Member Site › Forums › Rosetta 3 › Rosetta 3 – Applications › minirosetta: how to reserve (inter-chain) disulfides after relax ?
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January 24, 2015 at 6:56 pm #2130
Anonymous
(I would appreciate it very much if any of my questions can be answered.)
Dear friends,
I have been trying to reserve the disulfides of my target protein during the minirosetta comparative modelling. However, though I have gone through many posts, I still could not work it out after using several disulfide related flags.My situation is:
in the target protein:
Light chain (LC): residue 1-214
Heavy chain (HC): residue 215-442in the template Fab protein 4KMT:
residues 23 88 (light chain disulfide)
residues 134 194 (light chain disulfide)
residues 236 310 (heavy chain disulfide)
residues 356 412 (heavy chain disulfide)
residues 214 432 (inter-chain disulfide)In the target Fab protein C226S:
residues 23 88 (light chain disulfide)
residues 134 194 (light chain disulfide)
residues 236 310 (heavy chain disulfide)
residues 358 414 (heavy chain disulfide)
residues 214 434 (inter-chain disulfide)During the relax of minirosetta (option attached), the disulfide (especially the inter-chain disulfide) in target protein randomly exist. So I want to use some flags to keep them fixed.
PS: the last two pairs of disulfide are not of identical pose numbering between template and target.
PS: the last disulfide is inter-chain disulfide
PS: after minirosetta, the chain ID of “L” and “H” both change to “A”The following is the options for my minirosetta and my unsuccessful experience for the disulfide related flags.
-run:protocol threading
-in:file:alignment /home/lanselibai/Cheng/20150121_minirosetta/input/C226S.4KMT.aln
-cm:aln_format general
-frag3 /home/lanselibai/Cheng/20150121_minirosetta/input/aaC226S_03_05.200_v1_3
-frag9 /home/lanselibai/Cheng/20150121_minirosetta/input/aaC226S_09_05.200_v1_3
-in:file:fasta /home/lanselibai/Cheng/20150121_minirosetta/input/C226S.fasta
-in:file:fullatom
-loops:frag_sizes 9 3 1
-loops:frag_files /home/lanselibai/Cheng/20150121_minirosetta/input/aaC226S_09_05.200_v1_3 /home/lanselibai/Cheng/20150121_minirosetta/input/aaC226S_03_05.200_v1_3 none
-in:file:psipred_ss2 /home/lanselibai/Cheng/20150121_minirosetta/input/C226S.psipass2
-in:file:fullatom
-out:nstruct 10000
-in:file:template_pdb /home/lanselibai/Cheng/20150121_minirosetta/input/4KMT_FullAtom_missing_residue_in_HC.pdb
-loops:extended
-loops:build_initial
-loops:remodel quick_ccd
-loops:refine refine_ccd
-silent_decoytime
-random_grow_loops_by 4
-select_best_loop_from 1
-out:file:fullatom
-out:output
-out:pdb
-out:file:scorefile threaded_model.fasc
-overwrite
-loops:relax fastrelax
-relax:default_repeats 8#-in:fix_disulf /home/lanselibai/Cheng/20150121_minirosetta/input/disulfide_file
#-norepack_disulf
#-disulfide_file
#-detect_disulfide_before_relax#-disulf_filter true
#-rebuild_disulf false
#-detect_disulfides#-constraints:cst_fa_file /home/lanselibai/Cheng/20150121_minirosetta/input/inter_chain.cst
#-constraints:cst_file /home/lanselibai/Cheng/20150121_minirosetta/input/inter_chain.cst
Flag: -in:fix_disulf /home/lanselibai/Cheng/20150121_minirosetta/input/disulfide_fileThis seems to be the most related one (e.g. https://www.rosettacommons.org/node/3207).
However, by referring to “mainsourcesrccoreioraw_dataDisulfideFile.hh”, I tried to used the below in the “disulfidefile”:23 88
(a new blank line)But I got:
“Segmentation fault (core dumped)” and no record was in the log file.
The link below contains all my files.
https://copy.com/GOaBvrz3tBb4JSz2
When the “-in:fix_disulf” is disabled, it works all fine.
Can I ask why? I was trying if it can work for just one disulfide.
Flags not available:-norepack_disulf
-disulfide_fileI got the error message:
ERROR: ERROR: Option matching -norepack_disulf not found in command line top-level context
ERROR: ERROR: Option matching –disulfide not found in command line top-level context
Flag: -detect_disulfide_before_relaxWith this flag, the disulfide bond still randomly exist after the minirosetta. What is this flag supposed to do?
Flags that I still do not know:-disulf_filter true
-rebuild_disulf false
-detect_disulfidesThese flags appeared in some posts. But what do they really do?
How to filter outputs based on disulfide?According to Dr R Moretti said on https://www.rosettacommons.org/node/3207 , I think he refers to “dslf_fa13” in the score_jd2 method. However, as I rank the score based on “dslf_fa13” for 76 outputs (“score.sc” attached), some of the high dslf_fa13 score ones (e.g. 3.634) are also of all the disulfide while some of the low dslf_fa13 score ones (e.g. -2.877) are not of all the disulfide. Therefore, we cannot simply filter out the outputs with all the 5 disulfide based on “dslf_fa13”. As a result, the rule of “the lower of the dslf_fa13, the more disulfide exist” does not exist.
What is the proper constraint to use?I think SiteConstraint should be the most related one as the residue 214 in LC should interact with HC by the inter-chain disulfide. So I write the below in the “inter_chain.cst” file and apply “-constraints:cst_fa_file” and “-constraints:cst_file” in the option file (https://www.rosettacommons.org/docs/latest/constraint-file.html):
SiteConstraint CA 214L H SIGMOID 5.0 2.0
However, I got “Segmentation fault (core dumped)” and the log file is attached.
I also do not understand the meaning of “AmbiguousConstraint” as smlewis suggested on https://www.rosettacommons.org/node/3207.
I would be very grateful if someone can help me for any of the problems I have encountered. Thank you very much.Yours sincerely
Cheng
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