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RAbD with N-term，Proline-like ncaa returns segmentation fault during CCDEndsGraftMover

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RAbD with N-term，Proline-like ncaa returns segmentation fault during CCDEndsGraftMover
#1
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Recently I'm stumbling with rosetta RAbD application (version 2020.08.61146) in hope to modify an antibody whose antigen containing a non-canonical,N-term, and PRO-like aa ,namely the pyroglutamic acid in its epitope. 

I generated params file according tutorials in :https://new.rosettacommons.org/demos/latest/public/design_with_ncaa/README,with input mol file below.

the product params runs properly during dock,relax,and RAbD's seq_design mode (except for wrong number in N-term hydrogen adding). but when doing graft design ,the application failed during CCDEndsGraftMover with a segmentation fault.

 

I had a series of attempts to solve the problem:

I fixed the patch file in  database/chemical/residue_type_sets/fa_standard( and centroid ) /patches .

I tried to use Pro's centroid Params for this one. I tried to modify the  ICOOR_INTERNAL lines so as to make the fold tree grows from CA (rather than from N , as the python script generated)  ,adding virtual N and cut_bonds(as Pro's params does) .

I tried to change the value of NBR_RADIUS.  

I even tried to modifying the params directly from PRO's by adding an acyl-oxgen atoms. 

 

Below are two files I finally got. Sadly neither of them works.  Due to limit error message, it's quite hard for me to debug the 'segmentation fault'.

 

Can anyone give it a look and help me out ?

thank you very much.

AttachmentSize
params_modified_from_PRO.txt2.36 KB
params_gen_and_modified_manually.txt1.99 KB
starting_pose_for_design.txt145.47 KB
Category: 
Design
Chemically Modified Residues
Fragment Generation
Post Situation: 
Unsolved
Sun, 2022-02-06 19:09
CordycepsLin
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This will be tricky.  Can you attach a full log of the crash?  I shoud be able to track it down from that.  

Sun, 2022-02-06 20:23
jadolfbr
(Reply to #3)
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here is the crash log. 

please note that  warning in line 17-19,30,109-112,129 and 292-296 existed even if I had removed the NCAA

File attachments: 
full_crash_log.txt
Mon, 2022-02-07 09:04
CordycepsLin
(Reply to #4)
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Thanks.  Is there anything in Rosetta_crash.log?  Are you able to try running it in debug mode? 

Mon, 2022-02-07 09:40
jadolfbr
(Reply to #5)
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crash log is here.  seems no valuable information.

File attachments: 
ROSETTA_CRASH.txt
Tue, 2022-02-08 18:14
CordycepsLin
(Reply to #6)
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I don't know how to run a debug mode. I'm searching for how to run it.

btw,I can offer the files needed  for bug-repeating, would you need them?  

Mon, 2022-02-07 22:33
CordycepsLin
(Reply to #7)
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Hi,

I added a flag: -out:level 500 to my original commands(/opt/rosetta_bin_linux_2020.08.61146_bundle/main/source/bin/antibody_designer.mpi.linuxgccrelease -s complex_3.pdb -primary_cdrs H1 -graft_design_cdrs H1 -do_dock -nstruct 1 -overwrite -constant_seed ).

but the file is larger than the size-limit of rosetta common. here is the last 7500 lines.

Is there anything useful? would you mind if I send the full log  to your mailbox?

thank you for your kindly guide! 

File attachments: 
tail7500_debug_log.txt
Tue, 2022-02-08 18:13
CordycepsLin
(Reply to #8)
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Hi, I checked the logfile and find some creepy traces:

here are some lines indicating trouble with processing my ncaa: 

core.chemical.Patch: (0) successfully patched: PCA to: DPCA
core.chemical.GlobalResidueTypeSet: (0) Residue 'PCA' not found in pdb components file, but is in regular ResidueTypeSet. Ignoring components.
core.chemical.GlobalResidueTypeSet: (0) Attempted to load 'PCA' from PDB components dictionary, but the appropriate entry wasn't found.
core.io.pose_from_sfr.PoseFromSFRBuilder: (0) Match: 'PCA:NtermProteinFull'; missing 7 coordinates
core.io.pose_from_sfr.PoseFromSFRBuilder: (0) [ WARNING ] discarding 1 atoms at position 123 in file complex_3.pdb. Best match rsd_type:  PCA:NtermProteinFull
Warning!  Unable to find countpair representatives for restype PCA

and here is the last error-like message  before segmentation fault: 

core.conformation.Conformation: (0) Residue 148 is not a branch point!

core.conformation.Conformation: (0) Residue 221 is not a branch point!

may they have anything to do with the bug?

Wed, 2022-02-09 19:39
CordycepsLin
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I would wager that the pyro-glu is an artifact of crystallization. Unless you have specific knowledge that the pyro-glu is critical to the function of the antigen, I suggest you mutate it back to Gln and proceed on your way with the natural AA.

Mon, 2022-02-07 09:39
nannemdp
(Reply to #10)
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there are  actually experimental evidences on the critical role of  this pyro-glu . After the enzyme catalysing this PTM knocked out , this antigen almost loses its function. 

See:https://pubmed.ncbi.nlm.nih.gov/31462760/

 

File attachments: 
PTM.png
Mon, 2022-02-07 22:28
CordycepsLin