LigPlot+ v.1.4

Graphical User Interface for the LIGPLOT and DIMPLOT programs

Operating Manual

Contents

LIGPLOT of Val-Trp dipeptide
in 3tmn
1.   Introduction
DIMPLOT of interface between
chains A and B in 3sdp
2.   Paths and directories
3.   Generating a LIGPLOT/DIMPLOT diagram
4.   Editing the plot
5.   Multiple plots
6.   Plot parameters
7.   Runtime parameters
8.   Adding missing H-bonds
9.   Removing unwanted H-bonds
10.   Plots from structural alignments
11.   Structural alignment file formats

1. Introduction

LigPlot+ is a graphical front-end to the LIGPLOT and DIMPLOT programs. The plots generated by these programs can be interactively edited on screen and then written to a PostScript file for printing or conversion to various image formats. Additionally, you can superpose related LIGPLOTs to highlight similarities and differences between related proteins binding the same/similar ligand, or the same/similar ligand binding to different proteins. The very first time you run LigPlot+ on your computer, you will need to define several paths and directories so that the program knows where to find things like PDB files, the Het Group Dictionary and the RasMol/PyMOL programs (if you have them). LigPlot+ will detect that it is being run for the first time and will pop up an entry-form, described in the section below, for you to fill in.

2. Paths and directories

The entry-form for defining paths and directories looks as shown below.

Edit the text fields as follows:

i. PDB paths
PDB files are most conveniently identified simply by the 4-character PDB code or identifier. If your system contains one or more locations where PDB files are stored, you can define a "template" path which will allow LigPlot+ to find the relevant PDB file from its 4-character code alone. The files can be gzipped or not.

Alternatively, if you are connected to the internet when you run LigPlot+, the required file can be retrieved by ftp from a given ftp site.

The 4 characters of the PDB code in each template are represented as abcd. These appear in the template within square brackets.

For example, the template

C:/roman/pdbsum/pdb/pdb[abcd].ent
means that, for example, PDB code 1ral would be translated as:
C:/roman/pdbsum/pdb/pdb1ral.ent
Similarly, the ftp template
ftp://ftp.ebi.ac.uk/pub/databases/rcsb/pdb-remediated/data/structures/divided/pdb/[bc]/pdb[abcd].ent.gz
would map to PDB code 1ral as:
ftp://ftp.ebi.ac.uk/pub/databases/rcsb/pdb-remediated/data/structures/divided/pdb/ra/pdb1ral.ent.gz
Note that here the PDB files are stored in subdirectories whose names correspond to the middle part of the PDB code (here shown as "[bc]").

The above ftp address retrieves gzipped PDB files from the PDBe ftp site at the EBI. A full list of possible ftp addresses is given below:

PDBe     ftp://ftp.ebi.ac.uk/pub/databases/rcsb/pdb-remediated/data/structures/divided/pdb/[bc]/pdb[abcd].ent.gz
PDBj     ftp://ftp.pdbj.org/pub/pdb/data/structures/divided/pdb/[bc]/pdb[abcd].ent.gz
RCSB PDB     ftp://ftp.wwpdb.org/pub/pdb/data/structures/divided/pdb/[bc]/pdb[abcd].ent.gz
You can add others.

ii. The Het Group Dictionary
LigPlot+ gets its information about ligand molecules from the Het Group Dictionary. This defines the atom names, connectivities and bond orders of every Het Group in the PDB. You can download a copy of this dictionary from the wwPDB at:

ftp://ftp.wwpdb.org/pub/pdb/data/monomers/components.cif

It is a good idea to download it regularly to always have an up-to-date copy.

Record the full path and filename of the Het Group Dictionary here so that the programs know where to find it.

SDF Dictionary. Alternatively, if your ligand definitions are in a .sdf file, you can provide the name of your file here. There are two important considerations:

  1. The file must have a .sdf extension.
  2. The 3-character "residue name" of the ligand, as it appears in your PDB file(s), must be given in the molecule's description. For example:
    > <PDB_HET_ID>
    PLP
The exact name of the field (in this case "PDB_HET_ID") is not important as different people will use different conventions. The program will look for potential identifiers and, if they match one of the Het Group names in the PDB file will take the corresponding molecule definition.

iii. Temporary directory
LigPlot+ needs a directory where it can puts its temporary working files (which it deletes once it has done with them). Use this parameter to define a directory to which you have write-access and which LigPlot+ can use for its working files.

iv. Name of RasMol executable
Enter the full path and name of the RasMol executable file. This will allow you to display the 3D coords of the given LIGPLOT/DIMPLOT diagrams. If you do not have RasMol, enter NONE here, although it is recommended that you download and install a copy of the program.

Linux users might find it more convenient to enter the RasMol path using the following format:

xterm -e rasmol -script
This will open up an X-term window as well as a RasMol window. The former can be used for entering RasMol commands.

v. Name of PyMOL executable
Similarly, if you have PyMOL installed, enter the full path and name of the executable here. As in the RasMol option, this will allow you to display the 3D coords of the given LIGPLOT/DIMPLOT diagrams. If you do not have PyMOL, enter NONE here.

Save. Once you have entered all the parameters, press the Save button to store your paths. Should you need to alter the paths at some later date, you can do so via the Program paths menu item in the Edit menu:

3. Generating a LIGPLOT/DIMPLOT diagram

To generate a new LIGPLOT or DIMPLOT diagram, select the PDB file of interest using File-Open-PDB file from the menu bar.
 
Enter the 4-character PDB code into the dialogue window that pops up. The program will search for the PDB file using the location templates defined in your Paths and Directories parameters described above.

Alternatively, you can browse for a PDB file in your local directory system by clicking the Browse button.

When the file has been loaded, you will see a summary of the ligands and metals in the PDB file, if any, as shown below for PDB entry 1a95.
 

Clicking on the LIGPLOT or DIMPLOT tabs allows you to choose which of the two programs to run.

Then to run either program, click on the Run button.

When the plot has been generated it will appear on screen as shown below. This shows the PCP 301 ligand in PDB entry 1a95.

Key

The meaning of the items on the plot is as follows:

You can change colours, sizes, etc as described later.

Sometimes a plot contains thin purple lines. These are either covalent bonds between protein and ligand, or "elastic" bonds within the ligand. The latter occur when the ligand is a cyclic peptide; the program has to make one of the bonds "elastic" in order to be able to flatten the ligand into 2D.

The plot can now be edited interactively, as described next.

4. Editing the plot

a. Moving residues, atoms, and text items

To move any of the objects on the plot click and drag the object using the left mouse button.

Note, that to switch between moving whole residues and single atoms click the Move residue/move atom button at the bottom-right of the frame to toggle between the two modes.

 

Note also that the hydrophobic contact groups cannot be moved in the Move atom mode.

b. Panning and zooming

Use the left mouse button to click-and-drag on any blank area of the plot to pan around the plot.

Use the right mouse button to click-and-drag on any blank area of the plot to zoom in and out of the plot.

Note for mac users. If your mouse only has a single button, use the SHIFT key with your button-click to get the same functionality as a right button click.

c. Recentre

You can recentre the plot at any time by clicking on the Recentre button at the bottom left of the LigPlot+ window.

d. Rotating residues

You can rotate a residue about any of its atoms by right-clicking on the atom you want to pivot about. A marker will appear over the selected atom to identify it.
 

To rotate about this atom, click-and-drag any other atom in the residue. Release the button when you have reached the desired position.

To deselect the atom, right- or left-click on any blank part of the plot, or on any other residue.

e. Flipping about a bond

You can flip a residue, or part or a residue, about any of its bonds.

Firstly, select the bond by clicking on it with the right mouse button. A marker will appear over the selected bond.
 

To flip about this bond, click with the left mouse-button on any atom on the side of the residue to be flipped. A single click will perform the flip. Clicking a second time on any of the flipped atoms will reverse the flip.

To "unselect" the flip-bond, click with the right or middle mouse button on any blank area of the plot. The marker will disappear.

f. Editing text labels

You can edit any of the text labels on the plot.

Select the text item by right-clicking on it. A box containing the text string will appear. You can edit the text as required. Click the OK button to accept the new version, or the Cancel button to retain the previous text label.

5. Multiple plots

You can generate several related plots which the program will endeavour to overlay as best it can. For example, you might wish to plot several structures of the same protein with a different ligand bound. Or similar proteins with the same or similar ligands bound. Alternatively, it might be useful to see the interactions the same ligand makes when binding to completely different progteins.

After the plots have been generated and overlaid, you can edit them by moving their components independently and switching between them. The currently active plot is shown in full colour in the foreground, while all background plots are greyed out. Before printing you can "switch off" the display of the background plots such that only the foreground plot appears. This allows you to produce a set of clean plots with equivalent components in equivalent positions on them. Alternatively, if you use the "Write PostScript" option, you can have each plot printed on a separate page.

The first plot is generated in the usual way. Each subsequent plot is fitted to the first by a sequence-based comparison of the two binding sites. This identifies equivalent residues in the 3D structures and these equivalences drive the generation of the second LIGPLOT. If the program is unable to match the binding sites based on their sequences, it tries to fit the ligands using a graph matching procedure. For very distantly related proteins it is better to use a structural alignment. Section 7 describes how to import such an alignment.

a. Loading additional plots

In the simplest case, additional plots are loaded/generated in the same way as the initial plot was. Use either File-Add or File-Open-PDB file to fit a new LIGPLOT on top of the current one on screen.

The new plot, when generated, will become the foreground plot; any current plot(s) will go into the background.

 

The left-hand example above shows a plot of the interactions between guanine 600 in PDB entry 2pwu and the protein residues. The right-hand plot shows superposed on it a plot of a similar molecule (9DG) bound to PDB entry 1q2r. The red circles and ellipses identify the residue on the latter plot that are equivalent to the underlying residues from the first plot.

LigPlot+ attempts, as best it can, to place residues in the new plot on top of the equivalent residues in the old one. It does this by first performing a simple sequence alignment between the set of interacting residues from each structure. The equivalenced residues in this alignment are then used to superpose the 3D coordinates from both PDB files. This may throw up more pairs of equivalenced residues, being those that significantly overlap in the superposition. The residue equivalences are then used to "drive" the generation of the new LIGPLOT diagram; the 2D locations from the first plot restrain the positions of the corresponding residues in the second.

The superposition may not always be successful if either the two proteins are quite different, or the second ligand is very large relative to the first.

The "Split screen" button at the bottom of the frame separates the two (or more) plots, and arranges them in the window.

Two plots, as in the above example, are shown side by side:

The "Merge back" button will restore the plots to overlap mode.

You can change how the equivalent residues are shown on the plot by using the On/Off parameters to switch off the red circles and, instead, ticking the "Highlight equivalent side chains" option. The resultant plot will look like:

Here, the equivalent residues have a red underlay beneath their bonds and atoms. Equivalent residues engaged in hydrophobic interactions are shown in thicker lines.

Inverting the highlights

In some cases, it might be useful to highlight the residues in the proteins that are different, rather those that are equivalent. In this case, one of the On/Off parameters allows you to select the non-equivalenced residue for highlighting.

b. Moving any of the split plots relative to others

When the plots are in split-screen mode, as above, you can move one plot relative to the other(s) by selecting the "Move plot" option from the button at the bottom right of the screen:

You can then move any of the plots on screen by clicking and dragging any of their components (ie atoms, bonds, text items, etc). Remember to revert to "Move residue" mode when you're done to return to normal editing. When you merge the plots back, the "Move plot" option is lost.

c. Selecting the active plot

When the plots are merged, you can bring any of the background plots to the foreground either by clicking on their greyed-out label in the top right-hand corner, or by clicking on any of their components in the plot itself (ie atoms, bonds, text items, etc).

d. Viewing structures in RasMol or PyMOL

If you have either RasMol or PyMOL installed, and have defined the path to the program(s) in Paths and Directories above (Section 2), then there will be a RasMol and/or PyMOL button at the bottom of the frame.

Clicking on one of these buttons will pop up a RasMol or PyMOL window containing the superposed ligands, in 3D, with H-bonds added in cyan and atoms involved in non-bonded contacts represented by dot surfaces (in RasMol) or transparent surfaces (in PyMOL).

e. Switching off the background plots

For printing, you might wish to switch off the background plots. You can do this either by choosing Edit-On/off selection from the menu bar, or by clicking on the On/off button at the bottom of the frame.

     

Then, deselect the Show inactive plots option, circled in red below.

6. Plot parameters

You can adjust a number of parameters that define the appearance of the plot. These can be accessed either by using the Edit option in the menu bar at the top of the LigPlot+ window, or clicking one of the buttons at the bottom left. These are shown in the table below.

           
     

The colour options differ slightly depending on whether your plot is a LIGPLOT or a DIMPLOT. For the former, bonds and atoms are classified as ligand or non-ligand. For the latter, the two classes are: interface 1 and interface 2.

If you have two or more plots overlaid, there is an extra checkbox (see example on the left below) which allows you to apply the new parameters to the currently selected plot only, or to all plots. Thus you can, say, have the bonds or labels shown in a different colour in each plot. The background parameters always apply to all plots.

a. On/off selection

 
The on/off selection dialogue allows you to switch various components of the plot on or off. If the box is checked, that item is switched on. The left-hand panel above shows the LIGPLOT options while the right-hand panel contains the DIMPLOT options.

The items are grouped into: Labels, Atoms, Bonds and Miscellaneous.

b. Sizes selection

 
This dialogue allows you to alter the sizes of various objects on the plot. The sizes are given in "Ångströms" so as to relate to the scale of the coordinates and bond lengths in the original molecules.

The items are grouped into: Label sizes, Atom and residue sizes and Bond widths.

The left-hand panel above shows the LIGPLOT options while the right-hand panel contains the DIMPLOT options.

c. Colour selection

 
This dialogue controls the colours of the objects on the plot. The tabs give the colour selections for different item types:

Background, Labels, Atoms and Bonds.

The left-hand panel above shows the LIGPLOT options while the right-hand panel contains the DIMPLOT options.

Note that, for ligand and non-ligand bonds there is a special extra colour labelled "ATOM". If this "colour" is selected, the bonds will be coloured such that each half is of the colour of the atom bonded at that end.

7. Runtime parameters

The runtime parameters allow you to alter some of the parameters that are used in generating the LigPlot+ diagrams. The parameters are accessed from the Edit option in the menu bar at the top of the LigPlot+ window.

     

The four numeric parameters relate to the HBPLUS program used by LigPlot+ to compute all the potential hydrogen bonds and non-bonded contacts. The first two values correspond to the maximum hydrogen-acceptor and donor-acceptor distances for defining what is a hydrogen bond. By increasing these values you can "bring in" additional interactions which may be just outside the program's criteria for an H-bond.

The next two numeric parameters correspond to the range of distances defining non-bonded contacts (ie contacts between atoms that are neither covalently bonded, nor interacting via hydrogen bonds). The default range is 2.9-3.9 Å

Below the numeric parameters are two groups of radio buttons. The first set allows you to specify which atom-atom contacts are to be included as non-bonded contacts: a) between hydrophobic atoms only (ie C or S), b) between a hydrophobic atom and any other, or c) between any type of atom.

The second group of radio buttons relates to the treatment of any CONECT records that may be in your PDB file. The CONECT records define which atoms are covalently bonded to one another. They are generally used for defining the connectivity of the ligand. Sometimes, these records are incorrect and give unfeasibly long bonds.

You can choose how the program should treat the CONECT records. The default is to use them if they look sensible - that is, if they don't give ridiculously long bond lengths. The second option is to ignore these records altogether; the program will compute the ligand's covalent bonds itself, using set distance cut-offs. The third option is to accept all CONECT records, irrespective of the bond lengths they give.

8. Adding missing H-bonds

The H-bonds and non-bonded contacts shown by LIGPLOT are calculated by HBPLUS. Occasionally, HBPLUS can miss an H-bond, particularly when it encounters a ligand it does not recognize.

If you need your plot to have an H-bond that HBPLUS has missed, you can add it as follows:

  1. Edit your PDB file and add the missing H-bonds, or non-bonded contacts, as "HHB" and "NNB" records, respectively. These records should come before the ATOM records in the file.

    The format of each "HHB" or "NNB" record should be formatted exactly as shown below, where the dots represent blank spaces.

    Key   <----Atom 1 --->     <----Atom 2 --->    Dist
    HHB...RRR.C.NNNNI.AAAA.....RRR.C.NNNNI.AAAA....DDDD
    
    where   HHB   defines the record type: HHB for H-bonds, NNB for non-bonded contacts
      RRR   is the 3-character residue name
      C   is chain identifier (which may be blank)
      NNNN   is the right-justified residue number (1-9999)
      I   is the insertion code, or blank if none
      AAAA   is the 4-character atom name in standard PDB format
      DDDD   is the distance between the two atoms (to 2 places of decimals)

    Some example lines are given below:

    HHB...RRR.C.NNNNI.AAAA.....RRR.C.NNNNI.AAAA....DDDD
    HHB   HIS A  231   N       ASP A  226   OD2    2.77
    HHB   HIS A  231   ND1     ASP A  226   OD1    2.76
    HHB   HIS A  231   NE2     THR A 1317   O      2.80
    HHB   THR A 1317   N       ALA A  113   O      2.72
    HHB   ALA A  113   N       PHQ A  317   O2     3.31
    
  2. After editing any required H-bonds or non-bonded contacts into your PDB file, rerun LigPlot+ on this new PDB file.

9. Removing unwanted H-bonds

To remove any unwanted H-bonds or non-bonded contacts from your plot, follow the steps above, but annotating the unwanted bonds by "-HB" and "-NB" records in your PDB file, respectively. The value you put in the "Dist" is not important and can be "0.00".

So, for example, to remove the H-bond between the NH2 of Arg286(A) and the O7 of your ligand, LIG1(B), you would add the following line to your PDB file:

-HB...RRR.C.NNNNI.AAAA.....RRR.C.NNNNI.AAAA....DDDD
-HB   ARG A  286   NH2     LIG B    1   O7     0.00

10. Plots from structural alignments

For distantly related proteins it may be difficult for LigPlot+ to reliably identify equivalent residues in the binding sites. Thus an overlaid plot of two or more structures might not give a good alignment.

One way to help the program is to supply it with a structural alignment between the proteins, letting it know which residues are equivalent in 3D.

At present, LigPlot+ only accepts structural alignments in the following formats:

The formats are described later.

Import the alignment

Import using File-Import from the menu bar.
Locate the appropriate .cora, .caf or .fasta file and Open it.

A pop-up window will show you the list of PDB codes in the file and allow you to select which one you want to plot first.

After the first plot has been generated, you can add others on top of it via either File-Add or File-Open-PDB file.

11. Structural alignment file formats

The following are the structural alignment file formats currently accepted by LigPlot+:

1. CORA format (.cora or .aln)

The format must be CORA v.1.1, which is as follows:

#FM CORA_FORMAT 1.1
3
6insE0 1igl00 1bqt00
73
   1    0    1    0  0  0    1  A  0    0  0  0   0    0    0   0
   2    0    1    0  0  0    2  Y  0    0  0  0   0    0    0   0
   3    0    2    1B F  0    3  R  0    0  0  0   0    0    0   0
   4    0    3    2B V  H    4  P  0    1  G  0   0    1    0   2
   5    1    3    3B N  H    5  S  0    2  P  0   0    1    0   6
   6    0    3    4B Q  H    6  E  0    3  E  0   0    1    0   2
   7    2    3    5B H  H    7  T  0    4  T  0   0    1    0   5
---------+---------+---------+---------+---------+---------+---------+
1234567890123456789012345678901234567890123456789012345678901234567890
         1         2         3         4         5         6         7

2. CAF format (.caf)

This is one of the formats output by the CATHEDRAL structural alignment program.

HH CATHEDRAL Alignment 2.02
CC Date:    Tue Feb 22 21:07:49 2011
CC Author:  cathedral
CC Protein1 Protein2 Len1 Len2 Score  Align %Ov  %Seq RMSD
RR   1byq    2wi7  213  209  93.05  208   97   99   1.38
RR   1byq    3d36  213  121  72.64  115   53   17   9.95
RR   2wi7    3d36  209  121  72.58  114   54   16   9.68

                 1         11        21        31        41        51        
pdb|3d36         --------------------VDIQATLAPFSVIGEREKFRQCLLNVMKNAIEAMPN----
pdb|3d36                              SSSSS         HHHHHHHHHHHHHHHHH        
pdb|1byq         PMEEEEVETFAFQAEIAQLMSLIINTFYS-------NK-EIFLRELISNSSDALDKIRYE
pdb|1byq               SSSSS  HHHHHHHHHHHH               HHHHHHHHHHHHHHHHHHHH
pdb|2wi7         -----EVETFAFQAEIAQLMSLIINTFYS-------NK-EIFLRELISNSSDALDKIRYE
pdb|2wi7                SSSS  HHHHHHHHHHHH               HHHHHHHHHHHHHHHHHHHH
                      ...............:::::::::       :: :::::::::*:::*:::....

---------+---------+---------+---------+---------+---------+---------+
1234567890123456789012345678901234567890123456789012345678901234567890
         1         2         3         4         5         6         7

3. Multiple FASTA format (.fasta)

The standard FASTA multiple alignment format:

>pdb|3d36
--------------------VDIQATLAPFSVIGEREKFRQCLLNVMKNAIEAMPN----
------------GGTLQVYVSI---DNGRVLIRIADTGVGMTKEQLERLGEPYFTTKGVK
G---------------TGLGMMVVYRIIES-MNGTIRIESEIH-----------------
----------------KGTTVSIYLPLAS-------------------------------
-------------
>pdb|1z5a
----------KEKFTSLSPAEFFKRNPELAGFPNPARALYQTVRELIENSLDATDVHGI-
------------LPNIKITIDLIDDARQIYKVNVVDNGIGIPPQEVPNAFGRVLYSSKYV
NRQTR---------GMYGLGVKAAVLYSQMHQDKPIEIETSPVNSKRIYTFKLKIDINKN
EPIIVERGSVENTRGFHGTSVAISI--PGDWPKAKSRIYEYIKRTYIITPYAEFIFKDPE
GNVTYYPRLTNKI
>pdb|1byq
PMEEEEVETFAFQAEIAQLMSLIINTFYS-------NK-EIFLRELISNSSDALDKIRYE
TLTDPSKLDSGKELHINLIPNKQD-----RTLTIVDTGIGMTKADLINNLGTIAKSGTKA
FMEALQAGADISMIGQFGVGFYSAYLVA-----EKVTVITKHNDD-EQYAWESSAG----
--GSFTVRTDTGEPMGRGTKVILHLKEDQTEYLEERRIKEIVKKHSQFI-GYPITLFVE-
-------------
The key thing here is the format of the protein name line which must take one of the following forms:
>pdb|1z5a
>pdb|1z5aA
>pdb|1z5aA01
where "A" is the chain identifier and "01" is the domain number.