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 pressNext.

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:

·   5A

·   10A (default)

·   20A

·   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 pressBack 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).

The window includes:

·   Name - name of the ligand

·   IC50 uMol/L - IC50 value

·   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.