Difference between revisions of "CD task2 protocol"

From Bioinformatikpedia
(PsiBlast)
(PsiBlast)
Line 111: Line 111:
   
 
#hits GO terms
 
#hits GO terms
  +
564 hydrolase activity, acting on ester bonds
754 metabolic process
 
  +
564 metabolic process
752 hydrolase activity, acting on ester bonds
 
599 hydrolase activity
+
443 hydrolase activity
588 metal ion binding
+
433 metal ion binding
256 zinc ion binding
+
160 zinc ion binding
  +
114 arginine metabolic process
121 proteolysis
 
  +
114 arginine catabolic process to glutamate
118 metallocarboxypeptidase activity
 
  +
113 succinylglutamate desuccinylase activity
117 arginine catabolic process to glutamate
 
  +
88 hydrolase activity, acting on carbon-nitrogen (but not peptide) bonds, in linear amides
117 arginine metabolic process
 
116 succinylglutamate desuccinylase activity
+
60 aspartoacylase activity
  +
28 proteolysis
88 hydrolase activity, acting on carbon-nitrogen (but not peptide) bonds, in linear amides
 
60 aspartoacylase activity
+
27 metallocarboxypeptidase activity
  +
21 arginine catabolic process to succinate
57 carboxypeptidase activity
 
  +
12 carboxypeptidase activity
22 arginine catabolic process to succinate
 
9 cytoplasm
+
8 cytoplasm
  +
4 apical plasma membrane
6 ATP binding
 
  +
4 aminoacylase activity
5 membrane
 
  +
3 plasma membrane
5 transferase activity
 
  +
3 identical protein binding
4 methyltransferase activity
 
  +
3 membrane
4 methylation
 
  +
2 nucleus
4 apical plasma membrane
 
  +
2 arginine catabolic process
4 aminoacylase activity
 
  +
2 transferase activity
4 regulation of transcription, DNA-dependent
 
  +
3 identical protein binding
 
3 nucleotide binding
 
3 plasma membrane
 
3 nucleus
 
3 catalytic activity
 
2 sequence-specific DNA binding transcription factor activity
 
2 intracellular
 
2 kinase activity
 
2 phosphorylation
 
2 peptidase activity
 
2 molecular_function
 
2 signal transducer activity
 
2 arginine catabolic process
 
2 DNA binding
 
2 biological_process
 
2 signal transduction
 
2 integral to membrane
 
   
   

Revision as of 16:14, 29 August 2012

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

GO term enrichment

<source lang="java">

for(int i = 0; i< seq_id.length; i++ ){

   // URL for annotations from QuickGO for one protein
   URL u=new URL("http://www.ebi.ac.uk/QuickGO/GAnnotation?protein="+seq_id[i]+"&format=tsv");
   // Connect
   HttpURLConnection urlConnection = (HttpURLConnection) u.openConnection();
   // Get data
   BufferedReader rd=new BufferedReader(new InputStreamReader(urlConnection.getInputStream())); 
   List<String> columns=Arrays.asList(rd.readLine().split("\t"));
   int idIndex=columns.indexOf("GO ID");
   int nameIndex=columns.indexOf("GO Name");
   String line;
   if(rd.ready()) count_go_prots++;
   Set<String> names = new HashSet<String>();
   while ((line=rd.readLine())!=null) {

// Split them into fields String[] fields=line.split("\t"); if(!names.contains(fields[nameIndex])){ names.add(fields[nameIndex]); if(this.go_ids.containsKey(fields[idIndex])) { int count = Integer.parseInt(this.go_ids.get(fields[idIndex])[1]); this.go_ids.put(fields[idIndex], new String[]{fields[nameIndex], String.valueOf(count+1)}); } else{ this.go_ids.put(fields[idIndex], new String[]{fields[nameIndex],"1"}); }

       }
   }
   // close input when finished
   rd.close();

} </source>

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>

default E-Value 10 - GO Term Enrichment (hit more than once)

#hits   GO term
185	 metabolic process
184	 hydrolase activity, acting on ester bonds
133	 hydrolase activity
125	 metal ion binding
88	 hydrolase activity, acting on carbon-nitrogen (but not peptide) bonds, in linear amides
60	 aspartoacylase activity
44	 zinc ion binding
24	 arginine metabolic process
24	 arginine catabolic process to glutamate
23	 succinylglutamate desuccinylase activity
8	 cytoplasm
5	 arginine catabolic process to succinate
4	 apical plasma membrane
4	 aminoacylase activity
4	 membrane
3	 plasma membrane
3	 identical protein binding
2	 nucleus
2	 intracellular
2	 exonuclease activity
2	 nucleotide binding
2	 nucleic acid binding
2	 oxidoreductase activity
2	 oxidation-reduction process

E-Value 10e-10 - GO Term Enrichment (hit more than once)

#hits   GO term
94	 hydrolase activity, acting on ester bonds
94	 metabolic process
88	 hydrolase activity, acting on carbon-nitrogen (but not peptide) bonds, in linear amides
66	 hydrolase activity
62	 metal ion binding
58	 aspartoacylase activity
19	 zinc ion binding
8	 cytoplasm
4	 apical plasma membrane
4	 aminoacylase activity
3	 plasma membrane
3	 identical protein binding
3	 membrane
2	 nucleus

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"> time blastpgp -d /mnt/project/pracstrucfunc12/data/big/big_80 -i p45381_wt.fa -o psiblast_it2_h0002_1000.out -j 2 -h 0.002 -v 1000 -b 1000</source>

GO Term Enrichment (all terms represented more than once)

#hits GO terms
564	 hydrolase activity, acting on ester bonds
564	 metabolic process
443	 hydrolase activity
433	 metal ion binding
160	 zinc ion binding
114	 arginine metabolic process
114	 arginine catabolic process to glutamate
113	 succinylglutamate desuccinylase activity
88	 hydrolase activity, acting on carbon-nitrogen (but not peptide) bonds, in linear amides
60	 aspartoacylase activity
28	 proteolysis
27	 metallocarboxypeptidase activity
21	 arginine catabolic process to succinate
12	 carboxypeptidase activity
8	 cytoplasm
4	 apical plasma membrane
4	 aminoacylase activity
3	 plasma membrane
3	 identical protein binding
3	 membrane
2	 nucleus
2	 arginine catabolic process
2	 transferase activity


2 iterations, more strict E-value cutoff of 10E-10 <source lang="bash"> time blastpgp -d /mnt/project/pracstrucfunc12/data/big/big_80 -i P45381_wt.fasta -o psiblast_it2_h10e-10_1000.out -j 2 -h 10e-10 -v 1000 -b 1000</source>

GO Term Enrichment (all terms represented more than once)

#hits  GO term
480	 hydrolase activity, acting on ester bonds
480	 metabolic process
374	 hydrolase activity
363	 metal ion binding
148	 zinc ion binding
108	 arginine metabolic process
108	 arginine catabolic process to glutamate
107	 succinylglutamate desuccinylase activity
88	 hydrolase activity, acting on carbon-nitrogen (but not peptide) bonds, in linear amides
60	 aspartoacylase activity
22	 proteolysis
21	 metallocarboxypeptidase activity
19	 arginine catabolic process to succinate
9	 carboxypeptidase activity
8	 cytoplasm
4	 apical plasma membrane
4	 aminoacylase activity
3	 plasma membrane
3	 identical protein binding
3	 membrane
2	 nucleus
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>