Pfam

To identify a set of motifs and active site comprised in a family through hidden markov’s based model.

PRINCIPLE :

PFAM is repository of motifs and active sites occuring in common set of multiple aligned protein sequence similar to prosite. In PFAM the patterns are summarized in the form of probabilistic model all the hidden Markov’s model (HMM) which can drive common features of hte multiple aligned sequences and can transform it into a memorial mode.

PROCEDURE :

The query sequence (protein) is retrieved from any protein database in FASTA format and can be submitted in the submission form of PFAM. Run the program.??

Prosite

To identify the active site patterns in a query sequence.

PRINCIPLE :

Prosite is a secondary database comprising of patterns derived from the multiple sequence alignment of conserved active site and motifs present in a set of homologous proteins. The derived information is stored in hte form of regular expressions which encompasses and summarises the multiple sequence alignment into a single line representation.

PROCEDURE :

Prosite can be quered by a scan prosite interface the query sequence (protein) is retrieved from any protein database in FASTA format and can be submitted in the submission from of prosite (EXPASY) run the program.??

Orf Finder Protocol

To predict the open reading frame of the given sequence using ORF finder.

PRINCIPLE :

The basic principle of the ORF FInder is to identify all open reading frames using the standard (or) alternative genetic codes. It is a graphical analysis tool which finds all open reading frames of a selectable minimum size in a users sequence (or) ina sequence already in the database. It searches for ORF’s in the DNA sequence use enter. The program returns the range of each ORF along with its protein translation.

PROCEDURE :

The sequence is given in FASTA format in text box (or) the GI (or) accession number is given. Then, the desired length of the sequence is given in the form with optimum parameters.

Quantitative Structure Activity Relationships Qsar

To calcualte and correlate structural or property descriptors of compounds with activities.

PRINCIPLE :

Qunatitative structure-activity relationships (QSAR) represent an attempt to correlate structural or property descriptors of compounds with activities. These physiochemical descriptors, which include parameters to account for hydrophobicity, topology, electronic properties, and steric effects, are determined emperically or, more recently, by computational methods. Activities used in QSAR include chemical measurements and biological assays.

A QSAR generally takes the form of a linear equation

Biological Activity = Const + (C1 . P2) + (C2 . P2)+(C3 . P3) +….

?? where the parameters P1 through Pn are computed for each molecule in the series and the coefficients C1 through Cn are calculated by fitting variations in the parameters and the biological activity.

A new computer program called BuildQSAR has been designed to help the QSAR practitioner on the task of building and analyzing quantitative models through regression analysis. The main part of the program is a spreadsheet, in which the user can enter with the data set composed by the structure defination of the compounds, one or more types of biological activity values and manu physiochemical proeperties.

PROCEDURE :

1. Open Dragon Window.

2. Upload the Chemical Structure.

3. Calculate the description.

4. View and Save it in Excel.

??

Building QSAR :

1. Open BuildQSAR window.

2. Upload the Dataset.

3. Select QSAR menu — Draw 2D Plot-Save the result.

4. Select Correlation Matrix Model–Save the result.

5. View the regression plot.

6. Save the result.

Molecular Docking And Interaction Analysis Autodock

To find out the binding pattern of receptor and interactions of Protein Vs Small molecules.

PRINCIPLE :

Docking is a method which predicts the preferred orientation of one molecule to a second when bound to each other to form a stable complex. Docking is frequently used to predict the binding orientation of small molecule drug candidates to their protein targets in order to in turn predict the affinity and activity of the small molecule. Hence docking plays an important role in the rational design of drugs.

Mechanics of Drug :

1. Search alhorithm

2. Scoring function

Autodock is an automated procedure for predicting the interaction of ligands with biomacromolecular targets. Autodock 4.2 uses a semiempirical free energy force field to evaluate conformations during docking simulations. The force field was parameterized using a large number of protein-inhibitor complexes for which both structure and inhibition constants, or Ki, are known.

Autodock and Autodock Tools, the graphical user interface for AutoDock are available on the WWW at http://autodock.scripps.edu/.

AutoDock supports the following file formats :

1. Preparation of Receptor and Ligand files :

Input –R.pdb, L.pdb

Output –R_right.pdbqt, R_flex.pdbqt, L.pdbqt

File Contains : Rigid part of the receptor, Flexible part of the receptor

2. Autogrid :

Input –R_rigid.pdbqt, R.gpf

Output –R.glg, R.*.map, R.map.fld, R.d.map, R.e.map

File Contains : Grid parameters, Grid log file(not used), Atom-specific affinity maps, Gird_data_file, Desolvation map, Electrostatic map

3. Preparation for Docking :

Input —R.rigid.pdbqt, R_flex.pdbqt, L.pdbqt

Output —L.pdf

File Contains : Docking parameters

4. Docking :

Input : R_rigid.pdbqt, R_flex.pdbqt, L.pdbqt, L.pdf, R.*.map, R.maps.fld, R.d.map, R.e.map

Output : L.dlg

File Contains : Log + coordinates + energies

PROCEDURE :

A. Preparing the Protein :

1. Preparing file : [Right -click “PMV molecules”] —>[choose file].

2. Color by atom : [Click?????????under “Atom”].??

3. Eliminate water :Select—–>Select from string—->[write ??HOH* in “Residue” line and * in the “Atom” line]—>Add—–>Dismiss——>Edit—–>Delete—–>Delete Atomset.

4. Find missing atom and repairing them : File —–>Load Module—–[Pmv;repairCommands]—->Edit—->Misc—–>Check for missing atoms—–>Edit—–>Misc. —->Repair missing atoms.

5. Add hydrogens :Edit —>Hydrogens—->Add—–>[choose “All hydrogen”, “no bond order”, and “yes” to renumbering].

6. Hide Protein : [Click on the gray_3_under “show Molecules”].

(Note : if you are planning rigid docking (i.e no flexible parts in the protein), save the protein as RH.pdb for now.

B. Preparing the ligand

1. Make sure the ligand has all hydrogen added before working with ADT.

2. [Toggle the “AutoDock Tools” button].

3. Opening file :Ligand —>Input—->Open—->All Files—->[choose file]—–>Open. (ADT now automatically computes Gasteiger Charges, merges nonpolar hydrogens, and assigns Autodock Type to each atom.)

(Note: if there are problem with the automatic charge alignment on any residue, this_could be addressed by Edit—>Charges—->Check Totals on residues. The molecule is now shown with all the assigned charges, and they could be changed manually.

Alternatively, the deficit charge can be spread over the entire residue.)

4. Define Torsions :*Ligand—->Torsion Tree—->Detect Root(this is the rigid part of the ligand)*Ligand—>Torsion Tree—->Choose Torsions—->[either choose from the viewer specific bonds, or use the widget to make certain bond types active(rotatable) or inactive (non-rotatable). Amide bonds should NOT be active(colored pink)]—->Done.

* Ligand—->Torsion Tree—->Set Number of Torsions——>[Choose the number of rotatable bonds that move the ??’fewest’ or ‘most’ atoms].

5. Save ligand file:*Ligand—->Output—–>Save as PDBQT—->[save with L.pdbqt].

6. Hide the length, as explained in(A5) for the protein.

C. Preparing the flexible residue file :

Grid—->Macromolecule—->Open—->[Choose RH.pdb]. AutoDock will automatically add charges and merge hydrogens. Save the object as RH.pdbqt and move to section D.)

1. Flexible residues—>Input—–>Choose molecule—–>[Choose the original protein R.pdb]—-> Yes to merge nonpolar hydrogen(AutoDock assigns charges + atom types to R.pdb, and merges nonpolar hydrogens).

2. Select the residues to flexible :Select—->Select from string——>ARG8—–>Add—–>Dismiss.

3. Define the rotatable bonds :Flexible residues—->Choose torsions in currently selected residues—–>[Click on rotatable bonds to inactivate them, or vice versa].

4. Save the rigid residues :Flexible residues—->Output—–>Save flexible PDBQT—–>[Save as R_flex.pdbqt].

5. Save the rigis residues : Flexible residues—->Output—–>Save rigid PDBQT—–>[save as R_rigid.pdbqt].

6. Delete this version of protein :Edit—–>Delete—–>Delete Moclecule——>[Choose protein(R)]—->Delete—->Dismiss.

D. Running AutoGrid Calaculation :

The purpose of this section is to define the search grid and produce grid maps used later by Autodock.

1. Open the rigid protein :Grid—->macromolecule—->Open—->[Choose the rigid protein]—>Yes to preserving the existing charges.

2. Prepare grid parameter file : Grid—–>Set Map Types—->Choose Ligand—->[Choose the ligand already opened]—->Accept.

3. Set grid properties :Grid—->Grid Box—->[set the grid dimensions, spacing and center]—->File—->Close Saving Current.

4. Save the grid settings as GPf file : Grid—->Output—->Save GPF—->[save as R.gpf].

5. [Make sure the AutoGrid executable is in the same directory as the input files].??

6. Running: Run—->Run AutoGrid—->[make sure the program name has the right path, and that it is where the input files are]—->Launch—–>[in the command prompt prompt, type “tail-f hsgl.glg” to follow the process]

E. Preparing the docking parameter file(.dpf)

1. specifying the rigid molecule :Docking—->Macromolecule—->Set Rigid Filename—->[choose R_rigid.pdbqt].(or RH.pdbqt ??for rigid docking)

2. Specifying the ligand:Docking —->Ligand—–>Choose—->[choose L.pdbqt]——>[here you can set the initial location of the ligand]—->Accept.

3. Specifying the flexible residues:Docking—->Macromolecule—->Set flexible Residues Filename—->[Choose R_flex.pdbqt].

4. Setting the parameters for the choosen docking method: Docking—->Search Parameters—->Genetic Algorithm—->[for 1st time, use the short number of evalutaions(250,000), and for other runs choose the medium or long]—->Accept.

5. Setting docking parameters:Docking—->Docking Parameters—>[choose the defaults].

6. Specifying the name of the ligand dpf file to be formed, contaning the docking instructions :Docking—->Output—>Lamarckain GA—->[type L.pdf].

7. Confirming the details of docking: Docking—->Edit DPF—->[make sure the right liagnd pdbqt file name appears after the word “move”, and that the right number of active torsions is specified].

F. Running the AutoDock4

1. [Make sure the AutoDock executable is in the same directory as the macromolecule, ligand, GPF, DPF and flex files(in case of flexible docking)].

2. Running: Run—>Run AutoDock…—->Launch.

??

??When RH and LH already exists??

1. Protein :

Grid—->Macromolecule—>Choose RH.pdb—->(Charges & atom types assigned, nonpolar hydrogen merged)—->File—->save—->write PDBQT——>save as RH.pdbqt.

2. Ligand :

Ligand—->Input—->Open—–>All Files—->Choose LH.pdb—–>(Charges & atom types assigned, nonpolar hydrogen merged)—->save as LH.pdbqt.

3. Set the rest of the grid parameters & Calcualte map

4. Setting Docking Parameters:

Docking—>Macromocleucle—->Set Rigid Filename—->choose either RH.pdbqt or RH_rigid.pdbqt—->Docking——>Ligand—–>Choose—–>Choose LH.pdbqt——>set the rest of the docking parameters.

5. Running docking simulation.

Viewing Docking Results :

A. Reading the docking log file(.dlg)

1. [Toggle the “AutoDock Tools” button].

2. Analyze —->Docking—–>Open—->[Choose L.dlg].

3. Analyze—–>Conformations——>Load—->[double-click on each conformation to view it on screen].

B. Visualizing docked conformations

1. Analyze—->Conformations—>Play….(Note: & allows changing the ligand’s color)??

Molecular Dynamics Simulation Using Simple Models And Continuous Potentials Tinker

To simulate the biomolecules using simple model and continuous potential.

PRINCIPLE :

Molecular dynamics ??(MD) is ??a form of computer simulation in which atoms and molecules are allowed to interact for a period of time by approximations of known physics, giving a view of the motion of the particles. This kind of simulation is frequently used in teh study of proteins and biomolecules.

?? TINKER is designed to be an easily used and flexible system of programs and routines for molecular mechanics and dynamics as well as other energy-based and structural manipulation calculations. It is intended to be modular enough to enable development of new computational methods and effiecient enough to meet most production calcualtions needs. Rather than incorporating all the functionality in one monolithic program. TINKER provides a set of relatively small programs that interoperate to perform complex computations.

The TINKER Analysis Facility can provide :

1. Total Potential Energy and its components (E)

2. Energy Breakdown over each of the Atoms (A )

3. List of the Large Individual Interactions (L)

4. Details for All Individual Interactions (D)

5. Radius of Gyration and Moments of Inertia (I)

6. Total Electrical Charge and Dipole Moment (M)

7. Force Field Parameters for Interaction (P)

??

PROCEDURE :

1. Open TINKER window.

2. Select file in the toolbar menu and upload a protein molecule.

3. Change the file format of .pdb to .xyz .

4. Select Modelling commands and choose a Dynamics.

5. Launch the TINKER Command.

6. Choose Force Field.

7. Choose Analyze, and then select all variables (E, A, L, D, I, M, P).

8. Select the Temperature & Pressure.

9. Launch the TINKER ??command

10. Calcualte the RMSD value of superimposed initial Protein and Simulated protein molecule.

11. Save the result.

??

RESULTS AND INTERPRETATION :

The protein (example 2YZL) was simulated in TINKER molecular dynamics environment with the parameters as follows.

Constant Temperature –300 C

Constant Pressure ——1ATM

Time Step ??———1.0 picoseconds

No. of steps ——Ten thousand

The initial and the simulated molecule were superimpose using SPDBV and the RMSD value were found to be 1.07 A.

Molecular Format Conversion Openbabel Protocol

To convert the many languages of chemical data.

??PRINCIPLE :

OpenBabel is free software, a chemical expert system mainly used for converting chemical file formats. Due to the strong relationship to informatics this program belongs more to the category cheminformatics than to moelcular modelling. It is available for Windows, UNIX, and Mace OS. It is distributed under the GNU GPL. It’s open, collaborative project allowing anyone to search, convert, analyze, or store data from. E-BABEL is on-line interactive version of it. For more information, check the Open Babel website : http://openbabel.sf.net .

??PROCEDURE :

1. Open the Babel window.

2. Choose the input format and output format.

3. Upload the chemical structure (PDB).

4. Select Convert MMCIF(Macromolecular Crystallographic Information File).

5. Save the result.??

Structural Superimposition

The given two protein structure can be superimposed on the graphic window.

PRINCIPLE :

Superimposition of proteins is usually done to find out the similarities between the proteins. By doing superimposition one can find the alignment between two proteins and the regions which shows similarity among two proteins.

Swiss PDB Viewer is an application that provides a user firendly interface allowing analysing several proteins at the same time. The proteins can be superimposed in order to deduce structure alignments and compare their active sites or any other relevant parts, amino acid mutation, hydrogen bonds, angles, and distance between atoms are easy to obtain.

??PROCEDURE :

1. Retrieve two protein structure from www.rcsb.org

2. Open swiss-PDB viewer window.

3. Select the file in the toolbar menu and upload two protein structures.

4. Change the color of each molecules for differentiating each other.

5. The file menu offers two commandsto superpose a molecule on to another.

6. Select the type of atom to be considered to superimpose on to another.

7. By invoking these commands, displays the RMS (Root Mean Square) value and alignment window.

8. Save the result.??

Ramachandran Plot Procheck

To draw a Ramachandran plot for a given protein structure using procheck.

PRINCIPLE :

PROCHECK is a program for assessing the “stereochemical quality” of a given protein structure. This program was developed by Laskowski et al. in 1993. The aim of procheck is to assess how normal, or conversely how unusual, the geometry of the residues in a given protein structure is, as compared with stereochemical parameters derived from well-refined, high resolution structures. The input to procheck is a single file containing the coordinates of your protein structure. This must be in Brookhaven file format. The plots produced by procheck can be customised by amending the parameter file called procheck.prm file. Ramachandran plot is one of the way to visualize dihedral angle???? against??????of aminoacid residues in protein structure.

PROCEDURE :

1. Download the protein molecule from www.rcsb.org

2. Open command line window.

3. Enter this syntax,

?? ?? ?? ?? ?? ?? ?? ?? ?? ??Pro Structurename resolution

4. Run the program

5. Results were saved in PS format.

6. Point browser to www.ps2pdf.com

7. Convert the file format which is in PS to pdf file.

8. Save the result.??

Protein Structure Calcualtion Torsion Angle

To find out the torsion angle between four atoms using Swiss-PDB viewer.

PRINCIPLE :

Swiss-PDB viewer (deep view) has been developed since 1994 by Nicolas Guiex.

It is tightly linked to Swiss model, an automated homology modelling server developed within the (SIB) at the structural bioinformatics group at the Biozentrum in Basel.

Swiss-PDB viewer is an application that provides a user friendly interface allowing analyzing several proteins at the same time. The proteins can be superimposed in order to deduce structure alignment and compare their active sites or any other relevant parts, amino acids mutations, hydrogen bonds, angels and distances between atoms are easy to obtain thanks to the intuitive graphic and menu interface.

PROCEDURE :

1. Retrieve the protein molecule www.rcsb.org

2. Open Swiss-PDB Viewer window.

3. Select file in the toolbar menu and upload a protein molecule(3HEO).

4. Change the display of the protein to wire frame model.

5. Select the option of torsion angle menu from Swiss-PDB Viewer window.

6. Zoom the protein molecule.

7. Save the result.??