CD task2 protocol

Sequence

The native ASPA sequence (UniProt: P45381):

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

BlastP

We ran BlastP on student machines with the big_80 as a reference database.

<source lang="bash"> blastall -p blastp -d /mnt/project/pracstrucfunc12/data/big/big_80 -i P45381_wt.fasta -o blastp_p45381_wt_big80.out </source>

GO Term Enrichment (hit more than once)

GO ID		GO name	                                        # hits
GO16788	 hydrolase activity, acting on ester bonds	94
GO8152	         metabolic process	                        94
GO16811	 hydrolase activity...                    	88
GO16787	 hydrolase activity                     	66
GO46872	 metal ion binding                      	62
GO19807	 aspartoacylase activity	                58
GO8270	         zinc ion binding                        	19
GO5737	         cytoplasm                               	8
GO16324	 apical plasma membrane                 	4
GO4046	         aminoacylase activity                   	4
GO5886	         plasma membrane                        	3
GO42802	 identical protein binding                 	3
GO16020	 membrane                               	3
GO5634	         nucleus                                	2

PsiBlast

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

2 iterations and default E-Value 0.002 <source lang="bash"> blastpgp -d /mnt/project/pracstrucfunc12/data/big/big_80 -i P45381_wt.fasta -o psiblast_it2_p45381_wt_big80.out -j 2 -v 700</source>

2 iterations, more strict E-value cutoff of 10E-10 <source lang="bash"> 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 </source>

GO Term Enrichment (all terms represented more than once)

766	hydrolase activity, acting on ester bonds
665	metabolic process
507	hydrolase activity
488	metal ion binding
294	zinc ion binding
234	arginine catabolic process to glutamate
176	hydrolase activity, acting on carbon-nitrogen (but not peptide) bonds, in linear amides
158	aspartoacylase activity
132	arginine metabolic process
132	succinylglutamate desuccinylase activity
30	cytoplasm
24	arginine catabolic process to succinate
23	proteolysis
22	metallocarboxypeptidase activity
12	apical plasma membrane
9	carboxypeptidase activity
8	nucleus
8	aminoacylase activity
8	membrane
6	identical protein binding
6	oxidation-reduction process
6	plasma membrane
5	oxidoreductase activity
2	malate synthase activity
2	glyoxylate cycle
2 	exonuclease activity
2	nucleotide binding
2	acetate metabolic process
2	arginine catabolic process
2	cell adhesion
2	virus-host interaction
2	integral to membrane
2	transferase activity 

10 iterations, default Evalue 0.002 <source lang="bash"> blastpgp -d /mnt/project/pracstrucfunc12/data/big/big_80 -i P45381_wt.fasta -o psiblast_it10_p45381_wt_big80.out -j 10 </source>

10 iterations, E-value cutoff 10E-10 <source lang="bash"> 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 </source>

HHBlits

Run HHBlits on student machines with Uniprot20 database.

• 2 iterations

 <source lang="bash">  hhblits -i P45381_wt.fasta -d /mnt/project/pracstrucfunc12/data/hhblits/uniprot20_current -o hhblits_p45381_def.out </source>

• 8 iterations

<source lang="bash"> hhblits -i P45381_wt.fasta -d /mnt/project/pracstrucfunc12/data/hhblits/uniprot20_current -n 8 -z 1000 -v 1000-o hhblits_p45381_n10.out </source>

• 2 iterations, -e 10e-10

 <source lang="bash">  hhblits -i P45381_wt.fasta -d /mnt/project/pracstrucfunc12/data/hhblits/uniprot20_current -e 10e-10 -o hhblits_p45381_def.out </source>

• 8 iterations, -e 10e-10

<source lang="bash"> hhblits -i P45381_wt.fasta -d /mnt/project/pracstrucfunc12/data/hhblits/uniprot20_current -n 8 -e 10e-10 -z 1000 -v 1000 -o hhblits_p45381_n10.out </source>