The scripts and input files that accompany this demo can be found in the demos/public directory of the Rosetta weekly releases.


Conceptual steps

  1. Create fragments for target sequence
  2. Find template structure
  3. Make a sequence alignment between template sequence and target sequence
  4. Download and clean the template pdb
  5. Make Rosetta flags file
  6. Execute Rosetta
  7. Analyze results

Detailed steps

  1. Create fragments for target sequence

    Obtain a FASTA file of your target sequence. You can get this from NCBI or you can make the file manually using the following format:


    Open a web browser and go to Paste the target sequence into the sequence input box and click submit. Wait until the server finishes and download all the files produced to your working directory

  2. Find template structure

    Finding a template can be done in multiple ways. One suggestion is to submit the target sequence to HHpred or similar. HHpred can be found here:

  3. Make a sequence alignment between the template sequence and the target sequence

    In order to generate the alignment, you need the primary sequences of both the target and the template. For the template, go to and search for the PDB code of your template (obtained in Step 2). In this case, we are using 2AST. In fact, we are using part of chain B from 2AST. On the top right, click the down arrow by "Download Files" and click on "FASTA sequence". Alternatively, you can get the FASTA sequence from NCBI (see Step 1) or make it manually.

    Align the sequences using ClustalW or the alignment tool of your choice (e.g., EMBOSS, FASTA, Nexus, etc.). Create a FASTA file (template_target.fasta) containing both primary sequences. Go to and upload the newly created fasta file. The default settings are a good place to start to generate a generally good sequence alignment. Use the slow alignment for better results Enter your email so you can results. Click submit at the bottom of the page. Depending on the size of the sequences being aligned, it will take anywhere from 5 minutes to some hours or days to run

    After you generate the alignment file, put it in a format like this:

    score 123.456

    where the score is a floating point number, column 1 starting on line 2 is the name of the target (or template, on line 3), column 2 is the beginning sequence position for the threading, and the rest of the line is the sequence.

  4. Download and clean the template PDB

    Once you have decided upon a template, search for the PDB ID at and go to "Download Files" and select to download the PDB. This file then needs to be "cleaned" to run properly in Rosetta. To avoid errors when Rosetta reads in the PDB file, the protein must be formatted correctly or “cleaned”. A correctly formatted PDB file includes removed non-ATOM records, renumbered residues from 1, renumbered atoms from 1, and corrected chain ID inconsistencies. The script located in the scripts directory will be used to format the template PDB file.

    The script requires that you have python2.2 (go to for instructions on download and installation) or higher installed, as well as biopython (

    Execute the script by typing:

    ./scripts/ template.pdb A

    The script will output two files:

  5. Make the Rosetta flags file

    Rosetta supports a threading protocol (minirosetta application in the bin) which needs a set of input files and specific commandline flags. The commandline flags can be stated in a file in which case the commandline reduces to

    minirosetta @flags

    The format of the flagsfile is key-value pairs and an example of a working flagsfile to perform thrading looks like:

    -run:protocol threading # run the rosetta threading, loopbuilding, and refinement protocol
    -in:file:alignment ./starting_files/template_target_short.aln # path to alignment file
    -cm:aln_format general # format of alignment file
    -frag3 ./starting_files/fragments/aat000_03_05.200_v1_3.gz # path in 9mer fragment file
    -frag9 ./starting_files/fragments/aat000_09_05.200_v1_3.gz # path to 3mer fragment file
    -in:file:fasta ./starting_files/fragments/t000_.fasta # path to target fasta file
    -in:file:fullatom # we have fullatom format for the input template
    -loops:frag_sizes 9 3 1 # which size fragments are you using?
    -loops:frag_files ./starting_files/fragments/aat000_09_05.200_v1_3.gz ./starting_files/fragments/aat000_03_05.200_v1_3.gz none  # paths to 9mer file, 3mer file, and say "none" for the 1mer file
    -in:file:psipred_ss2 ./starting_files/t000_.psipred_ss2 # path to psipred secondary structure prediction file
    -out:nstruct 1 # number of structures you want to build.  Should build at least 1000, but 10,000 would be better
    -in:file:template_pdb ./starting_files/2astB_66-105_renumbered.pdb # path to template pdb
    -database ./rosetta-3.3/rosetta_database/ # path to rosetta database
    -loops:extended # Force extended on loops, independent of loop input file
    -loops:build_initial # Precede loop-modeling with an initial round of just removing the missing densities and building simple loops
    -loops:remodel quick_ccd # closing loops by quick_ccd
    -loops:refine refine_ccd # small movements to remodel loop
    -silent_decoytime # Add time since the last structure was written to score line
    -random_grow_loops_by 4 # randomly grow loops by up to this number of residues on either side.
    -select_best_loop_from 1 # Keep building loops until N and choose best (by score)
    -out:file:fullatom # output in fullatom mode
    -out:file:silent threaded_model.out # silent file stores internal coordinates of the PDB
    -out:file:silent_struct_type binary # output the silent file in binary format
    -out:file:scorefile threaded_model.fasc # output a table of Rosetta scores
    -run:constant_seed # Use a constant seed (1111111 unless specified)
    -run:jran 1111111 # this is good for testing since you should always get the same result
    -overwrite # overwrite any already-existing results having the same name
  6. Execute Rosetta

    $> <path/to/Rosetta>/main/source/bin/minirosetta.macosgccrelease @flags
  7. Analyze results

    There are several options for things to do next, see the following demos: