Bcell Epitope prediction
We want to look for epitopes on the HIV GP120 protein. We will use both sequence-based and structure-based methods. Input data.
Go to the pdb homepage and look for the pdb entry 4LSP. It is GP120 complexed with a broadly neutralizing antibody. These antibodies are extremely powerful, since they are effective against many different strains.
Q1: Download both the structure (pdb) and the sequence (fasta) of the proteins in this entry. Which proteins are in chain G, L and H respectively?
hint: you can find this information in the structure web-page, in the PDB file itself (you can open it with a text editor), and, if you are more experienced, in the fasta file.
Q2: using pymol, can you identify which residues are part of the epitope? Is the epitope linear? Does it have a linear core?
Hints: remove the waters, then select the antibody and modify this selection to find residues in a 5Å sphere. You can find a quick video on how to do this in the hands-on prerequisites.
Q3: using pymol, identify the antibody in the structure, select and remove it (action -> remove atoms). Save the resulting molecule as antigen.pdb
You can save using the GUI (remember to select PDB as option) as well as typing "save antigen.pdb" in the command line
Now we are starting with a sequence-based prediction of the epitopes.
Bepipred 2/3
Go to the bepipred website and upload the sequence of GP120. Leave the default parameters and submit the job. Don't close the window because we will need it again!
Q4: does the Bepipred prediction overlap with the actual epitope?
Be careful: pdb file numbering does not always start from 1! Compare the numbering of the pdb with the one returned by Bepipred.
Repeat the prediction now using BepiPred3.
Q5: Does the prediction overlap with our epitope now?
Q6: What is different in BepiPred 2 compared to Bepipred 3? Discotope 2/3
Finally, let's use DTU Discotope server. Load the antigen.pdb file and submit the prediction. Save the output as discotope.pdb Once again, the prediction is in the b-factor field (but you can download it in other formats as well) Load it in pymol and colour according to the b-factor.
Q7: Does the prediction overlap with our epitope?
Q8: Which prediction best matches our epitope?
Q9: Use Discotope to predict the epitopes in the simian HIV GP120 homolog (pdb code 3FUS). If you want to save time, use this pre-cleaned PDB. Compare the results. Try and explain the differences. According to you is it a meaningful result or not? Please note that some bugs might occur, according to which file you use. If some strange results appear, report it in your answer :)
Q10: Compare the results obtained in Discotope 2 and 3