Canavan Task 2 - Sequence alignments

Task 2 : Canavans Disease

Sequence

The native ASPA sequence that we used for the current task is shown below:

UniProt: P45381

>hsa:443 ASPA, ACY2, ASP; aspartoacylase; K01437 aspartoacylase [EC:3.5.1.15] (A)
MTSCHIAEEHIQKVAIFGGTHGNELTGVFLVKHWLENGAEIQRTGLEVKPFITNPRAVKK
CTRYIDCDLNRIFDLENLGKKMSEDLPYEVRRAQEINHLFGPKDSEDSYDIIFDLHNTTS
NMGCTLILEDSRNNFLIQMFHYIKTSLAPLPCYVYLIEHPSLKYATTRSIAKYPVGIEVG
PQPQGVLRADILDQMRKMIKHALDFIHHFNEGKEFPPCAIEVYKIIEKVDYPRDENGEIA
AIIHPNLQDQDWKPLHPGDPMFLTLDGKTIPLGGDCTVYPVFVNEAAYYEKKEAFAKTTK
LTLNAKSIRCCLH

BLASTP

First, we ran BlastP on student machines with the big_80 as a reference database.

Command:

blastall -p blastp -d /mnt/project/pracstrucfunc12/data/big/big_80 -i P45381_wt.fasta -o blastp_p45381_wt_big80.out

We got 195 resulting sequences, out of which the last 101 have EValues below E^-15. Most of the resulting proteins are Aspartoacylases of other species. Most of the results with EValue > E^-15 are Succinylglutamate Desuccinylases, which catalyze a reaction similar to Aspartoacylase.

PSIBLAST

PSIBlast was used in the same fashion as BLAST, with the big_80 as the background database. Results:

• Running 2 iterations and default E-Value 0.002
  • blastpgp -d /mnt/project/pracstrucfunc12/data/big/big_80 -i P45381_wt.fasta -o psiblast_it2_p45381_wt_big80.out -j 2
  • time: ~2m30
  • 500 results
  • best E-Value: E^-142
  • worst E-Value: 3E^-4
  • Results with best EValues are mostly Aspartoacylases
  • Sequences previously not found are mostly Succinylglutamate Desuccinylases

• 2 iterations, more strict E-value cutoff of 10E-10
  • blastpgp -d /mnt/project/pracstrucfunc12/data/big/big_80 -i P45381_wt.fasta -o psiblast_it2_h10e10_p45381_wt_big80.out -j 2 -h 10e-10
  • time ~2m30
  • 93 results
  • best E Value: E^-145
  • worst E Value: 2E-29
  • results mainly Aspartoacylases

• 5 iterations, default Evalue 0.002
  • blastpgp -d /mnt/project/pracstrucfunc12/data/big/big_80 -i P45381_wt.fasta -o psiblast_it5_p45381_wt_big80.out -j 2
  • time ~5m
  • 197 results
  • Best E-Value E^-164
  • Worst E-Value 7.9
  • More significant results mostly Aspartoacylases, with higher E-Value (> 3E^-5) more Succinylglutamate Desuccinylases

• 10 iterations, default Evalue 0.002
  • blastpgp -d /mnt/project/pracstrucfunc12/data/big/big_80 -i P45381_wt.fasta -o psiblast_it10_p45381_wt_big80.out -j 10
  • time: ~10m
  • converged after 8 rounds
  • 500 results
  • best E-Value 5E^-70
  • worst E-Value 8E^-38
  • most significant results include more Succinylglutamate Desuccinylases than Aspartoacylases

• 10 iterations, E-value cutoff 10E-10
  • blastpgp -d /mnt/project/pracstrucfunc12/data/big/big_80 -i P45381_wt.fasta -o psiblast_it10_h10e10_p45381_wt_big80.out -j 10 -h 10e-10
  • time: ~10m
  • all 10 iterations were done (no early convergence)
  • 500 results
  • best E-Value: 7E^-70
  • worst E-Value: 1E^-38
  • aspartoacylases slightly more frequent in lower E-Values (< E-58), but no significant difference in E-Values for aspas and succis

HHBLITS

Run HHBlits on student machines with Uniprot20 database.

• hhblits -i P45381_wt.fasta -d /mnt/project/pracstrucfunc12/data/hhblits/uniprot20_current -o hhblits_p45381_def.out
  • time: 2m50
  • 274 results
  • best E-Value: 2E^-110
  • worst E-Value: 0.0011
  • mixed results with Aspa and Succi

• hhblits -i P45381_wt.fasta -d /mnt/project/pracstrucfunc12/data/hhblits/uniprot20_current -o hhblits_p45381_n10.out

-n number of iterations (def 2)
-e E Value CutOff (0.001)