PseudoKnot+Project

= = RNA Pseudoknot Project

Christina's research on RNA pseudoknot (Revised 082712)

 * 1) repeat Michael's analysis of 1F27.pdb validation using Biotin that has been prepared in Maestro Lig Prep (get Biotin from PubChem not PDB)
 * dock in ICM, GOLD, VINA
 * 1) do docking of Biotin + Chembridge Diversity in ICM vs. GOLD vs. VINA
 * using structure0001.pdb (**unfixed**) vs. **fixed**
 * (do Molprobity on these two structures to compare their Clashscores, etc.)
 * using **unfixed** 'Best' of the 1,000 structures from Molprobity assignment spreadsheet vs. **fixed** 'Best'
 * (do Molprobity on these two structures to compare their Clashscores, etc.)

EXTRA: Compare regular docking result of Biotin to those that have also been re-ranked with Schrodinger's Prime software in the Maestro Suite -- this software calculated the Free Energy of Binding of a ligand:protein complex (or ligand:RNA) Hypothesis: ??? Testing to see what Prime does to the ranking list. Does it changes significantly? Ideally we want experimental conditions (Ki, IC50, etc) to compare the ranking list to. (see below)

-- don't use our Pseudoknot structures, but rather use known PDB poses (or get ensembles form Molecular Dynamice or NMR data of Stelzer paper) -- take compounds from Stelzer paper (see above) with known Ki values. Then compare VINA docking score against these.(I thin this is just RNA not a 'psudoknot' exactly - but still useful) -- can we get the virtual ligands in the supplementary info? Can we get all of the Ki values? Hypothesis: Virtual Screening with GOLD, ICM and VINA will NOT be able to rank the ligands with a high correlation (Pearson's linear correlation R > 0.6) to experimental data
 * Christina**'s research on RNA screening validation **(OLD version)**

Compare regular docking results to those that have also been re-ranked with Schrodinger's Prime software in the Maestro Suite -- this software calculated the Free Energy of Binding of a ligand:protein complex (or ligand:RNA) Hypothesis: Prime will NOT be able to re-rank the ligands with a higher correlation to experimental data

LATER ????: add in molecular dynamics (using Maestro) of ICM and GOLD docked compounds to further refine selections

For each run, get a Score (either ICM or GOLD) and an image of the ligand docked
 * Michael's work on Pseudoknot (Spring 12)**

COMPARISONS to make
 * ICM vs. GOLD docking of Biotin into 1F27 (this is our POSITIVE CONTROL) - which program does better?
 * Docking of Biotin vs. a 'random' ligand - use either GOLD or ICM results - Is the docking specific?
 * Original vs. 'fixed' pseudoknot docking with biotin in ICM - Did our 'fix' help out?
 * Original vs. 'fixed' pseudoknot docking with biotin in GOLD - Did our 'fix' help out?


 * ICM**- use 'Interactive Docking' mode only - with single SDF ligands as object in the table. Do NOT use the scripts method to run these - fails to properly dock.


 * remove ligand by 'moving object' and then use ligand to define docking site
 * 1) Biotin into 1F27 (get from PubChem)
 * 2) Random Chembridge ligand into 1F27


 * (LOWER PRIORITY): - Can ICM find the 'proper' docking site?
 * ICM finds docking site
 * 1) Biotin into 1F27


 * ICM find binding site by itself
 * 1) Biotin into our original pseudoknot struct-15
 * 2) Biotin into our 'fixed' pseudoknot struct-15


 * GOLD**
 * Use sdf files that have been 'converted to 3D' in ICM - after conversion need to go back and manually re-add names to the top line of each ligand since ICM removes them and places an 'm'
 * Define active site as 10 Angstroms around the X, Y, Z coordinates of the Carbon in biotin that is between the 2 nitrogens and the one oxygen (most proximal side of the ligand)
 * 1) Biotin into 1F27
 * 2) Random Chembridge ligand into 1F27

GOLD ACtive site point form ICM (10.004, -1.153, -24.478) NItrogen (C6 N1) Cytosine 6 Nitrogen 1)
 * Define active site as 10 Angstroms around the X, Y, Z coordinates an atom in the site that ICM found


 * 1) Biotin into our original pseudoknot struct-15
 * 2) Biotin into our 'fixed' pseudoknot struct-15

IMAGES: 1F27 with original Biotin xray pose vs. Biotin from ICM docking 1F27 with original Biotin xray pose vs. Biotin from GOLD docking

TABLE:
 * Software || Ligand || Receptor || Score ||
 * ICM || Biotin || 1F27.pdb || -78.02 ||
 * || Random || 1F27.pdb ||  ||
 * || Biotin || Orig PseudoKnot ||  ||
 * || Biotin || Fixed PseudoKnot || -66.19 ||
 * GOLD || Biotin || 1F27.pdb || 55.14 ||
 * || Random || 1F27.pdb ||  ||
 * || Biotin || Orig PseudoKnot ||  ||
 * || Biotin || Fixed PseudoKnot ||  ||

Carry out Molecular Dynamics simulations of RNA pseudoknot structures to create an emsemble Carry out Molecular Dynamics simulations and re-scoring of best ligand poses to re-rank them.(see Malmstrom & Watowich Dengue Virus paper)

Pseudoknot Fix Protocol
STEPS TO FIX STRUCTURE: take original struct-000X.pdb Open in PyMol, 'A' tab, Remove Hydrogens Save Molecule as struct-000XnoH.pdb Add Crystal Line CRYST1 90.000 90.000 90.000 90.00 90.00 90.00 C 1 2 1 (if fail - copy CRYST line from 1F27.pdb and use it) Save Molecule as struct-000XnoHcryst.pdb ?? need this step or will Phenix Eng Min without Cryst line?? Copy to a folder on the **vdsclass.dyndns.org** (vdsclass login, password is same as GDocs) Go do VDSclass.dyndns.org and open Phenix (Go to the folder you want Then type in command line 'phenix') go to Refinement >> **ReadySet** struct-0001NoHCryst.pdb -input Adds H's -unchekc 'Generate Ligand restraints' struct-0001NoHcrystReadySet.pdb -output Select 'Other Tools' Browse for your file from Ready Set Choose **Energy Minimization** Supply an output file name: struct-000XnoHcrystReadySetEngMin.pdb

then take this to ICM or GOLD or VINA for docking on the DDFE





Aims: >>
 * find binders to an RNA pseudoknot for anti-viral potential
 * validation dock of biotin to an aptamer pseudoknot - in PDB already (@http://www.rcsb.org/pdb/explore/explore.do?structureId=1F27)
 * set up RNA receptor, assess model on MolProbity
 * prepare ligands - generate tautomers, convert to 3D, minimize (in ICM or Babel)

Instructions for Using Molprobity to Analyze Structure:
 * Find Structures here: @https://docs.google.com/open?id=0B_Gl3lMyhDsoYTEyNzJhMWEtMDA2Ny00Y2U3LTgzM2ItZWE1MTlmNjM4MmUz
 * Find assigned structures and download
 * Go to : @http://molprobity.biochem.duke.edu/
 * On the **Browse** button - go find your .pdb file.
 * Hit ' **Upload**'
 * You will see a short page of some results and hopefully see an image on the right hand side (rhs), hit '**Continue**'
 * Analyze all-atom contacts and geometry
 * Use defaults that are already selected --> '**Run programs to perform these analyses'**
 * Input Data in to the Gdocs spreadsheet under the folder Vdsbiooproject
 * Go back to the Molprobity and Click **'Multi-criterion chart'**
 * To save a copy of the page, in your web browser go to File >> Save As >> Web Page complete
 * give it a name that corresponds to the structure you have analyzed. For example: **structure-0016.htm**
 * Save this file to your desktop and then upload it to the Gdocs folder VDSbiooproject at the bottom as shown
 * You will need to open a new Molprobity for each structure

FOR STAFF - disregard below:

02/16/12 - received 1000 pdb structures. Uploaded to Google Docs Michael to generate spreedsheet on GDocs that has a line for each structure and then accompanying Analysis Data from MolProbity.

Fields to save to spreadsheet:
 * Researcher Name
 * Date
 * Name of Structure file e.g. **structure-0016.pdb**
 * All-Atom**
 * Contacts**
 * Clashscore, all atoms:
 * percentile


 * Nucleic Acid**
 * Geometry**
 * Probably wrong sugar puckers:
 * Bad backbone conformations # :
 * Residues with bad bonds:
 * Residues with bad angles:

**Links to Multi-criterion plots for each structure:**
put your cursor where you want to insert the link go to '**File'** button at the top of this wiki page, upload your file and select the **'Click to Link to'** button. Then click on your file name.
 * Instructions:**
 * click on 'Edit'** to edit the page - you must be logged in.