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

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>

GO Term Enrichment (all terms represented more than once)

#hits   GO term
965	 metabolic process 
964	 hydrolase activity, acting on ester bonds 
790	 hydrolase activity 
754	 metal ion binding 
639	 zinc ion binding 
504	 proteolysis 
502	 metallocarboxypeptidase activity 
337	 carboxypeptidase activity 
118	 arginine metabolic process 
118	 arginine catabolic process to glutamate 
116	 succinylglutamate desuccinylase activity 
88	 hydrolase activity, acting on carbon-nitrogen (but not peptide) bonds, in linear amides 
60	 aspartoacylase activity 
22	 arginine catabolic process to succinate 
11	 peptidase activity 
11	 cytoplasm 
7	 nucleus 
7	 membrane 
6	 plasma membrane 
6	 metallopeptidase activity 
5	 serine-type carboxypeptidase activity 
5	 biological_process 
4	 transferase activity 
4	 regulation of root meristem growth 
4	 cell wall macromolecule catabolic process 
4	 apical plasma membrane 
4	 aminoacylase activity 
3	 vacuole 
3	 molecular_function 
3	 methyltransferase activity 
3	 methylation 
3	 integral to membrane 
3	 identical protein binding 
3	 cellular_component 
3	 cellular cell wall organization 
2	 transferase activity, transferring glycosyl groups 
2	 purine-nucleoside phosphorylase activity 
2	 polysaccharide catabolic process 
2	 fungal-type vacuole 
2	 fungal-type cell wall organization 
2	 catalytic activity 
2	 arginine catabolic process

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>