AMPK Methods and Protocols

outline the routine for species represented in the QfO reference

proteome collection, as the sequences in these sets are available

pre-annotated from the HaMStR project page. Per default, this

information is stored in pathToHaMStR/weight_dir where

each species has its own directory.

6. Create a directory to store the output of the annotation proce-
   dure. Give this directory a meaningful name like pathTo-
   HaMStR/data/AMPK_annoDir.


7. For every seed–ortholog pair, extract the precomputed feature
   annotations. Run the scriptpathToHaMStR/bin/fas/annota-
   tion.plscript for this purpose. Create a new directorypathTo-
   HaMStR/data/AMPK_annoDir/singleProteins.


8. Extract now the feature architecture information for PRKAA1
   from human. Note that the species names used here might
   deviate from the ones in the actual distribution: pathTo-
   HaMStR/bin/fas/annotation.pl -path¼pathToHaMStR/
   weight_dir/HUMAN@9606 -name¼Q13131 -extract¼pathTo-
   HaMStR/data/AMPK_annoDir/singleProteins/
   homsa_PRKAA1.


9. Rerun the above command for the yeast ortholog of PRKAA1.
   To do so, simply replaceYEAST@559292forHUMAN@9606,
   set –name to P06782, and modify the “extract” path
   accordingly.


10. Use the feature architectures extracted in the previous steps as
    input for the script greedyFAS.py to calculate the FAS score
    between the two proteins.pathToHaMStR/bin/fas/greedyFAS.
    py -s pathToHaMStR/data/AMPK_annoDir/singleProteins/
    homsa_PRKAA1 -p pathToHaMStR/data/AMPK_annoDir/
    singleProteins/saccePRKAA1 -o 1 –r pathToHaMStR/weight-
    dir/HUMAN@9606 -j prkaa1_homsa_sacce_FAS_Result.xml.
    The parameters–rand–jspecify the species used for feature
    weighting and the output file name, respectively.


11. To extract the corresponding feature architectures for a later
    visualization, run the script pathToHaMStR/bin/visuals/par-
    seArchitecture.pl: pathToHaMStR/bin/visuals/parseArchitec-
    ture.pl -i prkaa1_homsa_sacce_FAS_Result.xml -g PRKAA.
    Upon completion, the script will create an output file named
    prkaa1_homsa_sacce_FAS_Result.xml.domains.

3.5 Visualizing
FAS-Enhanced
Phylogenetic Profile

Visualize and explore the FAS-enhanced phylogenetic profile with

the PhyloProfile application. This will result in a comprehensive

overview of the phyletic distribution of the pathway components.

1. Install the PhyloProfile application. A simple way to do it is
   using the git tools [37]:git clonehttps://github.com/BIONF/
   PhyloProfile. You will need an up-to-date R installation to run
   the PhyloProfile application.

Tracing AMPK Evolution 127