Release 3:
Start FlexX/-SIS in commandline mode
(flexx --commandline); then proceed as described below.

Release 2 and older:
Many people don't even know that FlexX can dock covalently bound ligands. Therefore, we found it all the more important to bring this topic to our readers' attention. In the following we describe how the information of a ligand covalently bound to the receptor in the binding site can be used in a docking experiment.

We will explain covalent docking using the example given in the figure below. The covalent bond is established between the ligand oxygen of the phosphorous group (atom 2) and the carbon of the receptor amino acid Ser70 (atom 3). This is done by mapping the ligand atoms 1 and 2 onto the amino acid atoms 3 and 4, respectively. Note that atoms mapped onto each other must be of the same type!

Workflow:

1. Use an external molecular modeling program in order to transform atoms 1 and 2 such that they are of the same type as atoms 3 and 4, respectively. For our example in the figure, replace the hydrogen bound to the oxygen with a carbon. This carbon is called the terminal atom. Reasonably protonate the ligand except for this terminal atom. No hydrogen may be attached to it!

As normal read the ligand and receptor into FlexX; make sure to switch off automated protonation of the ligand (set ADD_HYDROGENS to 0 in the file config.dat, or in FlexX Release 2, type selinit !4 from the command line in the LIGAND menu). This is necessary because otherwise FlexX would protonate the terminal atom, which must remain deprotonated.

2. Change to the DOCKING menu. Manually select the base fragment (type SELBAS m). You get a list of possible choices — choose the terminal atom. In our example, this is atom 1.

3. Type PLACEBAS c; this invokes the routine for covalent placement of the base fragment. FlexX then asks for two pairs of ligand and receptor atoms.

a.       From the given list, choose the terminal ligand atom (atom 1 in our example) first and then the one that is bound to it (atom 2, in our example the oxygen).

b.       Next you will get a list of amino acid numbers of the receptor. Don't get confused by the numbers: The first number is the FlexX-number (this is the one you have to use); the number after the three letter amino acid name is the original PDB-file number. In our example '39: (Ser, 70)' you select 39. Please note, if you use FlexV to help you select the correct amino acid, take into account that FlexV displays the PDB-numbers, so you still need to identify the FlexX-number, which is required here.

c.       Finally, specify the two receptor atoms. Again FlexX asks you to choose two adjacent atoms from a list. The first selected receptor atom (in our example atom 3) is mapped to the terminal ligand atom. The second receptor atom (atom 4) needs to be terminal and is mapped to the second selected ligand atom (atom 2).

Now the base fragment is placed by positioning the ligand atoms onto the protein atoms (see bottom of the figure above). The bond between the two atoms (O and C) is rotated using 10° increments in order to obtain a set of energetically favorable arrangements. Note that for the first and second neighbor atoms in the ligand (in our example: P and C, O-) no clash-test applies.

In order to simplify repeating this exercise (e.g. while tuning parameters until it works properly) we recommend you use FlexX's ability to log an interactive session (see command line parameter -l in the user guide) and then use the generated log-file as a basis for a batch-script that automates the whole procedure.