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 754 metabolic process 752 hydrolase activity, acting on ester bonds 599 hydrolase activity 588 metal ion binding 256 zinc ion binding 121 proteolysis 118 metallocarboxypeptidase activity 117 arginine catabolic process to glutamate 117 arginine metabolic process 116 succinylglutamate desuccinylase activity 88 hydrolase activity, acting on carbon-nitrogen (but not peptide) bonds, in linear amides 60 aspartoacylase activity 57 carboxypeptidase activity 22 arginine catabolic process to succinate 9 cytoplasm 6 ATP binding 5 membrane 5 transferase activity 4 methyltransferase activity 4 methylation 4 apical plasma membrane 4 aminoacylase 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
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>
