Contents

1. Introduction

NUCPLOT is a program that automatically generates schematic 2D representations of protein-nucleic acid interactions. The input is a standard PDB file and the output is a colour or black-and-white PostScript file which gives a simple, at-a-glance representation of the hydrogen bonds and hydrophobic contacts between proteins and nucleic acids.

2. Installation of programs

a) Decompress and untar the HBPLUS and NUCPLOT programs

• gunzip hbplus.tar.Z
  
• tar -xvf hbplus.tar
  
  
• gunzip nucplot.tar.Z
  
• tar -xvf nucplot.tar
  
  

b) Compiling the programs.

i) HBPLUS:

Change directories to hbplus/ and type the following command:

• make

ii) NUCPLOT:

Change directories to nucplot/ and type the following command:

• cc -o nucplot nucplot.c -lm
• cc -o hbadd hbadd.c -lm

-lm is an option that includes the Standard Maths library.

iii) Het Group Dictionary:

Place the Het Group Dictionary in the nucplot/ directory.

c) Assignment of aliases/logical names

Under UNIX, the following aliases need to be defined to run NUCPLOT and HBPLUS. They can be placed in the user's .cshrc file.

** nucplot-directory gives the full path to where the NUCPLOT program files are held. Please change accordingly.

After modifying the .cshrc file, type the following command to set up the aliases:

• source .cshrc

3. How to run NUCPLOT

We recommend running NUCPLOT outside the nucplot/ and hbplus/ directories.

To run NUCPLOT on a structure for the first time, simply type:

• nucplot [pdb filename]
  
  
• eg. nucplot /data/pdb/pdb1zaa.ent

This runs a script which automatically executes the HBADD, HBPLUS and NUCPLOT programs in turn to produce the plot.

For subsequent runs of NUCPLOT on the same structure (eg. after changing the parameters to alter the appearance of the plot - see below), simply type:

• nuconly [pdb filename]

This runs the NUCPLOT program only and cuts down on the processing time taken for running HBADD and HBPLUS. The *.hb2 and *.nb2 files for the structure must be placed in the same directory as the one in which you are running NUCPLOT

This script may also be used if you wish to supply your own list of hydrogen bonds and non bonded contacts.

4. NUCPLOT outputs

• nucplot.ps:
  - A PostScript file showing the schematic diagram. This may be viewed with GHOSTSCRIPT or GHOSTVIEW.
  
  
• *.bond:
  - A text file containing a list of all interactions to the nucleic acid. A new file is only created if a .bond file doesn't already exist for the structure.
  
  
• hbplus.rc:
  - The input file to HBPLUS created by HBADD.
  
  
• *.hb2, *.nb2:
  - Output files from HBPLUS containing all hydrogen bond and non bonded contact information for the structure.
  
  

5. Altering the appearance of the plot

The resulting plots can be altered by changing the plot parameters in the parameter file "nucplot.par which can be found in the current directory. When running NUCPLOT in a new directory, the default parameter file is copied from the nucplot directory. To return to the default parameters for the plot, simply remove the parameter file from the working directory and run the NUCPLOT script. The file can be edited with any text editor.

• Produce a colour PostScript file (Y/N)? - Defines whether the diagram is drawn in colour or black and white.
  
  
• Orientation of plot: (P)ortrait or (L)andscape? - Defines whether the orientation of the plot is to be portrait or landscape.
  
  
• Plot title (Y/N)? - Defines whether the PDB filename is shown on the plot as a title.
  
  
• Show key to plot (Y/N)? - Defines whether the key to the symbols on the plot are shown.
  
  
• Output filename = "nucplot.ps" (Y/N)? - Defines whether the output filename is nucplot.ps. The alternative format is pdbname_X.ps where pdbname is the name of the input file and X is the chain ID of the first DNA chain on the plot.
  
  

• Read bonding information from .bond file (Y/N)? - Defines whether the protein-DNA, water-DNA, and protein-water-DNA interaction information is read from a file in .bond format. A file listing all the interactions shown on the plot is automatically produced by NUCPLOT. This can be edited and used as an input if the you wish to display certain interactions only.
  
  
• Include protein residue H-bonds (Y/N)? - Defines whether protein-DNA hydrogen bonds should be included in the plot.
  
  
• Include non-bridging water H-bonds (Y/N)? - Defines whether non-bridging water-DNA hydrogen bonds should be included in the plot.
  
  
• Include bridging water H-bonds (Y/N)? - Defines whether protein-bridging water-DNA hydrogen bonds should be included in the plot.
  
  
• Include non-bonded contacts (Y/N)? - Defines whether van der Waals interactions should be included in the plot.
  
  
• Use one-letter amino acid codes for protein residue names (Y/N)? - Defines whether the residue names shown on the plot are represented by the single or three letter code.
  
  
• Residues only, rather than all atoms, for non-bonded contacts (Y/N)? - Defines whether the atom name is shown for the van der Waals bonds. Showing only residue names reduces crowding on the plot.
  
  

The colours of the background and various symbols of the plot can be defined. The definitions of the different colours are described below. The colour name must be one that is defined in the list of colour definitions.

The colour definitions table allows you to modify any of the default colour definitions. You canalso set up new colours of your own (up to 20 different colours are allowed).

Each entry contains three numbers, each between 0.0 and 1.0, giving the ratios of red, green and blue, respectively, making up the given colour.

Each colour has a name defined within the single quotes and may be altered. These names are referred to when assigning colours in the "Colour parameters" section (see above section).

The list allows you to include non-standard bases in the plot. New base types may be added by appending the name to the list as it appears in the PDB.

6. Editing the PostScript file

If you are familiar with PostScript files, you can make simple amendments to the plot by editing the nucplot.ps file.

The file is an ASCII text file, and so can be modified using any text editor. The sorts of amendments you can make are: changes to labels (in terms of size, colour and text), addition of other text, changes to colours, sizes, etc.

Some changes, of course, are easier by altering the nucplot.par parameter file and re-running NUCPLOT.

7. Missing bonds

There are several possible explanations:

As mentioned in the introduction, NUCPLOT uses a list of bonds supplied by the HBPLUS program by default. In this case, HBPLUS sometimes gives incorrect results as, when the program encounters a ligand, residue or base it does not recognize, it may be unable to correctly calculate all the hydrogen bonds the interacting entity makes.

HBPLUS does allow the user to define ligands in a separate input file as described in section 2.6 of the HBPLUS Operating Manual. The file describes the nature of hydrogen bonds each atom in the ligand is able to make and therefore allows HBPLUS to correctly calculate the interactions to it.

HBPLUS calculates hydrogen bonds in the following method. All possible hydrogen atom (H) positions are calculated for donor atoms (D) which satisfy specified geometrical criteria with acceptor atoms (A) in the vicinity. The criteria used are: the H-A distance is < 2.7Å, the D-A distance is < 3.35Å, the D-H-A angle is > 90° and the H-A-AA angle is > 90°, where AA is the atom attached to the acceptor.

For non-bonded contacts, all atoms within 3.9Å of each other are considered to be interacting by HBPLUS.

c) Missing water contacts

NUCPLOT only considers hydrogen bonds between water molcules and nucleic acids. Non bonded contacts are not included to prevent overcrowding in the diagram.

d) Using the *.bond file

Reference

Appendix A - PDB file format

NUCPLOT reads the atomic coordinates from standard PDB format files only.

In particular, NUCPLOT may not produce coherent outputs if the following are not observed:

• Chain ID
  - all protein and nucleic acid chains must be labelled with a chain ID.
  
  
• Protein residue and nucleic acid base names
  - protein residues must be named by their three-letter codes and bases by their one-letter codes. Some crystallographic programs generate PDB files with three-letter base names but unfortunately some of these codes are already taken up as HETATM codes (eg. GUA = glutaric acid).
  
  
• Water names
  - water should be labelled HOH.
  
  
• Atom names
  - all atoms should be named according to the standard PDB format eg. sugar atoms in the nucleic acid backbone should be named C5*, C4* etc not C5', C4'.
  
  

Appendix B - The *.bond file format

The table below shows the NUCPLOT file format for the *.bond file containing the list of hydrogen bonds, non bonded contacts and covalent bonds to be plotted. The file is automatically produced when the HBPLUS files are used as inputs.

Example file:

                             10         20         30         40  
                    12345678790123456787901234567879012345678790123
                    ===============================================
     
     Line 1:        NUCPLOT v.1.0  -  Bond file (pdb1zaa.bond)
     Line 2:        -----------------------------------------------
       |  
     Line 4:        **** Hydrogen Bonds ***************************
       |                  Donor               Acceptor     Distance
       |            ARG C   70    NH2      G A    2    O1P   2.87
       |            ARG C   80    NH2      G A    2    N7    2.86
       |            ARG C   80    NH1      G A    2    O6    2.99
     Line X:        HOH    319    O        G B    4    O6    2.60
       |  
       |  
     Line X+3:      **** Non Bonded Contacts **********************
       |                 protein                DNA        Distance
       |            THR C   56    CG2      C A    3    P     3.63
       |            THR C   56    CG2      C A    3    O1P   3.82
     Line Y:        THR C   56    CB       C A    3    O2P   3.38
       |  
       |  
     Line Y+3:      **** Covalent Bonds ***************************
       |                 protein                DNA        Distance
     

Explanation of columns:

       ------------------------------------------------------------
        Field |    Column    |   Description
          No. |     range    |              
       ------------------------------------------------------------
           1. |     1 - 3    |   Donor residue 3-letter code
           -  |     4 - 4    |   Blank
           2. |     5 - 5    |   Donor chain ID
           -  |     6 - 7    |   Blank
           3. |     8 - 10   |   Donor residue number
           -  |    11 - 13   |   Blank
           4. |    14 - 17   |   Donor residue atom name
           -  |    18 - 21   |   Blank
           5. |    22 - 24   |   Acceptor residue 3-letter code 
           -  |    25 - 25   |   Blank
           6. |    26 - 26   |   Acceptor chain ID
           -  |    27 - 28   |   Blank
           7. |    29 - 31   |   Acceptor residue number
           -  |    32 - 34   |   Blank
           8. |    35 - 38   |   Acceptor residue name
           -  |    39 - 41   |   Blank
           9. |    42 - 45   |   H-bond distance
       ------------------------------------------------------------

Appendix C - The HBADD program

a) Pre-processor for HBPLUS

b) The algorithm

c) Problems