Connecting 2 Domains Via Non-Flexible Linker

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    • #1022


        I originally posted my question regarding modeling of a multi-domain oligomer under a different thread. After getting a lot of useful answers and consulting Ingemar Andre off the list I am still facing one major problem.

        The protein at hand consists of two domains A and B connected by a structured linker. Structural models for all three “parts” are available. How would one connect these to obtain a full-length model?

        This refers specifically to Ingemar’s and Rocco’s following advise for obtaining a oligomeric structure of my protein:

        1) Make the domainA-linker-domainB model(s).
        2) Symmetrically dock the model(s)
        1) Separately dock domainA and domainB
        2) From a set of decent looking models from self-docking with domain A and domain B find combinations of dimers of A and B that would be able to connect with a linker.
        3) Place the domains in such a manner that a linker can connect A to B.
        4) Connect the linker to the proteins using loop modeling to close the covalent links between A, linker and B.

        Thanks once more in advance.

      • #6026

          I think I solved the problem. The assembly protocol and standard loop modeling either did not work at all or not satisfyingly.

          A combination of different forum posts and ideas finally helped me to come up with the following protocol which works like a charm in my case. Comparative modeling is the key. The following protocol is a good way to start.

          What you have and what you want to do:

          You have structures (pdb files) of individual domains of a target protein and want a model of the full-length protein.

          I use the term “target protein” to refer to the full-length protein you want to model. “Template” refers to the input pdb files of individual domains.

          What you need:
          – a fasta file of your target protein
          – fragment files of your target protein
          – alignment file
          – template pdb file –> pdb file containing the structures of all individual domains you have as template
          (-optional: constraints and/or symmetry definition file)

          The alignment file looks in general as described in this post to the forum. In this case the target line is the sequence of the full-length protein you want to model. Template is a pseudo-alignment of the amino acids of all the domains you have with respect to the full-length protein. The following example might help:

          template 1



          The following protocol runs with the minirosetta application.
          -run:protocol threading
          -in:file:fasta target.fasta
          -loops:frag_files ./fragments/target_09_06.200_v1_3 ./fragments/target_03_06.200_v1_3 none
          -loops:frag_sizes 9 3 1
          -loops:remodel quick_ccd
          -loops:relax fastrelax
          -relax:minirelax_repeats 8
          -score:weights score12_full
          -in:file:alignment alignment.aln
          -in:file:template_pdb template.pdb
          -database /opt/rosetta-3.2/rosetta_database
          -nstruct 1000
          -out:user_tag default
          -out:file:silent default.out
          -out:file:scorefile default.scr

          optional for constraints:

          -cst_file ./constraints/110805_ytva_constraints.wofmn.cen_cst
          -cst_fa_file ./constraints/110805_ytva_constraints.wofmn.fa_cst
          -cst_weight 1.0
          -cst_fa_weight 1.0

          optional for symmetry:

          -symmetry:symmetry_definition target_symdef

        • #11098

            hi, thanks for sharing your idea! Could you give an example how you included your domain coordinates in your “template.pdb”?

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