Computational Biology and Data Mining

This is the supplementary material for the "Secondary structure prediction and threading" lesson in Master in Molecular Medicine; Module IV: Functional Genomics; coordinator Hanspeter Herzel.

Software required

ClustalX: multiple sequence alignment tool (Des Higgins / Julie Thompson)

• Download software: ftp://ftp.ebi.ac.uk/pub/software/clustalw2
  • windows version: clustalx-2.0.11-win.msi
  • (unix and mac versions also available there)
• Documentation: http://bips.u-strasbg.fr/fr/Documentation/ClustalX/

• Firefox
  • Go to Tools/Options/Content
    • enable Java
    • enable JavaScript
• Explorer
  • Go to Internet options/Security/Custom level/Scripting/Scripting of Java applets (enable)

Verify that you can run BiasViz http://cbdm.mdc-berlin.de/tools/biasviz/

Exercises

Remember to ask the teacher if you get lost! His job is to help you! :-)

1. Predicting the domains of a protein sequence

• Obtain a sequence from zebrafish from here: 
  • http://www.uniprot.org/uniprot/Q4RWT9.fasta
• Analyse its domains using 
  • SMART: http://smart.embl.de/
  • PFAM: http://pfam.sanger.ac.uk/ (click on sequence search option)
  • CDD: http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi
• Compare the outputs. Can you spot any differences?

2A. Predicting the transmembrane helices of protein sequences

• Here you can see the entry in the UniProt database for a human GPCR protein:
  • http://www.uniprot.org/uniprot/Q5T6X5
• Examine its transmembrane annotation which includes protein orientation.
• Obtain the sequence of this protein from here:
  • http://www.uniprot.org/uniprot/Q5T6X5.fasta

• Run the sequence in TMHMM (http://www.cbs.dtu.dk/services/TMHMM/) and check the output.
• Is the number of TM helices predicted correct?
• And the orientation of the protein?

2B. Predicting the transmembrane helices of protein sequences

• Here you can see the UniProt entry of the E. coli Vitamin B12 transporter:
  • http://www.uniprot.org/uniprot/P06129
• Examine its transmembrane annotation.
• Obtain the sequence of this protein from here:
  • http://www.uniprot.org/uniprot/P06129.fasta

• Run the sequence in TMHMM (http://www.cbs.dtu.dk/services/TMHMM/) and check the output.
• Is the number of TM helices predicted correct?
• Do you have an explanation for this result?

3A. Collecting homologs and making a multiple sequence alignment

• Obtain the sequence of this fly protein of unknown function from here: 

  • http://www.uniprot.org/uniprot/Q28WW9.fasta

• Use BLAST to find similar sequences in the NCBI database:
  • http://blast.ncbi.nlm.nih.gov/Blast.cgi (use option protein BLAST and input the sequence. You will have to wait one or two minutes).
• Scroll down to the Alignments section then tick at least 8 hits from different organisms in the results list and then click on the Get selected sequences link at the bottom of the page. Try to include some mammals too!
• Look for the option Display and select FASTA. Look for the option Send to and select Text. Save the output to a file. Name it like “a.txt”. You will likely get lost here. Just ask the teacher!
• Input that file using the alignment program ClustalX and align the sequences (menu Alignment, option Do complete alignment). Save alignment in FASTA format (menu File, option Save sequences as, tick option FASTA). You should a file named like “a.fasta”.
  • In the alignment representation the asterisks above the alignment represent conserved amino acids and the colors conserved aminoacid properties.
  • Do you notice some conserved features that could indicate function?

3B. Studying secondary structure using a multiple sequence alignment

• Input your alignment a.fasta to Jpred 
  • http://www.compbio.dundee.ac.uk/www-jpred/advanced.html (use the Choose file option. Select type of input: Multiple Alignment, FASTA and tick option Skip searching PDB before prediction. Then hit the button Make prediction at the bottom of the page. (If you see that there are three or more jobs in the queue just delete the job and try again later / Go to next exercise)

• If you get an output hit button Start Jalview and examine the predictions. 
  • Find out the predicted structure for a proline rich region.

• Input your alignment to BiasViz to identify conserved amino acid biased regions
  • http://cbdm.mdc-berlin.de/tools/biasviz/
• Identify amino acid biased regions.
  • Try hydrophobic residues and window 10. What is the result suggesting (compare to secondary structure prediction)?
  • Can you verify your hypothesis with another tool we have used before?
  • Can you see other biased regions?