disulfide bonds using abinitio folding module

[Anonymous]

Hi everybody,

I’m trying to fold a protein sequence using the abinitio module in rosetta3. I know that two cysteins make a disulfide bond so I have tried to put distance constrains between CA atoms in a .cst file but the structures generated do not have this bond formed. How can I tell rosetta that two cys are forming a disulfide bond?

Thanks a lot

[Anonymous]

There is a “disulfide file”, passed in via in::fix_disulf.

Check the commentary for file core/io/raw_data/DisulfideFile.hh to see how it works. I’ve never used it.

You may also need an option “abinitio::detect_disulfide_before_relax”.

[Anonymous]

> Hi everybody,
>
> I’m trying to fold a protein sequence using the abinitio module in rosetta3. I know that two cysteins make a disulfide bond so I have tried to put distance constrains between CA atoms in a .cst file but the structures generated do not have this bond formed. How can I tell rosetta that two cys are forming a disulfide bond?
>
> Thanks a lot

I have a native structure and in the native structure I have the disulfide bond. I fix residues to native, I use a disulfide file giving -fix_disulf filename and -detect_disulfides in the flag file. I also use the cst file where I put some distance constrains but even like this during the folding the native conformation is not maintain and the disulfide bond is not there. I have also added cst_weigth 1000. I have taken a look to the log file and at the begining of the folding says: detected disulfide bond between residues 19 and 45 which is correct!!!
Have any idea?

thanks!!!

[Anonymous]

> Hi everybody,
>
> I’m trying to fold a protein sequence using the abinitio module in rosetta3. I know that two cysteins make a disulfide bond so I have tried to put distance constrains between CA atoms in a .cst file but the structures generated do not have this bond formed. How can I tell rosetta that two cys are forming a disulfide bond?
>
> Thanks a lot

It seems that the program is not reading the cst_weights as in the score I can’t find the term of the constrains.
My flag file is like this:
-in:file:frag3 ../frag/aaTAGS_03_05.200_v1_3
-in:file:frag9 ../frag/aaTAGS_09_05.200_v1_3
-out:nstruct 6
-detect_disulfides
-disulf_filter true
-fix_disulf ./disulf.ini
-cst_weight 100000
-norepack_disulf
-abinitio:fix_residues_to_native 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97
-database /home/fenollar/rosetta3/rosetta3_database/
-constraints:cst_file ./TAGS_.cst
-out:pdb

the log finds the constrains files and the disulfide bond:

core.conformation.Conformation: Found disulfide between residues 16 49
core.pack.task: Packer task: initialize from command line()
core.io.fragments: reading fragments from file: ../frag/aaTAGS_09_05.200_v1_3 …
core.io.fragments: rosetta++ fileformat detected! Calling legacy reader…
core.fragments.ConstantLengthFragSet: finished reading top 25 9mer fragments from file ../frag/aaTAGS_09_05.200_v1_3
core.io.fragments: reading fragments from file: ../frag/aaTAGS_03_05.200_v1_3 …
core.io.fragments: rosetta++ fileformat detected! Calling legacy reader…
core.fragments.ConstantLengthFragSet: finished reading top 200 3mer fragments from file ../frag/aaTAGS_03_05.200_v1_3
core.scoring.constraints: Constraint choice: ./TAGS_.cst
core.io.constraints: read constraints from ./TAGS_.cst
protocols.jumping: compute strand/loop fractions for 95 residues…
protocols.abinitio.AbrelaxApplication: run ClassicAbinitio…..
core.scoring.ScoreFunctionFactory: SCOREFUNCTION: standardcore.scoring.ScoreFunctionFactory: SCOREFUNCTION PATCH: score12

and then the scoring file:

SCORE: score vdw cenpack pair env cbeta rg hs_pair ss_pair rsigma sheet rms gdtmm maxsub clashes_total clashes_bb time us description
SCORE: 61.237 14.810 -5.252 -11.644 -19.121 39.519 42.926 0.000 0.000 0.000 0.000 10.622 0.460 58.000 3.000 1.000 54.000 01 S_01_00000001

so no constrains term.

i would appreciate any help.

THANKS!!!

Cris

[Anonymous]

I asked around on the developer’s list and nobody knows quite what’s wrong. The best suggestion was to ignore the disulfide code and use an explicit AtomPairConstraint instead – perhaps the constraint weights will work properly if you remove the disulfide flags from the command line?

[Anonymous]

Hi,

I had the same problem and using “-in:fix_disulf disulfidefile” in fullatom-mode without constraints and any other option regarding disulfides like “detect_disulfide_before_relax” it worked.

Just put the 2 numbers like 7 and 35 of the cysteines separated by a space-character and followed by a new-line (this seems to be necessary) in a text file and it should work!

Hope this helped!

JS

[Anonymous]

Hi JS and Everyone,
It seems that JS’s method is simple to use. However, when I use

-fix_disulf /home/lanselibai/Cheng/20150121_minirosetta/input/disulfidefile

I have always been told “Segmentation fault (core dumped)” without any information in the log file.

The disulfidefile is as below:

23 88
(a new blank line)

I also DO NOT have constraints and other disulfide associated flags (e.g. -detect_disulfide_before_relax, -detect_disulfides, -disulf_filter, -norepack_disulf).

Can someone help me? Thank you very much.

Yours sincerely
Cheng

[Anonymous]

So any time you get a Segmentation fault in a Rosetta release version, it’s a definite bug in Rosetta – at the very least it should give you an error message instead.

Could you put together a minimal example that shows that behavior, upload it to some file hosting server (DropBox or Google Drive or the like), and post a link here? I can take a look at it and see what might be going wrong.

[Anonymous]

Hi R Moretti,
Nice to see you again. The link below contains all my files.

https://copy.com/GOaBvrz3tBb4JSz2

When the “-in:fix_disulf” is disabled, it works all fine.

Thank you very much.

Yours sincerely
Cheng

[Anonymous]

The example you posted seems to work fine for me with a recent version of Rosetta, with or without the fix_disulf flag.

[Anonymous]

Hi R Moretti,
Thank you. What is the version you are using? I just installed rosetta_src_2015.05.57576_bundle and tested it again. However, I was told “Segmentation fault (core dumped)” again and the attachment is the log file. All of the input files can be found at

https://copy.com/DZLN3Yti1BZnbVP4

Do you know why is that?

(In the beginning, I was told “-silent_decoytime not found in command line top-level context”. So I disable it)

Thank you very much.

Yours sincerely
Cheng

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