View source for 2009 DOCK tutorial with neuraminidase

==About DOCK==

DOCK was developed by Irwin D. "Tack" Kuntz, Jr., PhD and colleagues at UCSF.  Please see the webpage at [http://dock.compbio.ucsf.edu/ UCSF DOCK].

DOCK is a molecular docking program used in drug discovery. This program, given a protein active site and a small molecule, tries to predict the correct binding mode of the small molecule in the active site, and the associated binding energy. Small molecules with highly favorable binding energies could be new drug leads. This makes DOCK a valuable drug discovery tool. DOCK is typically used to screen massive libraries of millions of compounds against a protein to isolate potential drug leads. These leads are then further studied, and could eventually result in a new, marketable drug.

In the following tutorial, commands to be entered at the terminal and command line of Chimera will be in dotted boxes, like this one:
 A dotted box

Whether at terminal or command line will be distinguished by text

Programs central to DOCK are:

'''SPHGEN'''- This program generates sets of overlapping spheres to describe the shape of a molecule or molecular surface. It identifies the active site and other sites of interest and generates spheres that fill that site.

'''GRID'''  - This program generates the grid files necessary for rapid score evaluation in DOCK (rapid scoring evaluation can be done using contact or energy scoring).

'''DOCK'''  - This program matches the spheres generated by '''SPHGEN''' with ligand atoms and use scoring grids which are previously calculated using '''GRID''' to evaluate the ligand orientations

==Making sure DOCK in your PATH==
Try the command 

 which dock6

If dock6 is not in your path, edit your .cshrc (or equivalent) to include the dock6/bin folder in your PATH.

==About Neuraminidase==
[[Image:Neuraminidas.tetramer.ribbons.png|200px|right|thumb| Neuramindase tetramer]]
Neuraminidase is an enzyme family which cleaves glycosidic linkages. In the process of Influenzavirus infection, viral Neuraminidase helps newly assembled virus to be released from host cells by hydrolyzing glycosidic bonds which connect the infected host cells and the hemagglutinin on the surface of progeny viruses.

==Downloading the PDB complex (1F8B)==

Download the neuraminidase file from [http://www.rcsb.org/pdb/explore/explore.do?structureId=1F8B here] into your working directory. To get the monomer, download using the blue file arrow icon. Choose biological unit gz under download files folder at the left side. In that way you can downlad a '''biological gz''' file. If you download it by using "fetch by ID" in Chimera, you can only download a '''pdb gz''' file.

If the '''pdb gz''' file is downloaded the monomeric structure of neuraminidase with the bound ligand is seen.
 
However if the '''biological gz''' file is downloaded a tetramer complex of neuraminidase is seen.

When docking, we will only choose a monomer of the whole protein and its ligands instead of the whole tetramer. And it will be painful if you follow the procedure below, use vi to prepare the enzyme and ligand. Because you need to make sure the ligand you extract from the whole pdb files is right the one that match the monomer you've chosen.

==Manipulating the PDB file using vi for DOCK==

The PDB file can be opened and manipulated in the text editor vi.

For example it is very easy to isolate a certain ligand and create a new pdb file without having to work through Chimera. This alternate method may be appropriate in certain cases when the pdb file was not carefully compiled resulting in odd grouping of certain molecules and atoms. It is best to use vi editing in conjunction with Chimera to achieve greatest efficiency.

For a simple example, we can isolate the ligand '''2-Deoxy-2,3-dehydro-N-acetyl-neuraminic Acid''' (tagged DAN in this pdb), by simply copy a few lines out of the original pdb and pasting them into a new one. To do this simply open up the original pdb file:

  vi 1F8B.pdb

Find the ligand and copy it into a separate pdb file. 

The ligand can now be easily prepared for DOCK by opening it with Chimera and using its built in DOCK prep program.

Many other functions such as grouping ligands and deleting atoms or residues may be done in a facile manner by simply editing the pdb file through the vi text editor.

==Preparing the Enzyme and Ligand in Chimera==

Open the pdb file in chimera using '''Open''' or '''Fetch by ID''' from the '''File''' menu.[[Image:1F8B_select_ligand.png|thumb|left|The selected ligand appears green]]
To visualize the protein and the ligand, click '''Presets''' &rarr; '''Interactive 1 (ribbons)'''.
To access the command line, go to '''Tools''' &rarr; '''General Controls''' &rarr; '''Command Line'''.


'''Before you begin''' make sure to delete the extra molecules from the pdb file.  To do so, in chimera open the 1F8B.pdb file.  '''Select->Residue->HOH''' in the command line type
  delete selected
repeat this for  MAN and NAG as well.
                                                      
'''Preparing the enzyme'''

First you want to delete the ligand and prepare the enzyme for DOCK by charging it.  

To delete the ligand:
In the command line of Chimera, type
 select ligand 

to select the ligand. To delete the ligand, type:
 delete ligand
Or alternatively, you can use the chimera tool bar directly.

To charge the enzyme:
*go to '''Tools &rarr; Structure Editing &rarr; Dock Prep''' 
*In the first dialogue box that appears select ok without making changes. In the second, select '''Unspecified determined by method''' for the histidine protonation then ok.
*You will then get a dialogue box asking you to enter the net charges of specific molecules. Hit ok to run without changing the calculated net charges.
*Save the .mol2 file as nacharged.mol2. This is your charged enzyme.
We will use the 'dms' program to generate the surface of the enzyme.  In order to use this program, the hydrogens on the enzyme must be removed. this pdb file is already stripped of H, but to do this:
*go to '''Select &rarr; Chemistry &rarr; Element &rarr; H'''
*then hit '''Actions &rarr; Atoms/Bonds &rarr; Delete'''
*Save the charged receptor in .pdb format by '''File &rarr; Save pdb''' name it naforDOCK.pdb
For more details about DockPrep, use http://www.cgl.ucsf.edu/chimera/1.2199/docs/ContributedSoftware/dockprep/dockprep.html
[[Image:Example.jpg]]

One need to notice that the structure downloaded form PDB usually contains substrate, ligands, ions cofactors and water etc. While when preparing enzyme for docking, we should only keep substrate and ions.




[[Image:1F8B-ligand.jpg|thumb|The isolated ligand from neuraminidase.]]
'''Preparing the Ligand'''

Now you want to focus on just the ligand, so you need to delete the enzyme.

Open a new session in chimera using the original '1f8b.pdb'

To delete the enzyme, in the command line type:

 select ligand
 select invert all models 
 delete selected

However, after this , NAG(which is not a ligand is still left), maybe because the Chimera is not clever enough, so you need to remove NAG manually:
 on the tool bar, click "select->residue->NAG"
 on the command line, type "delete selected"


To prepare the ligand:

*add hydrogens using '''Tools &rarr; Structure Editing &rarr; Add H'''.  Again, select '''Unspecified determined by method''' for the histidine protonation.
*add charge using '''Tools &rarr; Structure Editing &rarr; Add Charge'''. Choose the AMBER ff03.r1 charge model that is selected by default.  After pressing OK, use the AM1-BCC charge method. Unlike prior steps, this may take several minutes.
*Save the molecule in .mol2 format as 'chargedligand.mol2'

Alternatively, one can just apply '''dockprep''' to get the mol2 file of the charged ligand. 

For reference the total charge on the ligand''' DAN''' will be -1.

==Generation of Enzyme Surface, Spheres, and Grid for DOCK==


===Enzyme Surface===

To generate the enzyme surface, you use the enzyme stripped of hydrogens (this is your naforDOCK.pdb file that we generated previously) in the dms program.

the dms program is found at: /home/sudipto/dms_program

In terminal (you must be in the directory where you saved naforDOCK.pdb):
  /home/sudipto/dms_program naforDOCK.pdb -n -w 1.4 -v -o nasurface.ms

These commands specify the dms program for the naforDOCK file, -n calculates normals for surface points, -w changes probe radius to 1.4, -v verbose, and
-o specifies the output file name (in your case nasurface.ms).

===Spheres===

To generate spheres, you will use a program called sphgen.  Before using sphgen, you need to create a INSPH file.

To create an INSPH file, in terminal:
  vi INSPH

this will create a file called INSPH

type in

 nasurface.ms
 R
 X
 0.0
 4.0
 1.4
 naspheres.sph

when sphgen runs this, it will use the .ms surface file you just created using dms and give an output file of naforDOCK.sph

to run type in:
 sphgen -i INSPH -o OUTSPH
it would take several minutes

An example of new created OUTSPH file

 density type = X
 reading  nasurface.ms 
 type   R
 # of atoms =   3067   # of surf pts =  81632
 finding spheres for   nasurface.ms                                              
 dotlim =     0.000
 radmax =    4.000
 Minimum radius of acceptable spheres?
  1.39999998
 output to  naspheres.sph                                                        
 clustering is complete     53  clusters


NOTE:If the output file OUTSPH or receptor.sph or any of the temp files (temp1.ms, temp2.sph or temp3.atc) exist, a core dump will occur and sphgen will fail. Delete all these files before running sphgen.

now to isolate only spheres surrounding the ligand, you will use sphere_selector

type in:
  sphere_selector naspheres.sph chargedligand.mol2 10.0

To visualize the spheres surrounding the ligand that you have just created, you need to convert the spheres into pdb format.  You will use the showsphere program to do this.

type in:
 showsphere

when prompted type in:
 Enter name of sphere cluster file: naspheres.sph
 Enter cluster number to process (<0 = all): 53
 Generate surfaces as well as pdb files (<N>/Y)? Y 
 Enter name for output PDB file name: naligandspheres.pdb
 Enter name for output surface file name: Any name you want.


You can use Chimera to open nasurface.ms to visualize the surface.
[[Image:Suface.png|200px|left|thumb| Surface of the receptor]]

===Grid===

The last files you need to generate before you can run DOCK are grid files.  The grid is the energy map that allows scoring of the DOCK performed. To run grid you need to create 2 input files: showbox.in and grid.in using vi.

In terminal type 
 vi showbox.in
or 
 vi grid.in

and create the following files

showbox.in
 Y
 5.0                            
 naspheres.sph   
 1                       
 nabox.pdb

grid.in
 compute_grids                  yes
 grid_spacing                   0.5
 output_molecule                no
 contact_score                  no
 energy_score                   yes
 energy_cutoff_distance         9999
 atom_model                     a
 attractive_exponent            6
 repulsive_exponent             9
 distance_dielectric            yes
 dielectric_factor              4
 bump_filter                    yes
 bump_overlap                   0.75
 receptor_file                  nacharged.mol2
 box_file                       nabox.pdb
 vdw_definition_file            /home/rizzo/AMS536software/dock6/parameters/vdw_AMBER_parm99.defn
 score_grid_prefix              grid

For the sake of time, your grid spacing is 0.5 A.  For an actual experiment, there should be less space between the grid points (0.3A is recommended). Also, we have weakened the van der Waals interactions from 6-12 down to 6-9 in our attractive_exponent and repulsive_exponent parameters.  Be aware of these facts when going to set up your own DOCK experiment. This makes the repulsive 

now you are ready to run showbox and grid.  In terminal type:
 showbox < showbox.in

to generate a grid box.  To generate a grid, type:
 grid -i grid.in 
It would take 20-40 minutes. Have a cup of tea. You could also add '-o grid.out &' to save the grid progress statistics to a file instead and have the job run in the background while you do other things.

You have created two output files: grid.nrg and grid.bmp.  You are now ready to run DOCK. Make sure these files exist. The grid.nrg contains the energy grid and should be several MB.

==DOCKing==

DOCK can be done with a flexible ligand or a rigid ligand.  You choose which way you would like to run the DOCK depending on your system. Either way, you will need to create an input file.

For a rigid ligand, your input file is rigid.in which you will create using vi.

In terminal, type 
 vi rigid.in

the rigid.in file is:
 
 ligand_atom_file                                             chargedligand.mol2
 ligand_outfile_prefix                                        rigid
 limit_max_ligands                                            no
 read_mol_solvation                                           no
 write_orientations                                           no
 write_conformations                                          no
 skip_molecule                                                no
 calculate_rmsd                                               yes
 use_rmsd_reference_mol                                       no
 rank_ligands                                                 no
 num_scored_conformers_written                                1
 orient_ligand                                                yes
 automated_matching                                           yes
 receptor_site_file                                           naspheres.sph
 max_orientations                                             500
 critical_points                                              no
 chemical_matching                                            no
 use_ligand_spheres                                           no
 flexible_ligand                                              no
 bump_filter                                                  no
 score_molecules                                              yes
 contact_score_primary                                        no
 contact_score_secondary                                      no
 grid_score_primary                                           yes
 grid_score_secondary                                         no
 grid_score_vdw_scale                                         1
 grid_score_es_scale                                          1
 grid_score_grid_prefix                                       grid
 dock3.5_score_secondary                                      no
 continuous_score_secondary                                   no
 gbsa_zou_score_secondary                                     no
 gbsa_hawkins_score_secondary                                 no
 amber_score_secondary                                        no
 minimize_ligand                                              yes
 simplex_max_iterations                                       1000
 simplex_max_cycles                                           1
 simplex_score_converge                                       0.1
 simplex_cycle_converge                                       1.0
 simplex_trans_step                                           1.0
 simplex_rot_step                                             0.1
 simplex_tors_step                                            10.0
 simplex_final_min_add_internal                               no
 simplex_random_seed                                          0
 atom_model                                                   all
 vdw_defn_file                                                /home/rizzo/AMS536software/dock6/parameters/vdw_AMBER_parm99.defn
 flex_defn_file                                               /home/rizzo/AMS536software/dock6/parameters/flex.defn
 flex_drive_file                                              /home/rizzo/AMS536software/dock6/parameters/flex_drive.tbl


Now to perform rigid docking type in the command line

 dock6 -i rigid.in -o rigid.out

For flexible docking, first create the file flex.in using vi

 vi flex.in

which will contain the following details

 ligand_atom_file                                             ./chargedligand.mol2
 limit_max_ligands                                            no
 skip_molecule                                                no
 read_mol_solvation                                           no
 calculate_rmsd                                               yes
 use_rmsd_reference_mol                                       no
 orient_ligand                                                yes
 automated_matching                                           yes
 receptor_site_file                                           ./selected_spheres.sph
 max_orientations                                             500
 critical_points                                              no
 chemical_matching                                            no
 use_ligand_spheres                                           no
 flexible_ligand                                              yes
 min_anchor_size                                              40
 pruning_use_clustering                                       yes
 pruning_max_orients                                          100
 pruning_clustering_cutoff                                    100
 use_internal_energy                                          yes
 internal_energy_att_exp                                      6
 internal_energy_rep_exp                                      12
 internal_energy_dielectric                                   4.0
 use_clash_overlap                                            no
 bump_filter                                                  no
 score_molecules                                              yes
 contact_score_primary                                        no
 contact_score_secondary                                      no
 grid_score_primary                                           yes
 grid_score_secondary                                         no
 grid_score_rep_rad_scale                                     1
 grid_score_vdw_scale                                         1
 grid_score_es_scale                                          1
 grid_score_grid_prefix                                       grid
 dock3.5_score_secondary                                      no
 continuous_score_secondary                                   no
 gbsa_zou_score_secondary                                     no
 gbsa_hawkins_score_secondary                                 no
 amber_score_secondary                                        no
 minimize_ligand                                              yes
 minimize_anchor                                              yes
 minimize_flexible_growth                                     yes
 use_advanced_simplex_parameters                              no
 simplex_max_cycles                                           1
 simplex_score_converge                                       0.1
 simplex_cycle_converge                                       1.0
 simplex_trans_step                                           1.0
 simplex_rot_step                                             0.1
 simplex_tors_step                                            10.0
 simplex_anchor_max_iterations                                500
 simplex_grow_max_iterations                                  500
 simplex_final_min                                            no
 simplex_random_seed                                          0
 atom_model                                                   all
 vdw_defn_file                                                /home/rizzo/AMS536software/dock6/parameters/vdw_AMBER_parm99.defn
 flex_defn_file                                               /home/rizzo/AMS536software/dock6/parameters/flex.defn
 flex_drive_file                                              /home/rizzo/AMS536software/dock6/parameters/flex_drive.tbl
 ligand_outfile_prefix                                        flex
 write_orientations                                           no
 num_scored_conformers                                        50
 write_conformations                                          no
 cluster_conformations                                        yes
 cluster_rmsd_threshold                                       2.0
 rank_ligands                                                 no

The command to do flexible docking is 
 
 dock6 -i flex.in -o flex.out

==Using a .csh File to Generate Enzyme Surface, Spheres, and Grid for DOCK and to run DOCK with a Single Command==

The above tells you how to step by step go through and create all files necessary for DOCK.  Each program uses a file generated by the program run before it.  It is also possible to create a single .csh file which specifies all of the programs and files to use so that all you will need to do is type "compute" and the computer will go through all of the programs for you.

First you will need to create certain files that the programs need. These are the same files that you created if you followed the step by step protocol (although right now some files still have different names and are not all there yet).

'''Files needed'''

showsph.in
 naspheres.sph
 1
 N
 naligandspheres.pdb

INSPH
 nasurface.ms
 R
 X
 0.0
 4.0
 1.4
 naspheres.sph

showbox.in
 Y
 5.0                            
 selected_spheres.sph   
 1                       
 rec_box.pdb

grid.in
 compute_grids                  yes
 grid_spacing                   0.5
 output_molecule                no
 contact_score                  no
 energy_score                   yes
 energy_cutoff_distance         9999
 atom_model                     a
 attractive_exponent            6
 repulsive_exponent             9
 distance_dielectric            yes
 dielectric_factor              4
 bump_filter                    yes
 bump_overlap                   0.75
 receptor_file                  nacharged.mol2
 box_file                       rec_box.pdb
 vdw_definition_file            /home/rizzo/AMS536software/dock6/parameters/vdw_AMBER_parm99.defn
 score_grid_prefix              grid

rigid.in
 ligand_atom_file                                             ./chargedligand.mol2   
 limit_max_ligands                                            no
 skip_molecule                                                no
 read_mol_solvation                                           no
 calculate_rmsd                                               yes
 use_rmsd_reference_mol                                       no
 orient_ligand                                                yes
 automated_matching                                           yes
 receptor_site_file                                           ./selected_spheres.sph
 max_orientations                                             1000
 critical_points                                              no
 chemical_matching                                            no
 use_ligand_spheres                                           no
 flexible_ligand                                              no
 bump_filter                                                  no
 score_molecules                                              yes
 contact_score_primary                                        no
 contact_score_secondary                                      no
 grid_score_primary                                           yes
 grid_score_secondary                                         no
 grid_score_rep_rad_scale                                     1
 grid_score_vdw_scale                                         1
 grid_score_es_scale                                          1
 grid_score_grid_prefix                                       grid
 dock3.5_score_secondary                                      no
 continuous_score_secondary                                   no
 gbsa_zou_score_secondary                                     no
 gbsa_hawkins_score_secondary                                 no
 amber_score_secondary                                        no
 minimize_ligand                                              yes
 simplex_max_iterations                                       1000
 simplex_max_cycles                                           1
 simplex_score_converge                                       0.1
 simplex_cycle_converge                                       1.0
 simplex_trans_step                                           1.0
 simplex_rot_step                                             0.1
 simplex_tors_step                                            10.0
 simplex_final_min                                            no
 simplex_random_seed                                          0
 atom_model                                                   all
 vdw_defn_file                                                /home/rizzo/AMS536software/dock6/parameters/vdw_AMBER_parm99.defn
 flex_defn_file                                               /home/rizzo/AMS536software/dock6/parameters/flex.defn
 flex_drive_file                                              /home/rizzo/AMS536software/dock6/parameters/flex_drive.tbl
 ligand_outfile_prefix                                        rigid
 write_orientations                                           no
 num_scored_conformers                                        10
 write_conformations                                          no
 cluster_conformations                                        yes
 cluster_rmsd_threshold                                       2.0
 rank_ligands                                                 no

flex.in
 ligand_atom_file                                             ./chargedligand.mol2
 limit_max_ligands                                            no
 skip_molecule                                                no
 read_mol_solvation                                           no
 calculate_rmsd                                               yes
 use_rmsd_reference_mol                                       no
 orient_ligand                                                yes
 automated_matching                                           yes
 receptor_site_file                                           ./selected_spheres.sph
 max_orientations                                             500
 critical_points                                              no
 chemical_matching                                            no
 use_ligand_spheres                                           no
 flexible_ligand                                              yes
 min_anchor_size                                              40
 pruning_use_clustering                                       yes
 pruning_max_orients                                          100
 pruning_clustering_cutoff                                    100
 use_internal_energy                                          yes
 internal_energy_att_exp                                      6
 internal_energy_rep_exp                                      12
 internal_energy_dielectric                                   4.0
 use_clash_overlap                                            no
 bump_filter                                                  no
 score_molecules                                              yes
 contact_score_primary                                        no
 contact_score_secondary                                      no
 grid_score_primary                                           yes
 grid_score_secondary                                         no
 grid_score_rep_rad_scale                                     1
 grid_score_vdw_scale                                         1
 grid_score_es_scale                                          1
 grid_score_grid_prefix                                       grid
 dock3.5_score_secondary                                      no
 continuous_score_secondary                                   no
 gbsa_zou_score_secondary                                     no
 gbsa_hawkins_score_secondary                                 no
 amber_score_secondary                                        no
 minimize_ligand                                              yes
 minimize_anchor                                              yes
 minimize_flexible_growth                                     yes
 use_advanced_simplex_parameters                              no
 simplex_max_cycles                                           1
 simplex_score_converge                                       0.1
 simplex_cycle_converge                                       1.0
 simplex_trans_step                                           1.0
 simplex_rot_step                                             0.1
 simplex_tors_step                                            10.0
 simplex_anchor_max_iterations                                500
 simplex_grow_max_iterations                                  500
 simplex_final_min                                            no
 simplex_random_seed                                          0
 atom_model                                                   all
 vdw_defn_file                                                /home/rizzo/AMS536software/dock6/parameters/vdw_AMBER_parm99.defn
 flex_defn_file                                               /home/rizzo/AMS536software/dock6/parameters/flex.defn
 flex_drive_file                                              /home/rizzo/AMS536software/dock6/parameters/flex_drive.tbl
 ligand_outfile_prefix                                        flex
 write_orientations                                           no
 num_scored_conformers                                        50
 write_conformations                                          no
 cluster_conformations                                        yes
 cluster_rmsd_threshold                                       2.0
 rank_ligands                                                 no


Now you can create the script files, prep.csh, rigid.csh, and flex.csh.  Run prep.csh to generate sphere and grid information for use in docking.  Run rigid.csh to perform rigid docking and flex.csh to perform flexible docking.

prep.csh
 #!/bin/csh
 
 echo Setting up
 #setup
 set dock6bin = /opt/software/AMS536software/dock6/bin
 set workdir = /home/rizzo/dock_project
 mkdir -p $workdir
 cd $workdir
 
 echo Generating the molecular surface
 ~sudipto/dms_program naforDOCK.pdb -n -w 1.4 -v -o nasurface.ms
 
 echo Generating spheres
 #delete temp files
 rm OUTSPH naspheres.sph temp1.ms temp2.sph temp3.atc
 #generate spheres
 $dock6bin/sphgen -i INSPH -o OUTSPH
 #select spheres near binding site
 $dock6bin/sphere_selector naspheres.sph chargedligand.mol2  10.0
 $dock6bin/showsphere < showsph.in
 
 echo Generating grid
 #generate grid box
 $dock6bin/showbox < showbox.in
 #generate grid
 $dock6bin/grid -i grid.in

rigid.csh 
 #!/bin/csh
 
 echo Setting up
 #setup
 set dock6bin = /opt/software/AMS536software/dock6/bin
 set workdir = /home/rizzo/dock_project
 mkdir -p $workdir
 cd $workdir
 
 echo Performing rigid docking!
 #perform rigid docking
 $dock6bin/dock6 -i rigid.in -o rigid.out

flex.csh
 #!/bin/csh
 
 echo Setting up
 #setup
 set dock6bin = /opt/software/AMS536software/dock6/bin
 set workdir = /home/rizzo/dock_project
 mkdir -p $workdir
 cd $workdir
 
 echo Performing flexible docking!
 #perform flexible docking
 $dock6bin/dock6 -i flex.in -o flex.out

<nowiki>*</nowiki>Note: In all these scripts, you will not be able to modify Professor Rizzo's directory, so at the line
 set workdir = /home/rizzo/dock_project
replace rizzo with your directory.


Summarily, to perform a complete rigid and flexible docking, type:
 ./prep.csh
 ./rigid.csh
 ./flex.csh

When the process is complete, you should end with .out files detailing the calculation of your DOCKed ligand, as well as rigid_scored.mol2 and flex_scored.mol2 files that contain topology and coordination information about the best scored ligand for each method.

==Docking Results==
[[Image:Nadocktest.jpg|thumb|654px|left|Screen shot of crystal structure ligand (white) and docked ligand (purple)]]
[[Image:Rigid flexoverlaid.jpg|thumb|left|400px|Overlaid shot of the crystal structure ligand(yellow), flexible docked result(purple) and the rigid docked ligand(cyan)]]
[[Image:Nadocktestclose.jpg|thumb|left|400px|Close of up docked ligand with crystal structure in the binding cleft of neuraminidase]]