User:Mary Mendoza/Notebook/CHEM572 Exp. Biological Chemistry II/2013/01/30: Difference between revisions
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==Molecular Fingerprinting | ==Molecular Fingerprinting of Compounds to the ADA active site== | ||
* The objective for this laboratory period is obtain the molecular fingerprint of aspirin and dock compounds to the binding pocket of adenosine deaminase (ADA). | |||
A. Opening Maestro | |||
* From the assigned computer, a new terminal was created from either of the following steps: | * From the assigned computer, a new terminal was created from either of the following steps: | ||
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B. Energy Minimization | |||
* To initiate energy minimization, go to the Applications option > Impact > Minimization. | |||
* Minimization analyzes the molecule using Classical Physics. | |||
* A dialog box appears with the force field set on OPLS 2005. | |||
** Force field is a set of rules containing how atom types are connected angle and torsional angle | |||
** gives the constant measure of force | |||
** The Constraints tab contain NOE, NMR, indicating the closeness of atoms and information about the radius | |||
* 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. | |||
* 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. | |||
* After all the parameters were set for minimization, the host zorro was chosen and the minimization was started. | |||
* Double-click the finished minimization on the monitor jobs (from applications option), then label the partial charges of the molecule. | |||
* Save the project entry. | |||
C. Calculation of Fingerprint | |||
* Opened Canvas from the Finder by typing $Canvas | |||
* Created a new project and named it as Aspirin_fingerprint. | |||
* Imported the energy minimized aspirin saved earlier from maestro. | |||
* Under applications option, click binary fingerprints > molprint2D | |||
* Click on the imported molecule and incorporate. | |||
* Export the molecule by saving it with an extension of .fp | |||
* A dialog box will appear, choose remove all properties and click ok. | |||
* Close the project. | |||
D. Screen Fingerprint | |||
* After closing the previous aspirin project, open the zinc database. | |||
* Imported the aspirin.fp molecule and allow duplicate mappings. | |||
* Go to the Applications > similar/distance screen > select aspirin. | |||
** Tanimoto similarity | |||
* Selected the fingerprint column and choose the fingerprint (aspirin) | |||
* Screened the molecules and then incorporate ~ 15,001 | |||
* Clicked the fingerprint screen column and sort in descending order to show molecules closest to aspirin. | |||
* On the panel, select rows 1-15,001 | |||
* Saved the selection by exporting the project as aspirin_15001.mae along with its properties and click OK. | |||
** A noticeable common feature among the imported database is the benzene ring. | |||
* Closed Canvas | |||
E. Docking Preparation | |||
[[Image:Prepwizard2.png|thumb|right|Protein Preparation Wizard_Import and Process Tab]] | |||
* Opened the protein databank (PDB) website, www.rcsb.org, and acquired two ADA Bovine structures. The two following structures were chosen by desirable resolutions: | |||
**1KRM (resolution 2.5 angstroms) | |||
**2Z7G (resolution 2.52 angstroms) | |||
* The pdb.txt of these structures were downloaded. | |||
* On Maestro, opened the aspirin.prj and imported the downloaded structures of ADA. | |||
* The structures of ADA were protonated according to the pH of 7.4. This was executed from: | |||
** Workflows > Protein preparation Wizard | |||
** On the Import and Process Tab, add H<sup>+</sup> (see image on the right side of the protein preparation wizard dialog box) | |||
** Uncheck the delete waters on the dialog box and click preprocess. | |||
** Moving to the Refine Tab, changed the default pH of 7.0 to 7.4 as shown below. | |||
** Then click Optimize | |||
[[Image:Refine75.png|center]] | |||
F. Superimposition of Proteins | |||
* After Optimization, navigated through Tools > Protein Structure Alignment | |||
** All > align | |||
** From the Undisplay icon, remove waters for both structures | |||
* Added ribbons for all residues to view the entire structure | |||
* Upon scanning the structure, subtracted all portions leaving only the ligand on display at 5 Angstroms. | |||
* Compared both structures by superimposing them. | |||
* Verified structure 1KRM has a better resolution and removed 2Z7G from the project table. | |||
* Showed ribbons for 1KRM and colored element entry carbons | |||
G. Docking | |||
* Duplicated the 1KRM H-minimized and deleted water molecules by manually clicking on each. | |||
** Removed the water molecules to remove the rigidity of the protein. This also interferes with the docking of compounds by taking up space. | |||
* Changed ribbon color by residue position | |||
* Made a grid of the docking region by: | |||
** Application > Glide > Receptor Grid Generation | |||
** Select the ligand | |||
** Click site tab > center on ligand > change dock ligands to 15 Angstroms | |||
** Click Start | |||
* Changed the grid name and specified the host: zorro; click OK | |||
* Seeing the docking is done from the Monitor Jobs window: | |||
** Applications > Glide > Ligand Docking | |||
** Browse for the .zip grid previously created | |||
** Verify host: zorro and click start | |||
* Imported the glide.pv file | |||
* Excluded the protein structure leaving only the ligand | |||
* Superimposed the raw, crystallized PDB ADA ligand structure with the docked ligand | |||
* Imported flavonoids to compare with the docked ligand by superimposition | |||
* Database screening was initiated by going to Applications > Glide > Ligand Docking | |||
* Selected the entry with 15,000 compounds from the .pv file | |||
* Selected zorro host and clicked start | |||
==Notes== | |||
* The database chosen were compounds that did not cross the Blood Brain Barrier. | |||
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Revision as of 08:48, 8 February 2013
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Molecular Fingerprinting of Compounds to the ADA active site
1. Finder > Services > New terminal at folder -or- 2. press the provided shortcut key F5
Notes
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