1. Introduction

• LIGPLOT - automatically generates schematic diagrams of protein-ligand interactions for a given ligand in a PDB file.
• DIMPLOT - plots interactions across a selected protein-protein or domain-domain interface.

2. Paths and directories

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

C:/roman/pdbsum/pdb/pdb1ral.ent

ftp://ftp.ebi.ac.uk/pub/databases/rcsb/pdb-remediated/data/structures/divided/pdb/[bc]/pdb[abcd].ent.gz

ftp://ftp.ebi.ac.uk/pub/databases/rcsb/pdb-remediated/data/structures/divided/pdb/ra/pdb1ral.ent.gz

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

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

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

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

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.

• LIGPLOT
  1. Select which ligand you wish to plot from the list.

     Alternatively, you can enter your own residue range in the box. The residue range can be one of the following:

     Example		Description
     18 20 A		Residue range: start-residue-no. end-residue-no. chain-id If the chain-id is blank it should be omitted
     NAG 18 MAN 20 A		Residue names and numbers, plus chain id.
     -n818 108 -n818 108 A		Where the residue name is numeric, prefix it with "-n" to indicate that it is the residue name rather than the residue number.
     "MTE" 1 "  G" 2 B		Where a residue name includes leading blanks (eg as in the old definition of the nucleic acid bases, A, C, G and T), enclose it in quotes and include the spaces.
     "-nMTE" 1 "-n  G" 2 B		An alternative for the case above.

  2. To include waters on the plot, tick the checkbox next to the "Include waters" option.

  3. Tick the "Filter waters" checkbox to remove waters that don't make at least 2 H-bonds to ligand or form a bridge bewteen ligand and protein. This removes waters that interact only with another water, or with just the protein.

• DIMPLOT
  1. Select which protein chain pair you would like plotted, or enter the residue ranges of the chains or domains you want to plot. Enter each residue range as: N1-N2 C, where N1 is the start residue, N2 is the end residue, and C is the chain id. If a domain is split, you can define it using several residue ranges, separating each range with an ampersand. For example

     1-136 A & 338-371 A

     A domain can consist of residue ranges from more than one chain.
  2. To include waters on the plot, tick the checkbox next to the "Include waters" option.
  3. If you want the sequence order of the first chain/domain to be retained in the final plot, click the "Retain sequence order of first chain/domain" checkbox.

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

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 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

d. Rotating residues

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

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

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

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

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

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

d. Viewing structures in RasMol or PyMOL

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

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

6. Plot parameters

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

b. Sizes selection

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

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 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

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

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

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:

• CORA format (ie from the CATH team's CORA program)
• CAF format (also from the CATH team's CORA program)
• Multiple FASTA format

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

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

• Header information:
  • Format and version: '#FM CORA_FORMAT 1.1'
  • Total number of proteins in the alignment
  • PDB codes identifying the proteins in the alignment with, optionally, chain identifier and domain number - eg 3d36A01).
  • Total number of alignment positions
• Alignment:

       START       PROT 1     PROT 2     PROT N         END
  <------------><---------><---------><---------><---------------->
  ddddxddddxddddxddddcxcxxcxddddcxcxxcxddddcxcxxcxxxcxddddxddddxxdd
  
     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
  

  where

  • START (14 characters) - not used by LigPlot⁺
    • Column 1: Alignment Position (dddd)
    • Column 2: No. of position selected for structural template (dddd)
    • Column 3: No. of proteins aligned at this position (dddd)
  • PROT 1,2,3... (11 characters per protein)
    • Column 4 (7,10 etc): Residue number in PDB file (ddddc) 4 digit number + 1 character insert code
      Importantly the insert code is always in the same position not within the 4 characters reserved for the pdb number (see below)
    • Column 5 (8,11 etc): Amino Acid Code (c)
    • Column 6 (9,12 etc): Secondary Structure Assignment (c)
  • END (18 characters) - not used by LigPlot⁺
    • Last Column-3: Consensus Secondary Structure Assignment (c)
    • Last Column-2: No. of alpha residues at this position (dddd
    • Last Column-1: No. of beta residues at this position (dddd)
    • Last Column: Structural Conservation Score (dd)

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

• Header information - not used by LigPlot⁺
  All-against-all structural similarity statistics.
• The alignment given in chunks, with 2 lines per entry: the first gives the amino acid sequence of the protein and the second gives the secondary structure (which is not used by LigPlot⁺).

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-
-------------

>pdb|1z5a
>pdb|1z5aA
>pdb|1z5aA01