Question about wrong homology design

[Anonymous]

Hi everyone, I have been using rosetta 3.4 and 3.5 to build an homology model of a protein and the result is the same, the structure is broken just in one of the amino acids. I made a control test and made the homologue model of the same template and also the structure is broken. In figure A is the template an in figure B is the homologue model where appears the some fragments of one residue outside of the structure, How can I solve that?
there is my script:

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/home/rosetta-3.5/rosetta_source/bin/minirosetta.default.linuxgccrelease @/$DATA_PATH/comparative_model.options -database /home/rosetta-3.5/rosetta_database -overwrite >& /$DATA_PATH/comparative_model.log

comparative_model.options
-run:protocol threading
-in:file:alignment /home/Auroge.aln
-cm:aln_format grishin
-frag9 /home/aat000_09_05.200_v1_3
-frag3 /home/aat000_03_05.200_v1_3
-in:file:fasta /home/t000_.fasta
-in:file:fullatom
-loops:frag_sizes 9 3 1
-loops:frag_files /home/aat000_09_05.200_v1_3 /home/aat000_03_05.200_v1_3 none
-in:file:psipred_ss2 /home/t000_.psipred_ss2
-in:file:fullatom
-out:nstruct 1000
-in:file:template_pdb /home/AURO.pdb
-database /home/rosetta-3.5/rosetta_database/
-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:file:silent /home/file.out
-out:file:silent_struct_type binary
-out:file:scorefile /home/file.fasc
-run:constant_seed
-run:jran 1111101
-overwrite

cluster.options
in:file:fullatom
-run:shuffle
-cluster:radius 1.5
-cluster:input_score_filter 0

$ROSETTA_BIN/combine_silent.$ROSETTA_SUFFIX -database $ROSETTA_DATABASE -in:file:silent /home/01/file.out … /home/10/file.out -in:file:silent_struct_type binary -in:file:fullatom -out:output -out:file:silent file_all.out -out:file:silent_struct_type binary -out:file:fullatom -overwrite >& /home/combine_silent.log

python /home/rosetta-3.5/rosetta_tools/protein_tools/scripts/clustering.py –silent=file_all.out –rosetta=$ROSETTA_BIN/cluster.$ROSETTA_SUFFIX –database=$ROSETTA_DATABASE –options=cluster.options cluster_summarytxt cluster_histogram_S.txt

sort -rnk4 cluster_summary.txt > cluster_summary_sorted.txt
head -n10 cluster_summary_sorted.txt

$ROSETTA_BIN/score_jd2.$ROSETTA_SUFFIX -database $ROSETTA_DATABASE -in:file:silent /home/file_all.out -out:output -out:pdb -out:file:fullatom -in:file:tags S_X
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Other error is that the conservative sites in the homologue model are placed lightly in different position and the rotamers are directed in opposite positions (see figure C) What do you think that could be the cause of this kind of wrong design?, Is there any way to constraint the system to build the homologue model without affecting the conservative sites (position and rotamers direction)?

[Anonymous]

What does your alignment look like, particularly with the residues which are having problems? Comparative modeling depends heavily on the alignment provided – if there’s problems with the alignment, you’ll get problems with the resulting model. (For example, is the sequence of the template protein in the alignment exactly the same as the coordinates present in the PDB? Often the “sequence” record of the PDB will contain residues which don’t have coordinates, and this may mess up Rosetta. Likewise, make sure your input fasta is exactly the same sequence as what’s in the aligment.) With the information given, it’s kinda hard to tell, but I’d guess that you have an issue with your alignment, and that’s why you’re getting strange results.

If you think your alignment is okay, try doing an -nstruct 1 run with the option “-out:levels all:debug”. Posting the tracer output from that run here with the alignment, fasta and template PDB files will help in narrowing things down.

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