Fixing and Building disulfide bonds in homology modeling

[Anonymous]

Dear Friends!

I am new to rosetta and I would like to become proficient in running it. As a start I need to find out if it is feasible to force disulfide bonds on the N and C terminal of a protein that does not have any disulfide bonds in nature but will be relevant for fusing into another protein structure that forms disulfide bonds such as the fusion occurs at the disulfide bond branch. My template sequence and target are of different length and so i ran into the this error

“core.conformation.Conformation: [ERROR] Residue 246 is out of range.”

I did a work around using smlewis suggestion by commenting “core::pose::initialize_disulfide_bonds(pose); ” in “rosetta_source/src/core/io/pdb/file_data.cc” file and recompiling rosetta.

This enabled the job to run. However the best output models do not have the disulfide bond I was trying to build. Here are the flags that i used to run minirosetta.linuxgcc on rosetta 3.5

-run:protocol threading
-in:file:fasta file.fasta
-in:file:alignment file.ali
-in:file:template_pdb temp.pdb
-in:file:fullatom
-cm:aln_format general
-in:file:psipred_ss2 file.psipred_ss2
-in:fix_disulf file.ss
-loops:frag_files file.frag9 file.frag3 none
-loops:frag_sizes 9 3 1
-idealize_after_loop_close
-loops:extended
-loops:build_initial
-loops:remodel quick_ccd
-loops:refine refine_ccd
-loops:relax fastrelax
-relax:default_repeats 8
-out:nstruct 100
-out:file:silent out/out.silent.${SGE_TASK_ID}
-out:file:fullatom
-run:seed_offset ${SGE_TASK_ID}

I have several questions
1. Does commenting out “core::pose::initialize_disulfide_bonds(pose); ” and recompiling affect future modeling jobs where the template structure has disulfide bonds and if so what is the best way to make use of the work around if needed without compromising future modeling jobs

2. I recompiled rosetta by reinstalling it but i believe this is unnecessary. What is the best way to recompile after minor code change

3. What flags would be essential to add in order to generate models with built disulfide bonds

4. I ran the job without first relaxing the template(1.35 Å  resolution) and I am strategizing on doing so before performing the threading. Is this a good strategy and in general when is it necessary

5. How do you improve modeling speed without compromising accuracy

I appreciate any help and suggestion.
Thank you for your time

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