2. How to run LIGPLOT

The script file that runs LIGPLOT assumes the following:

That you have installed the HBPLUS program in accordance with the Installation Instructions.
That you have installed the Het Group Dictionary, as het_dictionary.txt or as components.cif, in the LIGPLOT directory, in accordance with the Installation Instructions.

If you wish to use a program other than HBPLUS for calculating hydrogen bonds and nonbonded contacts, then you will need to alter the script file that runs the LIGPLOT program, ligplot.scr, to take this into account. The files output by the program will need to be called filename.hhb and filename.nnb, respectively, where the name of your input PDB file is filename.pdb. Their format must be as defined in Appendix B.

a. Running LIGPLOT

To run LIGPLOT, type the following:

ligplot filename [residue1] [residue2] [chain_id] [-w] [-m] [-h]

where:

filename is the name of the file containing your protein structure and should contain the full path unless your structure is in the default directory. The file must be in PDB format (see Appendix A).

[residue1] and [residue2] is the residue range for the ligand (ie its first and last residues). Both [residue1] and [residue2] can be entered as just a residue number. Alternatively, they can be entered as a residue name and residue number. In other word, the range can be entered either as, say, the first and last residue numbers:-

18 20

or as the relevant names and numbers:-

NAG 18 MAN 20

In many PDB files, residues in the protein have the same residue numbers (and chain-ids) as residues in the ligand. Thus entering the residue names, as above, helps LIGPLOT identify what is to be plotted. The residue names must be in upper-case.

Note that, where the residue name is wholly, or even partly, numeric you will need to distinguish the residue name from the residue number by prefixing the former with a -n. For example, the PDB file 1fkd has its ligand residue named "818", whereas its residue number is 108. To define this ligand for plotting by LIGPLOT you would need to enter the residue range as:-

-n818 108 -n818 108

In some cases the residue name includes leading spaces (for example, the nucleic acid bases, A, C, G and T are defined as ¤¤A, ¤¤C, etc in the PDB files, where the ¤ symbol represents a blank space). To include these blank spaces in the residue names use double-quotes for the name.

For example, to plot residues MTE 1 and ¤¤G 2 (chain B) in PDB file 1cxt, you would enter

ligplot p1cxt.pdb "MTE" 1 "¤¤G" 2 B

ligplot p1cxt.pdb "-nMTE" 1 "-n¤¤G" 2 B

[chain_id] is the ligand's chain identifier, which must be entered unless the ligand's chain is blank (ie space).

[-w] indicates that the ligand is a single water molecule or group of water molecules.

[-m] indicates that the ligand is a single metal ion. This option is only really necessary when there is another residue in the PDB file which has exactly the same residue number and chain-id as that of the metal ion you want plotted.

[-h] indicates that you would like to enter a heading for the output plot (the program will prompt you to enter the heading).

For example, to run a LIGPLOT for 6tmn, in which the ligand has a range of 1 to 4 with chain identifier I, you might type:

ligplot /data/pdb/p6tmn.pdb 1 4 I

To define the range more specifically, you can include the residue names as:

ligplot /data/pdb/p6tmn.pdb CBZ 1 LEU 4 I

ligplot /data/pdb/p6tmn.pdb "CBZ" 1 "LEU" 4 I

ligplot /data/pdb/p6tmn.pdb -nCBZ 1 -nLEU 4 I

ligplot /data/pdb/p6tmn.pdb "-nCBZ" 1 "-nLEU" 4 I

Alternatively, if the ligand range is unknown, you can omit the optional parameters and enter:

ligplot /data/pdb/p6tmn.pdb

In this case, the program will search through the PDB file 6tmn and will produce a list of chain and ligand ranges. It will then prompt you to enter one of these residue ranges as the ligand.

b. LIGONLY - running LIGPLOT on its own

If you do not have HBPLUS, or wish to use your own list of hydrogen bonds and hydrophobic contacts (see Appendix B), you can run just LIGPLOT on its own by typing:

ligonly filename [residue1] [residue2] [chain_id] [-w] [-h]

The parameters are as before.

The LIGONLY script is also useful where you have just run LIGPLOT and now want to generate a new plot (say having slightly modified the parameters in the ligplot.prm file - see section 5). This saves running HBPLUS again to recalculate all the hydrogen bonds and hydrophobic contacts.

c. Showing atom accessibilities computed by NACCESS

One of the options in the parameter file, ligplot.prm, allows you to shade the atoms in the plot according to their atomic accessibilities (see, for example, the plot of the Gly-Ala-Trp inhibitor of chymotrypsin). Solvent accessibilities can be calculated by running the NACCESS program.

(Note that, if you have a version of NACCESS earlier than v.2.1, it is recommended that you get the latest version from the NACCESS Web site).

The procedure is as follows: run NACCESS on your PDB file and then run LIGPLOT on the .asa file generated by NACCESS. Make sure that the option for showing accessibility shading is set to Y in the ligplot.prm parameter file.

When running NACCESS, you need to include the -f and -h options. The former defines that the "full" output format is required, and the latter states that HETATMs are to be included in the accessibility calculations.

So, your command might be:-

naccess filename -h -f

This generates a .asa file, and LIGPLOT should then be run on this .asa file in place of the original .pdb file.

That is:-

ligplot filename.asa [residue1] [residue2] ... etc

Note that, if LIGPLOT is run on an ordinary PDB file, the values in the B-value column will be taken to be accessibilities.