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Ligand fitting |
(10 minutes)
When you start X-LIGAND, there will be a few seconds' delay. This is because the program must generate a sphere of symmetry atoms, and then make sure the map covers these atoms. The message line at the bottom of the graphics screen will indicate the progress of the setting up. The X-LIGAND palette will appear with only 5 tools that can be selected.
The graphics display will show the molecule of insulin, the ligand, and a blue sphere of symmetry atoms.
To complete this tutorial you need a licensed copy of QUANTA that includes this module:
This example requires the file ligand.tar.Z. <Shift>-Click on it to copy it to your area. Then click OK in the next window that comes up. Make a note of the directory into which the file is going to be saved.
Now cd to that directory. Uncompress and untar the file by typing:
uncompress ligand.tar.Z
tar xvof ligand.tar
Now type:
cd ligand
And start QUANTA:
quanta
When QUANTA has finished loading, select Applications / X-LIGAND. This starts the ligand fitting tutorial.
Click the palette tool Search for ligands (tool number 1). This searches the map within the displayed sphere for possible sites for the ligand, and sorts these in order of volume. This takes approximately 2 - 3 seconds. The display centers at the first (largest) possible ligand site, the map is displayed (at 1 and 2 sigma), along with a search volume that is indicated by yellow crosses.
At this point, the application has already fitted the ligand at this site as a rigid body, so you will find that it will be in the correct position already, but in an incorrect conformation and orientation. (This fitting takes 0.001 seconds). You can turn the map on and off with the 4th tool on the X-LIGAND palette, Map on/off; the display manipulation is much e asier when the map is off.
To make the fitting quicker, turn off the display of the map using the 4th tool on the palette. If you want to see the application really fly, turn off the symmetry and display of the molecule.
Click the X-LIGAND tool Search conformations; which is tool number 7.
As the ligand is currently a long way from the best solution, there is a delay of a few seconds, displaying each improving sollution as it is found. The actual computation runs at about 1000 conformations/second. Since the ligand is small, the program uses an exhaustive grid search. For ligand with more internal degrees of freedom, the program would use a Monte Carlo search algorithm.
There are 2160 conformations to search for this ligand. If you click the Search conformations tool again, the message line indicates the actual progress of the search at full speed now that the correct solution has been determined. The search should take approximately 10 seconds on an R4000 series machine.
The program returns the best 20 conformations. Use the tool ...all 20 conformations to view all 20. You will see for this ligand they are almost identical. You can flick through the different conformations using the tools ...previous conformation and ...next conformation.
You can now refine the ligand conformation, orientation, and position using the Refine tool on the X-LIGAND palette. This proceeds faster without the map displayed (this avoids redrawing the picture every 10 steps during the refinement).
The refinement method is torsion angle real space refinement using both linear and quadratic interpolation of the electron density. The final coordinates will be within 0.01 Å of the refinement carried out with standard refinement procedures, but the radius of convergence is much higher with this routine.
Use the tool Save ligand to MSF. This creates a new coordinate file containing the refined coordinates.
You can investigate the radius of convergence of the refinement algorithm by refining the ligand again.
Also try looking at other sites using Next site and Previous site.