1. Question to be solved:

>EWHA_PRACTICE
MAEVEEKGLLSYYFPGERIPDSIISQDGSIRLGPGLVFQKKDREEEIVVSKAGRLHQTGK
NAMLIDSRTKRYIPATNDQVIGQIISRFAEGYRVDIGSAHIAQLNALAFENVTRKSRPNL
NVGSLVYARVSLADRDMEPELECFDATTGKAAGYGELKNGYMITGLSLSHCRKLILPKNT
LLQTLGSYIPFEIAVGMNGRVWVNSENLSTTVLICTAIRNCEFMSDEEQIKYCKDLIKKL

Procedure for Homology/Comparative Modelling

1. Save the amino acid sequence above to your PC.
2. Find the template for homology/comparative modelling
   

   Use the template linked here in this practice. This is the coordinate of a protein with PDB ID 2PEH (chain B). Save the linked file to your PC.
   In the real world, you have to use BLAST/FASTA to search for the best template.

3. Build the alignment for homology/comparative modeling

   Get amino acid sequence of the template (This is sometimes very tricky). Then perform pairwise alignment of the two sequences. The following website can deal with all the trick. http://cib.cf.ocha.ac.jp/EWHA/buildalign.html
   Save the alignment to your PC.
   In the real world, you have to use ClustalW, ALAdeGAP or other related tools.
   

4. Build a coordinate of the target sequence.

   Let us use,

   • SWISS-MODEL

     Register yourself in myWorkspace and use Alignment Mode. The procedure takes some time, but you have to be patient.

     1. Register yourself
        
     2. Go into myWorkspace and select Modeling/AlignmentMode
        
     3. Paste your alignment. Be sure of the format of the alignment. The best way is to manually covert the alignment into FASTA format. Follow the link with a question mark and check the format.
        
     4. Assign the target and template sequences and PDB ID. You cannot upload a PDB file.
        
     5. Check the alignment, and press the submit button
        
     6. Now keep the window as is and wait for the result for a while.
        
     7. Now you have the result and download the model structure PDB file by following the link below the protein figure.
        
   • SCWRL

     This is fast, but an insertion loop will not be modelled.

   • MODWEB

     Get license key and use the tool, but you cannot upload your template and your alignment.

   • MODELLER

     You have to install the software locally.

5. Evaluate the accuracy of the modelled structure
   • Visualize the model structure by RasMol
   • Is the overall structure OK? [MOLProbity]
   • Are dihedral angles appropriate? (1) [DIHED]
   • Are dihedral angles appropriate? (2) [PROCHECK (NOT a web server)]
   • Is the structure protein like? [ProSA-web]
   • Is the modelled structure similar to the real structure? [MATRA]
     

2. Use different website to build an alignment and build a different 3D structure, based on the aligment.

• Align the sequences by ClustalW
• Align the sequences by ALAdeGAP
• Align the sequences by your favorite tool.

Build the 3D structures and compare the structures based on different alignments.