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WEEK 7 Prediction of functional sites (II) Ligand Binding Sites CASTp server http://sts-fw.bioengr.uic.edu/castp Q-SiteFinder - http://www.modelling.leeds.ac.uk/qsitefinder/ PDBSUM - http://www.ebi.ac.

uk/thornton-srv/databases/pdbsum/

Q-SiteFinder and CASTp 1) Q-SiteFinder is a method for ligand binding site prediction. It works by binding hydrophobic (CH3) probes to the protein, and finding clusters of probes with the most favorable binding energy. 2) During this exercise, we will use the Crystal structures of soybean beta-amylase bathed with small substrates (PDB ID 1byb). The advantage of using an entry with a ligand, is that we can validate the results of the tool. 3) Go to the Q-SiteFinder web site, enter the 1byb PDB ID and submit the job. Treat As Ligand selection will removes the ligand from the protein before binding site analysis, but retained in the final output. Submit the job. 4) After a few moments the calculation is done, you will get a nice JMOL interface with several sub-windows. Explore it, what do you think it is displayed in the JMOL window? What are those colored regions in different regions of the protein surface? 5) The 10 best predicted sites (interconnected dots) and any ligands (colored by atoms) are displayed. To rotate the molecule, click and drag with the left mouse button. 6) Briefly explore the output site, the representations etc. on the right upper corner of the page. Predicted binding site are listed in the Display Sites (atoms forming the binding Sites on the protein) window according to the likelihood (energy) of the cluster of probes (Site 1, Site 2, etc.). Clicking on the Toggle Surface checkbox, will display the site volume. Does the biggest cleft include the ligand? 7) But again, in order to take a more detailed look at the results, we will download the modified PDB produced by Q-SITEFINDER. For this purpose right click on the Download link, choose and choose Save target link as on the menu that opens, and choose a name and a location to save the file PDB file. Be sure to use a ".pdb" type for this file.

8) First, let's see how the PDB file looks like. Go to the folder where you just saved the modified PDB file, right click on it and Open with and choose EditPad Lite program or any other text editor. 9) This file look like a PDB formatted file, but there are some "special residues" can you see them? In order to understand what you the special residues on the modified PDB file, you need first to carefully read the Manipulation of Output Using Rasmol section on the Help! file of Q-SiteFinder. 10)So there are three new residue types on this file; Ligand atoms, Sites defined by protein atoms and Sites defined by probe clusters. What are they? 11)Now open a new chimera window and upload the modified PDB file you just downloaded from Q-SITEFINDE into it. This file includes all the information you see on the JMOL window of Q-SITEFINDER, which we will manipulate. 12)It will be easier to work with a reduce representation (only CA) of the protein and every pocket in wire or CPK. Use the chain @CA command to show CA of the protein. All other atoms (~protein), display them in full representation. Protein in stick and all other in wire. 13)Now color ONLY the ORIGINAL ligands that appear in the PDB (note that their original residue names have been modified!) in red color, you will need to use their NEW residue names to do that. Try to find the way that most clearly shows the ligand alone (use repr and color). 14)Now the most interesting part, looking where is the most favorable energetic cluster of probes. The probes were added to the original pdb as special residues with special names (as detailed in the Help! file). 15)To see them clearly, change the color and representation of Predicted site 1 only, so you can see the probes clearly. Color the protein and probe clusters differently, so you can see them. Does it match the positions of the ligand atoms? All of them? Most of them? 16)When you are able to see clearly the Site1 in cpk and red for example, the ligand in green, do you think the best predicted site is a good guess? Does it include the whole ligand? What about the second site? 17)You can also look at the proteins atoms surrounding the probes (Sites defined by protein atoms) by using the special residue names used for them. Can you see the difference between the protein and the probe clusters?

18)Where are all other clusters found? Is there any other close to the biggest one? What does it mean? 19)Now we will compare the ligand binding site prediction obtained from QSiteFinder and CASTp. CASTp identifies cavities and pockets on protein structures. 20)CASTp can be run from its database online (see www), but now we want to compare the result from CASTp with Q-SiteFinder, so we will import the CASTp calculation directly for 1byb using the File Fetch by ID menu on the Chimera windows already open. Choose the CASTp database, fill the PDB ID and click on the Fetch button. This should import the PDB and a new CASTp Pocket List menu. 21)You can also import the CASTp entry directly from the command line, using the following command: open castp:1byb. 22)In this PDB you have a maltose sugar ligand, in order to see it properly, color it and show it clearly so you can identify its location on the protein surface. Did you find it? 23)When the molecule and the CASTp windows appears, take a look at the pocket list menu. You can sort the data by any column, explore the different columns, can you understand their meaning? 24)Since we are generally interested in the biggest pocket, sort the list by area or volume to get the biggest pocket on the top of the list. Then double click on the biggest pocket, the second one, etc. In this way, you will select the residues forming the pocket the protein surface. Can you identify the larger cleft? Be sure to color the pocket in a distinctive color. 25)Does the biggest pocket include the ligand? Is the pocket a good match to its location and shape? 26)Now compare the largest pocket predicted by CASTP and the more energetic pocket predicted by Q-SiteFinder, what can you say about them? 27)Maybe the easiest way to do it is to broaden the atom based chimera prediction using the select up command, and show both pockets in stick representation. DONE!!

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