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| ==Molecular Fingerprinting of Compounds to the ADA active site==
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| * The objective for this laboratory period is obtain the molecular fingerprint of aspirin and dock compounds to the binding pocket of adenosine deaminase (ADA).
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| A. Opening Maestro
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| * From the assigned computer, a new terminal was created from either of the following steps:
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| 1. Finder > Services > New terminal at folder
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| -or-
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| 2. press the provided shortcut key F5
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| * Then enter $maestro to open the suite.
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| * Draw the molecule on the workspace area. Modifications can be made through the Edit panel > build > fragments > atom properties. Sponge the drawn molecule for clarity.
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| * To save the molecule, create this entry on the project table -or- from the workspace option > project entry > name and create the project and then save.
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| * The next step is to put the molecule under energy minimization to find a stable energetically conformation.
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| B. Energy Minimization
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| * To initiate energy minimization, go to the Applications option > Impact > Minimization.
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| * Minimization analyzes the molecule using Classical Physics.
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| * A dialog box appears with the force field set on OPLS 2005.
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| ** Force field is a set of rules containing how atom types are connected angle and torsional angle
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| ** gives the constant measure of force
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| ** The Constraints tab contain NOE, NMR, indicating the closeness of atoms and information about the radius
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| * Then click the Minimization tab. On the maximum cycles, this was changed to 10,000. The other standard parameters such as gradient criterion (0.01) and energy change criterion (1e-07) were kept. The pH was changed to the desired pH level of 7.4.
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| * Knowing there are amino acids present, by changing the pH of the parameters, the structure must also be converted to its alkoxide form at pH 7.4. As a result, through atom types O3 was changed to OM.
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| * After all the parameters were set for minimization, the host zorro was chosen and the minimization was started.
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| * Double-click the finished minimization on the monitor jobs (from applications option), then label the partial charges of the molecule.
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| * Save the project entry.
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| C. Calculation of Fingerprint
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| * Opened Canvas from the Finder by typing $Canvas
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| * Created a new project and named it as Aspirin_fingerprint.
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| * Imported the energy minimized aspirin saved earlier from maestro.
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| * Under applications option, click binary fingerprints > molprint2D
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| * Click on the imported molecule and incorporate.
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| * Export the molecule by saving it with an extension of .fp
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| * A dialog box will appear, choose remove all properties and click ok.
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| * Close the project.
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| D. Screen Fingerprint
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| * After closing the previous aspirin project, open the zinc database.
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| * Imported the aspirin.fp molecule and allow duplicate mappings.
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| * Go to the Applications > similar/distance screen > select aspirin.
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| ** Tanimoto similarity
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| * Selected the fingerprint column and choose the fingerprint (aspirin)
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| * Screened the molecules and then incorporate ~ 15,001
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| * Clicked the fingerprint screen column and sort in descending order to show molecules closest to aspirin.
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| * On the panel, select rows 1-15,001
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| * Saved the selection by exporting the project as aspirin_15001.mae along with its properties and click OK.
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| ** A noticeable common feature among the imported database is the benzene ring.
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| * Closed Canvas
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| E. Docking Preparation
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| [[Image:Prepwizard2.png|thumb|right|Protein Preparation Wizard_Import and Process Tab]]
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| * Opened the protein databank (PDB) website, www.rcsb.org, and acquired two ADA Bovine structures. The two following structures were chosen by desirable resolutions:
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| **1KRM (resolution 2.5 angstroms)
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| **2Z7G (resolution 2.52 angstroms)
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| * The pdb.txt of these structures were downloaded.
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| * On Maestro, opened the aspirin.prj and imported the downloaded structures of ADA.
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| * The structures of ADA were protonated according to the pH of 7.4. This was executed from:
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| ** Workflows > Protein preparation Wizard
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| ** On the Import and Process Tab, add H<sup>+</sup> (see image on the right side of the protein preparation wizard dialog box)
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| ** Uncheck the delete waters on the dialog box and click preprocess.
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| ** Moving to the Refine Tab, changed the default pH of 7.0 to 7.4 as shown below.
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| ** Then click Optimize
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| [[Image:Refine75.png|center]]
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| F. Superimposition of Proteins
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| * After Optimization, navigated through Tools > Protein Structure Alignment
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| ** All > align
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| ** From the Undisplay icon, remove waters for both structures
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| * Added ribbons for all residues to view the entire structure
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| * Upon scanning the structure, subtracted all portions leaving only the ligand on display at 5 Angstroms.
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| * Compared both structures by superimposing them.
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| * Verified structure 1KRM has a better resolution and removed 2Z7G from the project table.
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| * Showed ribbons for 1KRM and colored element entry carbons
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| G. Docking
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| * Duplicated the 1KRM H-minimized and deleted water molecules by manually clicking on each.
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| ** Removed the water molecules to remove the rigidity of the protein. This also interferes with the docking of compounds by taking up space.
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| * Changed ribbon color by residue position
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| * Made a grid of the docking region by:
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| ** Application > Glide > Receptor Grid Generation
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| ** Select the ligand
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| ** Click site tab > center on ligand > change dock ligands to 15 Angstroms
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| ** Click Start
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| * Changed the grid name and specified the host: zorro; click OK
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| * Seeing the docking is done from the Monitor Jobs window:
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| ** Applications > Glide > Ligand Docking
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| ** Browse for the .zip grid previously created
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| ** Verify host: zorro and click start
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| * Imported the glide.pv file
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| * Excluded the protein structure leaving only the ligand
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| * Superimposed the raw, crystallized PDB ADA ligand structure with the docked ligand
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| * Imported flavonoids to compare with the docked ligand by superimposition
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| * Database screening was initiated by going to Applications > Glide > Ligand Docking
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| * Selected the entry with 15,000 compounds from the .pv file
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| * Selected zorro host and clicked start
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| ==Notes==
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| * The database chosen were compounds that did not cross the Blood Brain Barrier.
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