Trouble enforcing a disulfide in loop building

[Anonymous]

Hello,

Like some others on this forum, I’m having some trouble getting disulfides to work. I pass

-in:fix_disulf ./disulf

where disulf is a text file containing the residue numbers of the cysteines I am trying to bridge like so:

269 280

With an empty line at the end, which others have mentioned may be necessary. Unfortunately, this has no discernable effect on the loop in question–I have done pretty extensive testing, and I never see a disulfide form. Should I try to encode this disulfide as a constraint, or is there something I’m clearly doing wrong?

EDIT:

Given that -in:fix_disulf has no effect by my lights, can any of you suggest a benchmark or standard to test whether this command is working properly in my installation? I’ve had success with other projects, so it would have to be a pretty specific issue.

Thanks,
CH

[Anonymous]

-in:fix_disulf (note the underscore and not a colon between fix and disulf) is handled at the application level, rather than at a general Rosetta level – depending on your protocol it may not even be read. Which protocol are you using?

If your protocol does not obey -in:fix_disulf, then (atom pair) constraints may be the way to go – many protocols, even if they don’t obey -in:fix_disulf, will automatically detect disulfides if the two cysteines are close enough.

For an example of -in:fix_disulf usage, you can take a look at Rosetta/demos/protocol_capture/2012/replica_docking/, which makes use of it.

[Anonymous]

Looking back at what I’ve done, I was passing -in:fix_disulf with correct punctuation, I just reproduced it incorrectly in my first post.

I’m using a homology modelling protocol to do loop building on a membrane protein, here’s the contents of the options file:

-in:file:fullatom
-s ./hTRPM8_full_c-term_threaded.pdb
-loops:loop_file ./test.loops ##loop file
-loops:extended true
-loops:build_initial
-loops:idealize_after_loop_close
-loops:relax fastrelax
-loops:fast
-out:file:fullatom
-use_input_sc
-ex1 #Include extra chi1 rotamers
-ex2
-loops:frag_sizes 9 3 1
-loops:frag_files ./aat000_09_05.200_v1_3 ./aat000_03_05.200_v1_3 none
-loops:remodel quick_ccd
-loops:refine refine_kic
-in:file:psipred_ss2 ./t000_.psipred_ss2
-in:file:spanfile ./modellerrebuild.span
-in:file:lipofile ./hTRP.lips4
-in:fix_disulf ./disulf
-score:weights /usr/local/apps/rosetta/rosetta-3.5/rosetta_database/scoring/weights/membrane_highres_Menv_smooth.wts
-overwrite

Any idea what the problem might be? I’ve begun to try using an atom pair constraint, we’ll see how that goes.

EDIT: For anyone with similar problems, I seem to have successfully generated disulfides by passing this constraint file:

AtomPair SG 269 SG 280 HARMONIC 1.800 2.05

with these options:

-cst_fa_file disulf.cst
-cst_fa_weight 1000

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