Tutorial 2. Generation of a new ligand description using
In the first tutorial you were able to refine you example
structure both globally against X-ray data using Refmac and
locally against electron density using Coot's real space
refinement. This was only possible because both programs knew
about geometrical restraints associated with all covalent bonds in
the structure. This is because both amino-acid residues and sugar
residues present in that particular structure, as well as covalent
linkages between monomers are described in CCP4 dictionary.
Obviously the dictionary does not include all possible chemical
compounds and sometimes it is necessary to generate new dictionary
entries describing covalent bonds in a new ligand.
In this tutorial we demonstrate how it can be done using JLigand
included in CCP4 suite. For simplicity, we assume a somewhat
artificial situation. Suppose we want to analyse a structure
downloaded from the PDB. In addition, we are going to
perform several cycles of refinement and examine the electron
density - to make sure that the structure of a ligand binding site
is trustworthy (see Notes, §1). In this example we'll
examine a recently deposited structure with PDB code 4do4. The
structure contains a ligand with PDB code DJN, which has not yet
been added to the CCP4 monomer library. Therefore, we will need to
generate an additional library containing the template restraints
for DJN before any refinement.
(1) The directory "jligand/2_newligand" contains the
tutorial data (files 4do4.mtz and 4do4.pdb, see also Notes, §2).
Set up ccp4i project "1_newligand" with this directory as the
(2) Run Refmac5 with 4do4.pdb and 4do4.mtz. Use default output
4do4_refmac1.mtz, 4do4_refmac1.pdb will not be created
Note that the job has quickly finished and is marked as FAILED in
the ccp4i job list. Double-click on the job to open result viewer.
It will show Refmac log-file because no graphical output has been
generated. You can find the following messages in the log-file
explaining why Refmac refused to refine the structure (see Notes,
"WARNING : residue:
510 chain:Aj - is not in the library"
"Refmac_5.7.0032: New ligand has been encountered. Stopping
(3) The drawing below shows the chemistry of the ligand DJN and
The simplest way to generate the description of DJN using JLigand
is detailed below. (If you wish, you may also draw the molecular
graph from scratch following instructions in JLigand > Help
> JLigand Help.)
• Launch JLigand (ccp4i > Refinement > Restraint Preparation
> Ligands and links with JLigand)
• Import DJN form the pdb-file into JLigand
- File > Open > PDB File; select 4do4.pdb
- Select "DJN" in "Choose Ligand Id" window
- Press "Submit" and JLigand will read coordinates of DJN atoms
from the pdb-file and suggest a possible chemical graph.
• Correct bond orders if necessary
- double-click on a bond
- select required bond order from a menu in "Edit Bond Type"
• Regularise (Ligand > Regularise > DJN)
• Check chiralities of carbon atoms. They should be correct as the
ligand has been imported from a structure that was well refined
against high resolution data. However, you may try to change one
- Double-click on a chiral carbon atom
- Select "make positive" or "make negative" from a menu in "Edit
Atom Details" window (as opposed to "keep negative" or "keep
- Regularise DJN for changes to have effect
- Do not forget to restore the original chirality
• Save ligand description as an additional library
- File > Save as Monomer > DJN
- Keep default name for the library file (DJN.cif)
(5) Rerun Refmac job with additional library DJN.cif
- Select Refmac job and press "Rerun job" button in the ccp4i main
- In Refmac task interface, populate "LIB in" with DJN.cif
- Change "Do 10 cycles ..." to "Do 1 cycles ..." in the
- "Run" > "Run now"
(6) Try real space refinement in Coot without and with the
additional library loaded
- When Refmac job has finished, open result page and launch Coot
from "Output Files" section.
- Navigate to one of the two copies of DJN (Draw > Go To Atom
> select A510 DJN from pull down menu)
- Click "Real Space Refine Zone" and then click two times on any
atom of the ligand. This does not work because the main monomer
library does not contain description of DJN
- File > Import CIF-dictionary; select the project
"2_newligand" in the menu initially showing "-Current Dir-" ;
- Try real space refinement again. Now, with the additional
library loaded, Coot knows how to refine the DJN ligand.
(1) The electron density maps for most of the PDB structures are
readily available from Uppsala Electron Density Server. In
practice, you would not normally need any refinement, but only a
quick look at the electron density map to make sure that ligand
position and conformation are well defined. This can be done in
Coot (File > Fetch PDB & Map using EDS). But, as you may
expect, real space refinement would not work for DJN unless you
"Import CIF dictionary" DJN.cif.
(2) The file 4do4.pdb was downloaded from the PDB. The file
4do4-sf.cif was downloaded from the PDB and converted to 4do4.mtz
using the task "Convert to/ modify/ extend MTZ" located in ccp4i
"Reflection Data Utilities" folder.
(3) Actually, DJN happened to be a different PDB code for the
compound, which already exists in the CCP4 monomer library under
the name NOK. Refmac will figure this out and continue refinement
if you select "Check only ligands against Refmac's dictionary
description" in the line starting from "Checking against
dictionary:", in the folder "Setup Geometric Restraints".