Lab Journal - Task 6 (PAH)
All files are stored in /mnt/home/student/waldraffs/masterpractical/Task6
- /ras: contains all files for H-RAS
- /pah: contains all files for PAH
Multiple Sequence Alignment
The multiple alignments are downloaded from the PFAM server and are converted into a freecontact readable format using a2m2aln.
- Protein H-RAS:
/usr/share/freecontact/a2m2aln -q '^RASH_HUMAN/(\d+)' --quiet < PF00071_full.txt > PF00071.aln
- For our protein PAH, we have two domains (ACT: PF01842, Biopterin: PF00351) and therefore used the hhblits result of Task2. The .a3m file is converted into stockholm format using
perl /usr/share/hhsuite/scripts/reformat.pl a3m sto PAH_2000.a3m PAH_2000.stockholm
After that the header is changed into # query=" and positions that have a gap in the query sequences are removed: PAH.aln.
- Freecontact is used to calculate CN-score for the multiple alignments:
freecontact -o evfold < '<FILE>.aln' > <FILE>.evfold
- contact_map.pl extracts all residue pairs with less than 5 Ångstrom minimum atom distance:
perl contact_map.pl -pdb <pdb-file> -out <output-file>
- extract_pairs.pl extracts all residue pairs with distance >5, if such a pair also is included in the output of contact_map.pl it is marked with 'TP' (true positive) else with 'FP' (false positive):
perl extract_pairs.pl -inp <FILE>.evfold -map <contact_map.pl output-file> -out <output-file>
- the results are sorted (CN-score descending) for both all and extracted residue pairs:
sort -k 6 -g -r <FILE> >sort_<FILE>
- CN_dist2.R makes histograms for the CN-Score distribution (for all and extracted pairs). 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)
- contact_map.R creates a contact map with the output-files of the two perl scripts above (pdb = reference structure, extracted = predicted).
Reference structure for Ras is 121p.
For the biopterin family we have to set the starting position to 106 to get a multiple alignment.
The perl and R scripts can be found in
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.