FastRelax Mover with symmetry, ligand and membrane

Dear all,

I am currently trying to run the FastRelax Mover in RosettaScripts to relax a C4-symmetric protein with 4 ligands (1 per monomer). My input structure is a single protomer and a ligand (the pdb file unfortunately exceeds the maximum file size). My xml and option files look as follows. Note that I don't actually use the MembranePositionFromTopologyMover because it fills up 16 GB of memory pretty quickly. This leads to my first question. Can I just omit this mover if the protein is reasonably aligned and I specify a spanfile in the (Symmetric)AddMembraneMover?

XML File:

<ROSETTASCRIPTS>
    <TASKOPERATIONS>
        <RestrictToRepacking name="restrict"/>
        <IncludeCurrent name="keep_curr"/>
    </TASKOPERATIONS>
    <SCOREFXNS>
        <ScoreFunction name="memb_hires" weights="%%sfxn_weights%%"/>
    </SCOREFXNS>
    <FILTERS>
    </FILTERS>
    <MOVERS>
        <SetupForSymmetry name="setup_symm" definition="mHCN2_from_6UQG_07_S_0001.symm" set_global_symmetry_at_parsetime="0"/>
        <SymmetricAddMembraneMover name="add_memb" spanfile="mHCN2_from_6UQG_A.span"/>
        <MembranePositionFromTopologyMover name="init_pos"/>
        <FastRelax name="fast_relax" scorefxn="memb_hires" repeats="8"/>
    </MOVERS>
    <APPLY_TO_POSE>
    </APPLY_TO_POSE>
    <PROTOCOLS>
        <Add mover="setup_symm"/>
        <Add mover="add_memb"/>
        <!--Add mover="init_pos"/-->
        <Add mover="fast_relax"/>
    </PROTOCOLS>
    <OUTPUT scorefxn="memb_hires"/>
</ROSETTASCRIPTS>

 Options file:

-in:file:s mHCN2_from_6UQG_07_S_0001_INPUT.pdb
-in:file:extra_res_fa ../04_Threading/04_Ligand_Parameters/CMP.fa.params
-parser:script_vars sfxn_weights=franklin2019
-mp:setup:spanfiles mHCN2_from_6UQG_A.span
-parser:protocol mHCN2_from_6UQG_relax.xml
-out:file:scorefile model_scores.sc
-mp:lipids:composition DLPC
-out:pdb
-relax:jump_move true
-relax:fast
-packing:pack_missing_sidechains false
-score:weights franklin2019

In any case, RosettaScript exits with a rather uninformative error unless I delete the ligand from the PDB file.

AN INTERNAL ERROR HAS OCCURED. PLEASE SEE THE CONTENTS OF ROSETTA_CRASH.log FOR DETAILS.


terminate called after throwing an instance of 'std::out_of_range'
  what():  map::at
Got some signal... It is:6
Signal 6 (SIGABRT) means that the process was aborted.  This usually means an internal Rosetta error caused by (often) bad inputs, (sometimes) developer error, or (rarely) hardware problems.

Next, I figured, I should add the Ligand to the pose using the AddMPLigandMover, so I changed my XML file accordingly:

[...]
    <MOVERS>
        <SetupForSymmetry name="setup_symm" definition="mHCN2_from_6UQG_07_S_0001.symm"/>
        <SymmetricAddMembraneMover name="add_memb" spanfile="mHCN2_from_6UQG_A.span"/>
        <AddMPLigandMover name="add_lig" ligand_seqpos="1" closest_rsd="450"/>
        <MembranePositionFromTopologyMover name="init_pos"/>
        <FastRelax name="fast_relax" scorefxn="memb_hires" repeats="8"/>
    </MOVERS>
    <APPLY_TO_POSE>
    </APPLY_TO_POSE>
    <PROTOCOLS>
        <Add mover="setup_symm"/>
        <Add mover="add_memb"/>
        <Add mover="add_lig"/>
        <!--Add mover="init_pos"/-->
        <Add mover="fast_relax"/>
    </PROTOCOLS>
    <OUTPUT scorefxn="memb_hires"/>
</ROSETTASCRIPTS>

Unfortunately, RosettaScripts didn't like the idea of adding a ligand to an already symmetric membrane pose. It seems the AddMPLigandMover can only add ligands to non-symmetric membrane poses (I checked this on the monomer and it worked).

In thinking of a workaround, I decided to add a membrane to the monomer pose first, add the Ligand with the AddMPLigandMover, then SetupForSymmetry and then run FastRelax. This worked, the protein looks ok (despite a lot of "inaccurate G! step" warnings) and the ligand is there in all four protomers. Here is the final working XML file:

<ROSETTASCRIPTS>
    <TASKOPERATIONS>
        <RestrictToRepacking name="restrict"/>
        <IncludeCurrent name="keep_curr"/>
    </TASKOPERATIONS>
    <SCOREFXNS>
        <ScoreFunction name="memb_hires" weights="%%sfxn_weights%%"/>
    </SCOREFXNS>
    <FILTERS>
    </FILTERS>
    <MOVERS>
        <AddMembraneMover name="add_memb" spanfile="mHCN2_from_6UQG_A.span"/>
        <AddMPLigandMover name="add_lig" ligand_seqpos="580" closest_rsd="450"/>
        <SetupForSymmetry name="setup_symm" definition="mHCN2_from_6UQG_07_S_0001.symm"/>
        <FastRelax name="fast_relax" scorefxn="memb_hires" repeats="1"/>
    </MOVERS>
    <APPLY_TO_POSE>
    </APPLY_TO_POSE>
    <PROTOCOLS>
        <Add mover="add_memb"/>
        <Add mover="add_lig"/>
        <Add mover="setup_symm"/>
        <Add mover="fast_relax"/>
    </PROTOCOLS>
    <OUTPUT scorefxn="memb_hires"/>
</ROSETTASCRIPTS>

Since i symmetrize after adding the membrane, I now have 4 MEM residues that seem slightly different (see attachment). Although the protein looks OK, I have doubts about the results because of this. Is there any "non-workaround" solution that I should clearly be using here? I couldn't find any AddMPLigandMover equivalent that works on symmetric poses.

I would appreciate any help!

Best regards

Michele