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clashes created with enzdes

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clashes created with enzdes



I'm working on de novo enzyme design. Utilizing the enzdes application through theozyme definition. While doing regular design procedures I have noticed that Rosetta inserts amino acid identities that create clashes with the ligand. Most notable is an Ile with a distance of 3A to the ligand, instead of the minimum 4. Images attached. As far as I know, I have not altered the default behavior of vdw radii at any point.  The energies for these residues do not sky-rocket and Rosetta scores these designs very favorably.  

Any idea where this behavior can come from? Attaching param file for the ligand, a typical theozyme cst file, xml and flags. 

Thank you in advance, 


The param file for the ligand is as follows:

ATOM  C3  aroC  X   0.03
ATOM  C1  aroC  X   0.05
ATOM  C2  aroC  X   0.17
ATOM  O1  ONH2  X   -0.35
ATOM  N1  Nhis  X   -0.09
ATOM  C7  aroC  X   0.08
ATOM  H4  Haro  X   0.09
ATOM  C5  aroC  X   -0.01
ATOM  C6  aroC  X   0.02
ATOM  C4  aroC  X   0.27
ATOM  N2  Npro  X   0.08
ATOM  O2  OOC   X   -0.58
ATOM  O3  ONH2  X   0.04
ATOM  H3  Haro  X   0.07
ATOM  H2  Haro  X   0.07
ATOM  H1  Haro  X   0.07
BOND_TYPE  O1   N1  4   
BOND_TYPE  O1   C2  4   
BOND_TYPE  O2   N2  1   
BOND_TYPE  O3   N2  2   
BOND_TYPE  N1   C7  4   
BOND_TYPE  N2   C4  1   
BOND_TYPE  C1   C2  4   
BOND_TYPE  C1   C3  4   
BOND_TYPE  C1   C7  4   
BOND_TYPE  C2   C5  4   
BOND_TYPE  C3   C4  4   
BOND_TYPE  C3   H1  1   
BOND_TYPE  C4   C6  4   
BOND_TYPE  C5   C6  4   
BOND_TYPE  C5   H2  1   
BOND_TYPE  C6   H3  1   
BOND_TYPE  C7   H4  1   
BOND_TYPE  X1   C1  1   
CHI 1  C6   C4   N2   O2 
NBR_RADIUS 4.077500
ICOOR_INTERNAL    C3     0.000000    0.000000    0.000000   C3    C1    C2 
ICOOR_INTERNAL    C1     0.000000  180.000000    1.402570   C3    C1    C2 
ICOOR_INTERNAL    C2     0.000000   60.943856    1.384027   C1    C3    C2 
ICOOR_INTERNAL    O1   180.000000   70.652342    1.361113   C2    C1    C3 
ICOOR_INTERNAL    N1     0.000000   70.603861    1.389387   O1    C2    C1 
ICOOR_INTERNAL    C7     0.000000   74.708206    1.336420   N1    O1    C2 
ICOOR_INTERNAL    H4  -180.000000   61.149946    1.080381   C7    N1    O1 
ICOOR_INTERNAL    C5   180.000000   55.698289    1.391715   C2    C1    O1 
ICOOR_INTERNAL    C6     0.000000   63.971895    1.398802   C5    C2    C1 
ICOOR_INTERNAL    C4     0.000000   58.688975    1.398790   C6    C5    C2 
ICOOR_INTERNAL    N2   180.000000   60.487217    1.420992   C4    C6    C5 
ICOOR_INTERNAL    O2     0.000000   61.054258    1.254218   N2    C4    C6 
ICOOR_INTERNAL    O3   180.000000   61.059244    1.254323   N2    C4    O2 
ICOOR_INTERNAL    H3  -180.000000   62.856989    1.088162   C6    C5    C4 
ICOOR_INTERNAL    H2   180.000000   57.890384    1.083155   C5    C2    C6 
ICOOR_INTERNAL    X1    -0.000000  119.179329    1.400833   C1    C3    C2 
ICOOR_INTERNAL    H1   180.000000   60.729221    1.085561   C3    C1    C2 


the theozyme definition: 

# cst constraint descriptor for kemp eliminaze.

#block 1 for glu/asp, interaction with ligand 

 TEMPLATE::   ATOM_MAP: 1 atom_name:  C7 C1 N1  
 TEMPLATE::   ATOM_MAP: 1 residue3: UNK

TEMPLATE::   ATOM_MAP: 2 atom_type: OOC ,
TEMPLATE::   ATOM_MAP: 2 residue1:  DE

 CONSTRAINT:: distanceAB:   2.73  0.2   100.  1    1
 CONSTRAINT::    angle_A:  127.6  5.0   80.0  360. 1
 CONSTRAINT::    angle_B:  120.0  5.0   80.0  360. 1
 CONSTRAINT::  torsion_A:  180.0  7.0   70.0  360. 1
 CONSTRAINT:: torsion_AB:    0.0  0.0   0.00    1. 1 
 CONSTRAINT::  torsion_B:  180.0 10.0   10.0  360. 1

#block2 for pi-stacking with the ligand
TEMPLATE::   ATOM_MAP: 1 atom_name: X1 C5 C6
TEMPLATE::   ATOM_MAP: 1 residue3: UNK

TEMPLATE::   ATOM_MAP: 2 atom_type: aroC
TEMPLATE::   ATOM_MAP: 2 residue1:  WFY

CONSTRAINT:: distanceAB:     4.5    0.2   80.0    0   1
CONSTRAINT::    angle_A:    90.0    5.0   10.0  360   1
CONSTRAINT::    angle_B:    90.0    5.0   10.0  360   1
CONSTRAINT::  torsion_A:    90.0   15.0   10.0  180   1
#CONSTRAINT:: torsion_AB:     0.0   10.0   10.0   60   1 
CONSTRAINT::  torsion_B:    90.0   15.0   10.0  180   1


#ALGORITHM_INFO:: match_positions
#     all


example of a typical xml:


		<ScoreFunction name="ref15_cst" weights="ref2015_cst.wts">
			<Reweight scoretype="res_type_constraint" weight="0.64"/>
		<ScoreFunction name="soft_rep_full" weights="soft_rep">
			<Reweight scoretype="coordinate_constraint" weight="0.64"/>
			<Reweight scoretype="res_type_constraint" weight="0.64"/>
			<Reweight scoretype="atom_pair_constraint" weight="1"/>
			<Reweight scoretype="angle_constraint" weight="1"/>
			<Reweight scoretype="dihedral_constraint" weight="1"/>
			<Reweight scoretype="metalbinding_constraint" weight="1"/>
		Index name="catres_res" resnums="%%catres%%"/>
		 <ResidueName name="Lig" residue_names="UNK"/>	

		<InitializeFromCommandline name="init"/>
		<IncludeCurrent name="include_curr"/>	
		<RestrictToRepacking name="rtr"/>
		<LimitAromaChi2 name="limchi2"/>
		<SeqprofConsensus name="seqprofcons" min_aa_probability="-1" probability_larger_than_current="0" keep_native="1" filename="%%pssm%%" ignore_pose_profile_length_mismatch="1" debug="1" convert_scores_to_probabilities="0" chain_num="1"/>
		<OperateOnResidueSubset name="LigFix" selector="Lig">
		<DetectProteinLigandInterface name="edto" cut1="6.0" cut2="8.1" cut3="8.2" cut4="8.3" design="1"/>
		<DetectProteinLigandInterface name="edto_repack" cut1="6.0" cut2="8.1" cut3="8.2" cut4="8.3" design="0"/>
		<SetCatalyticResPackBehavior name="fix_res" fix_catalytic_aa="1"/>
		<SetCatalyticResPackBehavior name="rres" fix_catalytic_aa="0"/>

		<EnzScore name="allcst" score_type="cstE" scorefxn="ref15_cst" whole_pose="1" confidence="0" />
		EnzScore name="ligfarep" score_type="fa_rep" res_num="0" scorefxn="ref15_cst" confidence="0"/>
		LigInterfaceEnergy name="interfE" scorefxn="ref15_cst" include_cstE="1" jump_number="1"/>
		<DSasa name="lsasa" lower_threshold="0.3" upper_threshold="0.95" confidence="0"/>
		<DesignableResidues name="designable" task_operations="seqprofcons" designable="1" packable="1"/>	
		<RepackWithoutLigand name="ddg_noLigand" scorefxn="ref15_cst" energy_threshold="10000" confidence="0"/>
		<RepackWithoutLigand name="rms_NL_allProt" scorefxn="ref15_cst" target_res="all_repacked" rms_threshold="10000" confidence="0"/>
		<RepackWithoutLigand name="rms_NL_carbox" scorefxn="ref15_cst" target_cstids="1B" rms_threshold="10000" confidence="0"/>
		<RepackWithoutLigand name="rms_NL_arom" scorefxn="ref15_cst" target_cstids="2B" rms_threshold="10000" confidence="0"/>
		<IRmsd name="int_RMSD" jump="1" threshold="50" scorefxn="ref15_cst" confidence="0"/>
		<ShapeComplementarity name="shape_comp" min_sc="0" jump="1" write_int_area="true" confidence="0"/>
		<ScoreType name="total_energy" scorefxn="ref15_cst" score_type="total_score" threshold="0" confidence="0"/>
		<Sasa name="inter_sasa" threshold="0.0" upper_threshold="10000000000.0" jump="1" confidence="0"/>
		<TotalSasa name="total_sasa" threshold="0.0" upper_threshold="10000000000.0" confidence="0"/>
		<AtomicContactCount name="contacts" confidence="0"/>
		<PackStat name="packStat" threshold="0" chain="0" repeats="1" confidence="0"/>
		<CavityVolume name="cavity_vol" /> A value of 20 is approximately equal to the volume of a carbon atom


	        <ConstraintSetMover name="add_CA_cst" cst_file="%%cst_full_path%%"/>
		<AddOrRemoveMatchCsts name="cstadd" cst_instruction="add_new"/>
		<PackRotamersMover name="soft_design_fix" scorefxn="soft_rep_full" task_operations="init,seqprofcons,fix_res,edto,LigFix,limchi2,include_curr"/>
		<PackRotamersMover name="hard_design_fix" scorefxn="ref15_cst" task_operations="init,seqprofcons,fix_res,LigFix,limchi2,edto,include_curr"/>

		<PackRotamersMover name="soft_design" scorefxn="soft_rep_full" task_operations="init,seqprofcons,rres,edto,LigFix,limchi2,include_curr"/>
		<PackRotamersMover name="hard_design" scorefxn="ref15_cst" task_operations="init,seqprofcons,rres,LigFix,limchi2,edto,include_curr"/>
		<MinMover name="hard_min" scorefxn="ref15_cst" chi="1" bb="1" jump="1"/>
		PackRotamersMover name="fin_repack" scorefxn="ref15_cst" task_operations="init,rtr,rres,LigFix,limchi2,include_curr,edto_repack"/>
		<PackRotamersMover name="fin_repack" scorefxn="ref15_cst" task_operations="init,rtr,fix_res,LigFix,limchi2,include_curr,edto_repack"/>

		<AddOrRemoveMatchCsts name="cstrem" cst_instruction="remove" keep_covalent="1"/>
		<AddOrRemoveMatchCsts name="fincstadd" cst_instruction="add_pregenerated"/>
		<ParsedProtocol name="design_block_fix">
			<Add mover="soft_design_fix"/>
			<Add mover="hard_min"/>
			<Add mover="hard_design_fix"/>
			<Add mover="hard_min"/>
		<LoopOver iterations="4" mover_name="design_block_fix" name="iter4"/>
		DumpPdb name="dump" fname="before_filters.pdb" scorefxn="ref15_cst" tag_time="1"/>
		<InterfaceScoreCalculator name="int_scores" chains="X" scorefxn="ref15_cst"/>

        <Add mover="add_CA_cst"/>
	<Add mover_name="cstadd"/>
	<Add mover_name="iter4"/>
	Add mover_name="cstrem"/>
	<Add mover_name="fin_repack"/>
	<Add mover_name="hard_min"/>
	Add mover_name="fincstadd"/>
	<Add mover_name="int_scores"/>
	<Add filter= "designable"/>
	<Add filter="allcst"/>
	<Add filter="ddg_noLigand"/>
	<Add filter="rms_NL_allProt"/>
	<Add filter="rms_NL_carbox"/>
	<Add filter="rms_NL_arom"/>
	<Add filter_name="lsasa"/>
	<Add filter="inter_sasa"/>
	<Add filter="total_sasa"/>
	<Add filter_name="int_RMSD"/>
	<Add filter_name="shape_comp"/>
	<Add filter="total_energy"/>
	<Add filter="contacts"/>
	<Add filter="packStat"/>
	<Add filter= "cavity_vol"/>
	<OUTPUT scorefxn="ref15_cst" />


and a typical flag file:

-database /home/labs/fleishman/sarel/Rosetta_source/main/database/
-lj_hbond_hdis 1.75
-lj_hbond_OH_donor_dis 2.6
 -extrachi_cutoff 1
 -no_optH false
 -optH_MCA false
 -linmem_ig 10
-parser:protocol /home/labs/fleishman/dinal/kemp_elim/Design/in/deNovo_design.xml 
-nstruct 1
-chemical:exclude_patches LowerDNA UpperDNA Cterm_amidation SpecialRotamer VirtualBB ShoveBB VirtualDNAPhosphate VirtualNTerm CTermConnect sc_orbitals pro_hydroxylated_case1 pro_hydroxylated_case2 ser_phosphorylated thr_phosphorylated tyr_phosphorylated tyr_sulfatedlys_dimethylated lys_monomethylated lys_trimethylated lys_acetylated glu_carboxylated cys_acetylated tyr_diiodinated N_acetylated C_methylamidated MethylatedProteinCterm
-enzdes:cstfile /home/labs/fleishman/dinal/kemp_elim/theozime.cst
-extra_res_fa /home/labs/fleishman/dinal/kemp_elim/UNK_virt.params


Post Situation: 
Mon, 2021-08-02 06:51