Lab Journal - Task 6 (PAH)

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Revision as of 14:51, 15 June 2013 by Waldraffs (talk | contribs) (Calculate structural model)

Multiple Sequence Alignment

The multiple alignments are downloaded from the PFAM server and are converted into a freecontact readable format using a2m2aln.

  1. Protein H-RAS:
    /usr/share/freecontact/a2m2aln -q '^RASH_HUMAN/(\d+)' --quiet < PF00071_full.txt > PF00071.aln
  2. For our protein PAH, we have two domains. As the Biopterin-domain is said to be causing PKU if damaged, we used the PFAM alignment of this domain:
    /usr/share/freecontact/a2m2aln -q '^PH4H_HUMAN/(\d+)' --quiet < PF00351_full.txt > PF00351.aln

Calculate and analyze correlated mutations

  1. Freecontact is used to calculate CN-score for the multiple alignments:
    freecontact -o evfold < '<PFAM-ID>.aln' > <PFAM-ID>.evfold
  2. extract_pairs.pl extracts all residue pairs with distance >5.
  3. the results are sorted (CN-score descending) for both all and extracted residue pairs:
    sort -k 6 -g -r <PFAM-ID>.evfold >sort_<PFAM-ID>.txt
  4. CN_dist.R makes histograms and multiple histograms for the CN-Score distribution. Furthermore it calculates the top L-Score (L = protein length) for each residue i that belongs to the top L:
    top L-Score(i) = (sum of CN scores for residue i)/mean(CN-Scores of top L)
  5. Evcouplings
    Reference structure for Ras is 121p.
    For the biopterin family we have to set the starting position to 106 to get a multiple alignment.

Calculate structural model

The length of Pfam alignment of H-Ras is 160, therefore we take following number of contacts: 64, 104, 160.
For biopterin the protein length is 346 as we only make an alignment with amino acids 106 to 452. So we take 138, 225 and 346 as number of contacts.