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

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. Commands:

• 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

• 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

• 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

• 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

Parameters	it2, def E-Value (0.002)	it2 E-Value 10e-10	it10 def E-Value (0.002)	it10 E-Value 10e-10
time	~2m30	~2m30	~10m	time: ~10m
results	500	93	500	500
best E-Value	1e-142	1e-145	5e-70	7e-70
worst E-Value	3e-4	2e-29	8e-38	1e-38
comments	Results with best EValues are mostly Aspartoacylases, Sequences previously not found are mostly Succinylglutamate Desuccinylases	results mainly Aspartoacylases	- converged after 8 rounds - most significant results include more Succinylglutamate Desuccinylases than Aspartoacylases	- all 10 iterations were done (no early convergence) - 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.

Commands

• 2 iterations:
  • hhblits -i P45381_wt.fasta -d /mnt/project/pracstrucfunc12/data/hhblits/uniprot20_current -o hhblits_p45381_def.out

• 8 iterations:
  • hhblits -i P45381_wt.fasta -d /mnt/project/pracstrucfunc12/data/hhblits/uniprot20_current -n 8 -o hhblits_p45381_n10.out

-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

BlastP

Comparison of distribution of Sequence Identiy between the two BlastP runs

A simple blast search yields only about 90 significant hits it one considers a threshold of 10e-10 as a significance cutoff. As one can see in Figure ??, the restriction of the Evalue results in less hits with a low sequence similarity.

Psi Blast

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.

Comparison of found sequences

Parameters	it 2	it 8
time	2m50	~6m
results	274	500
best E-Value	2e-110	2.9e-68
worst E-Value	0.0011	9.5e-09
comment	mixed results with Aspartoacylases and Succi	very varying results: Aspartoacylasen, Succinylasen, Zinc Proteins

Default E-Values: as could be expected, the normal BLAST search is mostly contained in the PsiBLAST search with two iterations. HHBlits found a large number of different hits, with only 48 out of 274 common hits in common with the BLAST searches.