunFolDes" fragments_id="auto" template_motif_selector="insert" >
      <Nub reference_name="motif_pose" residue_selector="motif" binder_selector="binder" />
    </NubInitioMover>

    # POSTPROCESSING: CONSTRAINTS
    <AddConstraints name="designCST" >
      <AtomPairConstraintGenerator name="atompairCST2" sd="1" ca_only="true"
        use_harmonic="true" unweighted="true" min_seq_sep="6" max_distance="40" residue_selector="design"
      />
    </AddConstraints>
    <ClearConstraintsMover name="clearCST" />

    # POSTPROCESSING: DESING
    <FastDesign name="DesignRelax" scorefxn="fullatom" clear_designable_residues="true"
      task_operations="FFLMOTIF_TASKOP,FFLFLEX_TASKOP,FFLTEMPLATE_TASKOP"
      repeats="3" delete_virtual_residues_after_FastRelax="true"
      movemap_factory="FFLSTANDARD_MOVEMAP" >
    </FastDesign>

    # POSTPROCESSING: LOOP CLOSURE
    <NubInitioLoopClosureMover name="loopC" fragments_id="auto"
      break_side_ramp="true" design="true" fullatom_scorefxn="fullatom" />
  </MOVERS>
  <PROTOCOLS>
    # PREPROCESSING
    <Add mover="makeFrags"   />
    <Add mover="foldingCST"  />
    # MAIN
    <Add mover="FunFolDes"  />
    <Add mover="clearCST"   />
    # POSTPROCESSING
    <Add mover="designCST"   />
    <Add mover="DesignRelax" />
    <Add mover="loopC"       />
  </PROTOCOLS>
</ROSETTASCRIPTS>