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The IC50 of a Protein-Ligand Complex
Calculates the free binding
energy and thus predicts the IC50 of a given protein-ligand complex.
BACKGROUND: The IC50 calculation requires 3D structures of a protein and
a ligand. The ligand should already be located in the protein's active site. In
particular, such structures can be downloaded from the Protein Data Bank, as
well as from other sources of 3D biological macromolecular structure data. If
the position of ligand inside of the protein's active site is not known, use
the Ligand Docking Module of the
program.
In order to perform IC50 calculations, you have two options: download a
file in PDB format with molecular structure or download a file in PRJ format (a
Quantum format for saving projects). In the first case (PDB format), you should
manually prepare given complexes. This includes adding hydrogen atoms and
setting charges and the atom's types. PRJ files contain the structures of
protein-ligand complexes with all necessary data, so you do not need to take
the preliminary steps and can proceed with the IC50 calculations right after
opening the PRJ file. PRJ files can be downloaded from our web site (about 300
structures).
After selecting Tools->IC50 of a protein-ligand complex,
you must take the following steps:
Selecting
a Ligand
Make sure that the protein-ligand structure is displayed.
Choose the ligand by clicking on a 3D structure.
Note: You can also do this in a
different way: press on the Sequence viewer button, and select the
ligand on the 1D structure of the complex.
After selecting a ligand, the viewer zooms in on the ligand.
Press the Next button of the Wizard.
The selected small molecule is added as a separate object under the name
"ligand".
Setting the Protonation State and Adding Explicit
Hydrogen Atoms to the Ligand
BACKGROUND: Most sources of 3D structures provide molecules only with
heavy atoms and without hydrogen atoms. There is also no information on bond
types (single, double, etc.) and the protonation state (adding or extracting a
hydrogen atom depending on the pH and the chemical group). But it is essential for Quantum's calculations to have
the right number of hydrogen atoms and the bond types in the molecules. This
procedure helps with this. Then the program will automatically define the
coordinates of the hydrogen atoms.
This step begins by adding hydrogen atoms and setting the protonation
state to the ligand.
To cancel the procedure of adding hydrogen atoms and setting the
protonation state, set the Build Model option of the wizard to OFF. The
previous ligand structure wil appear.
If you turn on the Build Model option, you again get the
structure with hydrogen atoms added by a default procedure.
No matter whether the Build model option was ON or OFF, you can manually
add/remove hydrogen atoms and/or bond types to the ligand by using Builder (see
the Build
molecules section).
Note: We recommend that you use the Build
Model option. Build Model analyzes the geometry of the molecule (the bond
lengths and the bond and dihedral angles) and adds missing hydrogen atoms. The
Build Model procedure also sets the right protonation states.However, you
should take into account that in some cases, molecules from the Protein Data
Bank do not have the right geometry, and you will have to fix them without
assistance from the Build Model procedure, or you will have to at least
manually correct them after Build Model.
Press the Next button of the Wizard.
If the total charge of the ligand exceeds 5e, a warning will appear.
This means that the ligand can have the wrong number of hydrogen atoms. You can
press Back and correct the number of hydrogen atoms. If you think that the
number is right press Next.
Selecting Other
Structures Near the Ligand (if any)
BACKGROUND: Very often a ligand interacts with other ligands and/or ions
in the active site. The correct energy calculation should involve all such
structures. On the other hand, the more atoms involved in a calculation, the
longer it takes. A practical compromise is to exclude the structures that are
too far from the ligand so that their influence on the ligand binding can be
neglected.
Imagine a figure consisting of spheres with centers in the ligand atoms
position. All spheres have the same radius. All molecule structures, which are
partly, even if by one atom, inside of this imagined figure, are considered to
be near the ligand. Now you should choose the radius.
Go to the Wizard (Figure 21) and choose the selection radius.
Figure 21 - Choosing a
Radius
When selecting the radius you have the following options:
·
none (no structures will be selected)
·
all (all structures will be selected)
In the next step, you will see all structures within the selected
radius. For instance, for the 1CQP complex, the following structures will be
selected within 10A radius (Figure 22):
Figure 22 - Active Site
Structures
We have three new objects here: protein, metal and hetatom. Make sure
that these objects are the structures of the protein-ligand complex.
Note: You must exclude water molecules
in modeling since Quantum has its own model of water.
You can remove any structure from the list.
Increase the radius to add structure if necessary.
Adding
Hydrogen Atoms to the Protein and to the Hetatoms
This step begins by adding hydrogen atoms and setting the protonation
state to the protein and the hetatoms.
To cancel the procedure of adding hydrogen atoms and setting the
protonation state, set the Build Model option of the wizard to OFF. The
previous protein and hetatoms structures will appear.
If you turn on the Build Model option, you again get the
structures with hydrogen atoms added by a default procedure.
No matter whether the Build model option was ON or OFF, you can manually
add/remove hydrogen atoms to the protein and the hetatoms.
Press the Next button of the Wizard.
If the total charge of the protein exceeds 30e, a warning will appear.
This means that the protein can have the wrong number of hydrogen atoms. You
can press Back and correct the number of hydrogen atoms. If you think
that the number is right press Next.
Select the option regarding protein flexibility (Figure 23), which is
off by default.
Figure 23 - Wizard
If this option is OFF, then the protein is treated as a rigid structure.
If it is ON, then full protein flexibility will be taken into account.
Note: Protein flexibility capability
depends on the license you purchased. This manual describes all possible
functions, and some of them may not be accessible in your installation.
You can finally start modeling.
Press the Calculate IC50 button.
Calculations and Results
All stages of the process are displayed on the Progress Bar and in the
Information Panel.
When the calculation is complete, you can see the results in a window,
that will appear instead of the information panel (Figure 24).
·
E bind, kJ/mol - binding free energy
·
E es, kJ/mol - electrostatic and solvation
energy
·
E vdw, kJ/mol - short range electrostatic
and exchange and Van der Waals energies
· TdS, kJ/mol - entropy contribution
·
E tor, kJ/mol - ligand internal energy change
·
Charge, Mass, Flex.bonds - total charge,
mass and number of flexible bonds of the ligand
·
RMSD, A - root mean square distance
between the initial and final positions
Note: Free binding energy is equal to the
sum of all listed contributions (E es, E vdw, TdS and E tor). In our
calculations,
.
Figure 24 - Results
You can compare the initial and final positions of the ligand by using
Viewer. The procedure will create the object ligand_pos with final coordinates.
You can also save the report in HTML format, which is readable for most
spreadsheet applications.
Press Save
Project to save the given protein-ligand structure with set hydrogen atoms,
bond types, and electric charges.
In order
to exit the IC50 procedure, press the Close button in the window or the Finish
button in the Wizard.