JLigand tutorial (link)

Biological macromolecules are polymers and, therefore, the stereochemical restraints for macromolecular refinement can be subdivided into two sets, restraints that are applied to the atoms that are all from the same monomer and restraints associated with the covalent bonds between monomers. Refmac dictionary contains three types of data blocks defining restraints: descriptions of monomers, their modifications (both for intra-monomer restraints) and links (inter-monomer restraints). Generic links for proteins, DNA, RNA and for some sugars are there in the standard Refmac library distributed with CCP4. Non-standard links can be defined in an additional user's library. Any non-standard link must also be explicitly specified in the LINK record of the PDB-file used as input for Refmac.

In general, if the restraints for compounds X and Y are already defined, there are two ways of defining restraints for the compound X-Y. Existing definitions for X and Y can be ignored and all restraints for X-Y can be put into a single description of monomer. Alternatively, necessary modifications to intra-monomer restraints for X and Y and inter-monomer restraints can be described in three additional data blocks. The second way is preferable for structural biology as it makes it possible to follow name conventions for monomers and atoms.

This tutorial uses a 1.6 Å resolution P212121 crystal structure of pig cytosolic aspartate aminotransferase in complex with 2-methylaspartate (Rhee et al., 1997; PDB code 1ajs). The asymmetric unit contains a dimer formed by subunits A and B. The coenzyme pyridoxal-5'-phosphate is present as the external aldimine in subunit A, where it forms an Schiff base with the substrate analogue 2-methylaspartate. At the same time, the coenzyme in subunit B forms the internal aldimine with the side chain of Lys 258. The two aldimines will be defined as a monomer and as a link, respectively.

Useful schemes can be found in Okamoto et al., (1994): Scheme 1 presents formula and conventional atom names for 2-methylaspartate and Scheme 2 shows transaldimination from the internal to the external aldimine. Some other relevant schemes can be found in e.g. Vacca et al. (1997) and Jeffery et al. (2000). The last two are free access articles.

Before starting

DATA for this tutorial are in the directory JLigand_link_tutorial and include three files described below.

The coordinate file model.pdb and the X-ray data file data.mtz were generated from the PDB entry 1ajs. The coordinate file was modified as follows,

Our goal is to produce the complete structure including ligands and refine it. This will include the following steps:

JLigand, Coot and REFMAC will be used for generation of library-file, docking and refinement, respectively. The library entries for the ligands will be created from scratch and compounds LLP and PLA defined in the standard library will be ignored.

Making description of a link

Actions (indented text) are performed in JLigand window except when the window is explicitly specified (underlined text).

Terminal: cd JLigand_link_tutorial

Terminal: jligand

Load Ligand: type in LYS, press enter and wait a bit

Help > JLigand Mouse Help or

Help > JLigand Keypad Help

Tick Atom Ids check box (the second horizontal panel from the top)

Note: Only the first letter of atom label is clickable; it is underlined when cursor is moved to it.

Press right mouse button and drag

Press Del in the left panel and click on the first letter of the atom label

Tick Hydrogens check box

Select Link in the left panel, click on N of NZ label and then on C of C4A label

Note: linked monomers are not editable except for the link's bond order; undo link to edit.

Double-click on the bond

Edit Bond Type: Bond Type: select double > Submit

Ligand > Regularise > LYS-PLP

File > Save as Link > LYS-PLP

Save as CIF-library: File: type in refmac.lib and press Save

Info: OK

Save Link Record: Yes

Save PDB Link Record: Save

Info: OK

NOTE: We will not need coordinates of the whole compound for docking because the coordinates of its constituents will be available from the standard library.

Merging two ligands into a single ligand

Ligand > Delete > LYS-PLP

Delete Ligand: Yes

Select C in the left-margin panel, click on CA to make it active (the active atom is underlined), press shift and click on canvas to place the new atom.

Double click on C of the CB label

Edit Atom Details: Atom Id: type in CB1 and press Submit

Double click on the ASP-PLP label under the picture of the ligand (you may need to move up the ligand to see the label)

Edit Ligand Id: Ligand Id: type in MSP and press Submit

File > Append as Monomer > MSP

Append to CIF-library: Select refmac.lib and press Append

Info: OK

View > View File ...

View File: select refmac.lib and press View

NB. The ligand MSP is defined in a single data block, data_comp_MSP. The previously created restraints for LYS-PLP are defined in three data blocks. Two data block, data_mod_LYSmod1 and data_mod_PLPmod1, define modifications to be applied to monomers LYS and PLP from the standard library, and one data block, data_link_LYS-PLP, defines extra restraints associated with the covalent bond between modified LYS and PLP.

Currently Coot reads coordinates of a ligand from a PDB-file, not from a CIF-library file. Therefore we need to read the monomer MSP and write its coordinates in a separate PDB-file.

File > Open > CIF File

Import Ligand from CIF-library: select refmac.lib, press Open and wait a few seconds

File > Save Coordinates > MSP

Save Coordinates: select Exclude Hydrogens and press Submit

Save Coordinates in PDB-file: keep the default selection File: MSP.pdb and press Save Coordinates

Info: OK

JLigand > Quit

Select an Option: Yes

Calculation of phases

At this point we need to generation an MTZ-file with phases. This file will be used to draw the electron density maps in Coot.

refmac5 xyzin model.pdb hklin data.mtz xyzout refmac1.pdb hklout refmac1.mtz

make hydr no

ncyc 0


Docking ligands into the electron density

Terminal: coot

Coot: File > Open Coordinates...

Select Coordinates File: select refmac1.pdb; OK

Coot: File > Auto Open Mtz...

Select Dataset File: select refmac1.mtz; OK

Coot: File > Import CIF dictionary

Select File: select refmac.lib; OK

Coot: File > Open Coordinates

Select Coordinates File: select MSP.pdb; OK

Coot: Calculate > Other Modelling Tools

Other Modelling tools: Find Ligands; Close

Find Ligands: Enlarge this window; in Select Ligands to Search for: tick .../MSP.pdb and Flexible? and then press Find'em! button

New Ligands: OK

Fitted Ligands: press Fitted Ligand #0 and then OK

Coot: Calculate > Model/Fit/Refine

Model/Fit/Refine: Select Map

Select Map for Fitting: OK

Model/Fit/Refine: Real Space Refine Zone

Coot: Click twice on the ligand.

Coot: Keep left mouse button pressed and drag atoms into their density.

Coot: To handle an individual atom, move cursor to it, keep ctrl-key and left mouse button pressed and drag the atom into its density.

Accept refinement: Accept

Model/Fit/Refine: Close

Coot: Calculate > Merge Molecules...

Merge Molecules: Enlarge this window; in Append/Insert Molecule(s): tick Fitted ligand # 0, and then press Merge

Coot: Display Manager

Display Manager: Enlarge this window to see all the molecules and Delete Molecule buttons.

Display Manager: Unselect Display check box for Fitted ligand # 0. If the copy of fitted ligand is there in the graphics window then previous steps have been done correctly and you can proceed and delete used objects.

Display Manager: In section Molecules: press Delete Molecule next to MSP.pdb, Fitted ligand # 0 and Fitted ligand # 1. In section Maps: press Delete Molecule next to Masked (by protein).

Display Manager: Close

Coot: File > Get Monomer...

Get monomer molecule from libcheck: 3 Letter Code: enter PLP and press OK

Coot: find O4A and centre on it; you may need to zoom in, as this atom can be very close to NZ of LYS

Model/Fit/Refine: Delete

Delete item: tick Atom

Coot: click on O4A

Coot: File > Save Coordinates...

Save Coordinates Molecule Selector: keep default selection .../refmac1.pdb and press Select Filename

Select Filename for Saved Coordinates: keep default selection refmac1-coot-0.pdb and press OK

Coot: File > Exit

Structure refinement with ligand and link

If you have followed instructions precisely, refmac1-coot-0.pdb will contain PLP as residue No 1 in chain D ("PLP D 1"). Check if this is correct using a text editor or coot.


Edit chain IDs and residue numbers in linkrecord.pdb. These should be: "LYS B 258" and "PLP D   1". Keep format of the record as it was, i.e. four characters for the residue number etc.

refmac5 xyzin refmac1-coot-0.pdb hklin data.mtz libin refmac.lib xyzout refmac2.pdb hklout refmac2.mtz

make hydr no

ncyc 5


NB. If you want Coot to display link, substitute " " for "R" in the LINKR record in refmac2.pdb. If this does not work, replace the LINKR record with the LINK record from linkrecord.pdb.