Error while running Enzyme Design

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- October 21, 2011 at 10:46 pm #1061
  Anonymous
  Hello,
  I am attempting to use the Enzyme Design application to redesign a ligand binding site to accept a different ligand. I am running the following command:
  
  $ROSETTA33/bin/enzyme_design.linuxgccrelease -database $ROSETTA33/rosetta_database -in:file:s input.pdb -in:file:extra_res_fa LG2.cen.params LG2.fa.params LG1.cen.params LG1.fa.params -enzdes:cstfile constraints.cst -enzdes:detect_design_interface -enzdes:cst_design
  
  where LG1 is a cofactor bound to the protein and LG2 is the ligand we are attempting to redesign the binding site for. When I run this, I am receiving the following error message:
  
  core.init: Mini-Rosetta version Release 3.3, from SVN 42942 from https://svn.rosettacommons.org/source/branches/releases/rosetta-3.3/rosetta_source core.init: command: /jgi/tools/edu_outreach/rosetta/3.3/rosetta_source/bin/enzyme_design.linuxgccrelease -database /jgi/tools/edu_outreach/rosetta/3.3/rosetta_database -in:file:s input.pdb -in:file:extra_res_fa LG2.cen.params LG2.fa.params LG1.cen.params LG1.fa.params -enzdes:cstfile constraints.cst -enzdes:detect_design_interface -enzdes:cst_design core.init: 'RNG device' seed mode, using '/dev/urandom', seed=838643726 seed_offset=0 real_seed=838643726 core.init.random: RandomGenerator:init: Normal mode, seed=838643726 RG_type=mt19937 core.scoring.etable: Starting energy table calculation core.scoring.etable: smooth_etable: changing atr/rep split to bottom of energy well core.scoring.etable: smooth_etable: spline smoothing lj etables (maxdis = 6) core.scoring.etable: smooth_etable: spline smoothing solvation etables (max_dis = 6) core.scoring.etable: Finished calculating energy tables. basic.io.database: Database file opened: pdb_pair_stats_fine basic.io.database: Database file opened: scoring/score_functions/hbonds/standard_params/HBPoly1D.csv basic.io.database: Database file opened: scoring/score_functions/hbonds/standard_params/HBFadeIntervals.csv basic.io.database: Database file opened: scoring/score_functions/hbonds/standard_params/HBEval.csv basic.io.database: Database file opened: env_log.txt basic.io.database: Database file opened: cbeta_den.txt basic.io.database: Database file opened: pair_log.txt basic.io.database: Database file opened: cenpack_log.txt basic.io.database: Database file opened: env_log.txt basic.io.database: Database file opened: cbeta_den.txt basic.io.database: Database file opened: pair_log.txt basic.io.database: Database file opened: cenpack_log.txt basic.io.database: Database file opened: CEN6_mem_env_log.txt basic.io.database: Database file opened: CEN10_mem_env_log.txt basic.io.database: Database file opened: memcbeta_den.txt basic.io.database: Database file opened: mem_pair_log.txt basic.io.database: Database file opened: P_AA basic.io.database: Database file opened: P_AA_n basic.io.database: Database file opened: P_AA_pp basic.io.database: Database file opened: Rama_smooth_dyn.dat_ss_6.4 core.scoring.etable: Using alternate parameters: LJ_RADIUS_SOFT in Etable construction. core.scoring.etable: Starting energy table calculation core.scoring.etable: smooth_etable: changing atr/rep split to bottom of energy well core.scoring.etable: smooth_etable: spline smoothing lj etables (maxdis = 6) core.scoring.etable: smooth_etable: spline smoothing solvation etables (max_dis = 6) core.scoring.etable: Finished calculating energy tables.
  
  ERROR: Residue names must be unique! ERROR:: Exit from: src/core/chemical/ResidueTypeSet.cc line: 374
  
  Do you have any ideas as to what we might be doing wrong? Thanks!
  Seth
- October 21, 2011 at 11:18 pm #6158
  Anonymous
  It very likely means that your new residue types share a name either with each other, or some residue type already in the database. Post the top 5 lines or so of your param files and I can diagnose further, if that isn’t enough to help you fix it.
- October 21, 2011 at 11:27 pm #6159
  Anonymous
  Hi Steven,
  Here are the top 5 lines from each of my params files:
  
  LG1.fa.params:
  NAME RET IO_STRING RET Z TYPE LIGAND AA UNK ATOM C12 aroC X -0.05
  
  LG1.cen.params:
  NAME RET IO_STRING RET Z TYPE LIGAND AA UNK ATOM C12 CAbb X -0.05
  
  LG2.fa.params:
  NAME BCT IO_STRING BCT Z TYPE LIGAND AA UNK ATOM C1 COO X 0.33
  
  LG2.cen.params:
  NAME BCT IO_STRING BCT Z TYPE LIGAND AA UNK ATOM C1 CAbb X 0.33
  
  Seth
- October 21, 2011 at 11:34 pm #6160
  Anonymous
  This should have been more obvious to me from your command line…
  
  You’ve passed ALL your parameters, centroid and fullatom, to extra_res_fa. This duplicates RET and BCT in the fullatom set. Pass the centroid versions to -extra_res_cen instead. (I think.)
- October 24, 2011 at 6:50 pm #6167
  Anonymous
  Thanks Steven and rmoretti,
  That problem was indeed caused by passing the centroid params to extra_res_fa. So I removed the centroid params files and reran it, and I’m getting a different error now:
  core.init: Mini-Rosetta version Release 3.3, from SVN 42942 from https://svn.rosettacommons.org/source/branches/releases/rosetta-3.3/rosetta_source core.init: command: /jgi/tools/edu_outreach/rosetta/3.3/rosetta_source/bin/enzyme_design.linuxgccrelease -database /jgi/tools/edu_outreach/rosetta/3.3/rosetta_database -in:file:s input.pdb -in:file:extra_res_fa LG2.fa.params LG1.fa.params -enzdes:cstfile constraints.cst -enzdes:detect_design_interface -enzdes:cst_design core.init: 'RNG device' seed mode, using '/dev/urandom', seed=1335275468 seed_offset=0 real_seed=1335275468 core.init.random: RandomGenerator:init: Normal mode, seed=1335275468 RG_type=mt19937 core.scoring.etable: Starting energy table calculation core.scoring.etable: smooth_etable: changing atr/rep split to bottom of energy well core.scoring.etable: smooth_etable: spline smoothing lj etables (maxdis = 6) core.scoring.etable: smooth_etable: spline smoothing solvation etables (max_dis = 6) core.scoring.etable: Finished calculating energy tables. basic.io.database: Database file opened: pdb_pair_stats_fine basic.io.database: Database file opened: scoring/score_functions/hbonds/standard_params/HBPoly1D.csv basic.io.database: Database file opened: scoring/score_functions/hbonds/standard_params/HBFadeIntervals.csv basic.io.database: Database file opened: scoring/score_functions/hbonds/standard_params/HBEval.csv basic.io.database: Database file opened: env_log.txt basic.io.database: Database file opened: cbeta_den.txt basic.io.database: Database file opened: pair_log.txt basic.io.database: Database file opened: cenpack_log.txt basic.io.database: Database file opened: env_log.txt basic.io.database: Database file opened: cbeta_den.txt basic.io.database: Database file opened: pair_log.txt basic.io.database: Database file opened: cenpack_log.txt basic.io.database: Database file opened: CEN6_mem_env_log.txt basic.io.database: Database file opened: CEN10_mem_env_log.txt basic.io.database: Database file opened: memcbeta_den.txt basic.io.database: Database file opened: mem_pair_log.txt basic.io.database: Database file opened: P_AA basic.io.database: Database file opened: P_AA_n basic.io.database: Database file opened: P_AA_pp basic.io.database: Database file opened: Rama_smooth_dyn.dat_ss_6.4 core.scoring.etable: Using alternate parameters: LJ_RADIUS_SOFT in Etable construction. core.scoring.etable: Starting energy table calculation core.scoring.etable: smooth_etable: changing atr/rep split to bottom of energy well core.scoring.etable: smooth_etable: spline smoothing lj etables (maxdis = 6) core.scoring.etable: smooth_etable: spline smoothing solvation etables (max_dis = 6) core.scoring.etable: Finished calculating energy tables. core.chemical.ResidueTypeSet: Finished initializing fa_standard residue type set. Created 4220 residue types protocols.jobdist.JobDistributors: Looking for an available job: 1 1 input.pdb 1 apps.public.enzdes.enzyme_design: Starting input_0001 ... core.conformation.Conformation: [ WARNING ] missing heavyatom: OXT on residue SER_p:CtermProteinFull 259 core.pack.task: Packer task: initialize from command line() protocols.enzdes.EnzdesFixBBProtocol: starting apply function... core.pack.dunbrack: Dunbrack library took 0.02 seconds to load from binary protocols.toolbox.match_enzdes_util.EnzConstraintIO: read enzyme constraints from retinalconstraints.cst ... done, 1 cst blocks were read. protocols.toolbox.match_enzdes_util.EnzConstraintIO: Generating constraints for pose... Error: residue RET260found in pdb header is not allowed by data in cstfile. ERROR:: Exit from: src/protocols/toolbox/match_enzdes_util/EnzCstTemplateRes.cc line: 272 protocols.toolbox.match_enzdes_util.EnzConstraintIO: checking cst data consistency for block 1... protocols.toolbox.match_enzdes_util.EnzConstraintIO: WARNING: Message(s) above was printed in the end instead of proper place because this Tracer object has some contents left in inner buffer when destructor was called. Explicit call Tracer::flush() or end your IO with std::endl to disable this warning.
  
  Here is the header of the PDB file:
  REMARK 666 MATCH TEMPLATE X RET 1 MATCH MOTIF A LYS 256 1 1
  
  And here are the contents of the constraint file (The constraint file is only constraining the cofactor to be in a position where it would be covalently bound to the protein):
  CST::BEGIN TEMPLATE:: ATOM_MAP: 1 atom_name: NZ TEMPLATE:: ATOM_MAP: 1 residue1: K
  
  TEMPLATE:: ATOM_MAP: 2 atom_name: C15 TEMPLATE:: ATOM_MAP: 2 residue3: RET
  
  CONSTRAINT:: distanceAB: 1.4 0.30 100.00 0 1 CST::END
- October 24, 2011 at 8:52 pm #6168
  Anonymous
  hey seth,
  i think the numbering in your cstfile is wrong.
  in the remarks, the RET ligand is listed before the lysine, but in the cstfile, the lysine is template 1 and ret is template 2.
  
  also, i think there are a few more problems with your setup
  1) if you’re constraining a covalent interaction, i would also constrain the angles in addition to the distance
  2) if you’re not marking the design positions in a resfile, you should specify values for the cut1-cut4 options (refer to documentation)
  3) i’d add -cst_min -chi_bin -bb_min -ex1 -ex2 -use_input_sc -design_min_cycles 3 to your commandline (refer to documentation)
  
  how are you placing the new ligand into the active site? the enzyme_design app won’t do this for you, you have to either use the matcher or do it manually.
  
  cheers,
  florian
- October 25, 2011 at 12:21 am #6175
  Anonymous
  Hi Florian,
  Thanks for the help! Changing the numbering in the cstfile did the trick. Also, I am using the other flags you specified, but I left them out during this trouble-shooting run to identify the minimal set of flags that produced the error.
  
  The ligand we’re trying to redesign the binding site for is bicarbonate, while the original ligand was chlorine. We just manually placed the bicarbonate where chlorine was in the active site. Do you think we should run the matcher anyways?
  
  A few additional questions:
  1) Should we prepack the starting structure before running it through enzyme design?
  2) We’re currently using the cut-offs -enzdes:cut1 6 -enzdes:cut2 8 -enzdes:cut3 10 -enzdes:cut4 12, but this causes the residue specified in our constraints which binds covalently to the cofactor to be repacked in the cst_min step. As a result, in the final structure, K265 has adopted a rotamer that places the amino group too far to covalently interact with the cofactor. Would you agree that the solution to this would be to also add a resfile that specifies the AUTO option and also specifies that this residue should not be repackable?
  3) We turned on the option -cst_opt to optimize the location of the ligand before running the design cycles, and we get the following error:
  protocols.enzdes.EnzdesFixBBProtocol: starting cst_opt minimization... ERROR: unknown atom_name: BCT CA ERROR:: Exit from: src/core/chemical/ResidueType.cc line: 1645
  Any ideas why we are getting this error?
  
  Thanks for your help!
  Seth
- November 10, 2011 at 8:22 pm #6248
  Anonymous
  hey seth,
  sorry for taking so long to get back to you, i was out of town for a bit.
  your questions
  
  1) does the starting structure already have your ligand in it, i.e. you want to pack the native sequence with the design ligand? that’s probably not necessary
  
  2) there’s another problem in your constraint file
  your line
  CONSTRAINT:: distanceAB: 1.4 0.30 100.00 0 1
  does not decalre the constraint to be covalent, but you’re clearly trying to enforce a covalent interaction. to declare the constraint covalent, you have to change the 4th column to 1 (refer to documentation), the line should read
  CONSTRAINT:: distanceAB: 1.4 0.30 100.00 1 1
  i would also add some angles to the constraint, to make sure you’re specifying the right interaction.
  
  3) how many ligands do you have in your pdb? you can also just email me your pdb+cstfile+params file and i’ll take a look at it? the error you’re getting means that rosetta is trying to mutate a non protein residue to ala
  
  cheers,
  florian
- May 11, 2012 at 12:33 pm #7096
  Anonymous
  Hello
  I was trying the enzyme design demo provided with the Rosetta package..
  I came across this problem.-
  
  core.pack.task: (1) Packer task: initialize from command line()
  protocols.enzdes.EnzdesFixBBProtocol: (1) Finished after design unconstrained repack/minimization…
  output file name: .//outdir_1/UM_1_D41H116K189_1tml_11_mocktim_1__DE_1.pdb
  ERROR: Unable to open file: .//outdir_1/UM_1_D41H116K189_1tml_11_mocktim_1__DE_1.pdb
  ERROR:: Exit from: src/protocols/jobdist/JobDistributors.cc line: 727
  
  Kindly help me on this.
- May 15, 2012 at 8:05 am #7113
  Anonymous
  Hello,
  THanks for the reply.
  I just created a dir ‘outdir_1’, and then it worked fine.
  Thanks once again.
- October 22, 2011 at 6:32 pm #6163
  Anonymous
  Standard enzyme design doesn’t need centroid parameters – it’s a full-atom only protocol. (There are some (currently unpublished, I believe) extensions which include things like loop remodeling which do go through centroid mode, though.)
  
  Of course, including centroid parameters with -extra_res_cen shouldn’t hurt, even if they aren’t needed.
- May 11, 2012 at 1:25 pm #7097
  Anonymous
  Does outdir_1 exist, and do you have write permissions for it? Rosetta generally can’t make missing directories for output.
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