Canavan Task 2 - Sequence alignments
Contents
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
| Parameters | default E-Value = 10 | E-Value 10e-10 |
| results | 196 | 94 |
| best E-Value | 1e-155 | 1e-155 |
| worst E-Value | 9.6 | e-15 |
| comment | 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. | 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 -n 8 -o hhblits_p45381_n10.out- time: 6m
- 500 results
- best E-Value: 2.9e-68
- worst E-Value: 9.5e-09
- very varying results: Aspartoacylasen, Succinylasen, Zinc Proteins
-n number of iterations (def 2)
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.
