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

---

Search

BLASTP

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

• default E-Value cutoff (10)
  • 196 resulting sequences, out of which the last 101 have EValues below e-15.
  • best E-Value: e-155
  • worst E-Value: 9.6
  • 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.

• more strict E-Value cutoff of 10e-10
  • 94 results
  • best E-Value: e-155
  • worst E-Value: e-15
  • The results are the same as for the first run, just with an earlier cutoff

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

• 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)

---

Summary and Comparison

Along with the expactations one can find more hits with Psi-Blast than with a simple Blast search.

In general, one can distinguish between two kinds of proteins, that frequently are identified by the sequence searches:

• Aspartoacylases
• Succinylglutamate Desuccinylases

Increasing the amount of iterations performed in a PSI-Blast search, obviously increases the running time. One can see, that the best ranked hits have lower E-Values than the best Hits of the runs with less iterations. Yet, there are more hits found with better E-Values, which is not surprising because more homologues of significant profile sequences will be found.

When restricting the E-Value Cutoff for the profile built-up, we found that more hits are classified as Aspartoacylases than as Succinylglutamate Desuccinylases. The running time, as well as the E-Values of the resulting hits did not change significantly.