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August 2, 2021 at 1:51 pm #3821
Anonymous
Hello,
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:
NAME UNK
IO_STRING UNK Z
TYPE LIGAND
AA UNK
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 X1 VIRT VIRT 0.53
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_ATOM C3
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
CST::BEGIN
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
CST::END
#block2 for pi-stacking with the ligand
CST::BEGIN
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
SECONDARY_MATCH: DOWNSTREAM
ALGORITHM_INFO::END
#ALGORITHM_INFO:: match_positions
# all
#ALGORITHM_INFO::END
CST::END
example of a typical xml:
<ROSETTASCRIPTS>
<SCOREFXNS>
<ScoreFunction name="ref15_cst" weights="ref2015_cst.wts">
<Reweight scoretype="res_type_constraint" weight="0.64"/>
</ScoreFunction>
<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"/>
</ScoreFunction>
</SCOREFXNS>
<RESIDUE_SELECTORS>
Index name="catres_res" resnums="%%catres%%"/>
<ResidueName name="Lig" residue_names="UNK"/>
</RESIDUE_SELECTORS>
<TASKOPERATIONS>
<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">
<PreventRepackingRLT/>
</OperateOnResidueSubset>
<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"/>
</TASKOPERATIONS>
<FILTERS>
<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
</FILTERS>
<MOVERS>
<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"/>
</ParsedProtocol>
<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"/>
</MOVERS>
<PROTOCOLS>
<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"/>
</PROTOCOLS>
<OUTPUT scorefxn="ref15_cst" />
</ROSETTASCRIPTS>
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
-enzdes
-parser_read_cloud_pdb
-detect_design_interface
-packing
-extrachi_cutoff 1
-use_input_sc
-flip_HNQ
-no_optH false
-optH_MCA false
-linmem_ig 10
-nblist_autoupdate
-parser:protocol /home/labs/fleishman/dinal/kemp_elim/Design/in/deNovo_design.xml
-run:preserve_header
-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
-pdb_gz
-overwrite
-out:no_nstruct_label
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