Tutorial 3. Using a new ligand and link descriptions

1. Introduction

This tutorial uses a 1.6 resolution P212121 crystal structure of pig cytosolic aspartate aminotransferase in complex with 2-methylaspartat  (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.

The directory "jligand/3_newlink" contains the tutorial data (files 1ajs.mtz and model.pdb).
Set up ccp4i project "3_newlink" with this directory as the project directory.

2. Refine incomplete structure and analyse the electron density

Run Refmac
Input files: 1ajs.mtz, model.pdb
Output files: 1ajs_refmac1.mtz, model_refmac1.pdb
Launch Coot from Refmac result page.
Validate > Difference Map Peaks
Two top peaks should correspond to
PLP covalently bound to lysine
PLP covalently bound to alpha-methyl-aspartate.
The two compounds are shown below. The first one is incorporated into the polypeptide chain and we will define it as a link in the additio al library; the second is an isolated ligand and will be defined as a monomer.

3. Generate description of link LYS-PLP

ccp4i > Refinement > Restraint Preparation > Ligands and links with JLigand
In JLigand window, load LYS and PLP from "Load Ligand" text box
Check "Atom Ids" check-box
Select "Del" in the left bar and delete O4A atom in PLP
Select "Link" and connect NZ (LYS) and C4A (PLP)
Double-click on the new bond and change bond type to "double"
Ligand > Regularise > LYS-PLP
File > Save as Link > LYS-PLP
In "Save as CIF-library" window select filename refmac.cif
Read notes in "Save Link Record" window and press "Yes" button
Save "linkrecord.pdb"
Output files: refmac.cif, linkrecord.pdb

4. Generate description and coordinates of PLP bound to Methyl-ASP

Examine molecular graph editing Help > JLigand Help
Load PLP and extend the molecular graph following the figure below
Rename atoms according to the figure
Rename ligand to MSP
Regularise MSP
To avoid confusion, make sure that chirality of CA is set to  "allow both"
Double-click on CA to open "Edit atom details"
Check selection in "Chirality" menu and change it if necessary
This will make it possible to correct chirality of the atom later in Coot
File > Append as Monomer > MSP
In "Append to CIF-library" window select refmac.cif and press "Append"
File > Save Coordinates > MSP
In "Save coordinates" window select "Exclude Hydrogens"
In "Save coordinates in PDB-file" window, keep the default filename MSP.pdb
Output files: refmac.cif, MSP.pdb

5. Dock MSP into electron density

Files 1ajs_refmac1.mtz and model_refmac1.pdb are already opened in Coot
Load the description and coordinates of MSP
File > Import CIF dictionary; select project 3_newlink and file refmac.cif
File > Open Coordinates; select project 3_newlink and file MSP.pdb
Fit MSP into its density,
Calculate > Other Modelling Tools > Find Ligands
Find Ligands window, section "Select Ligands to Search for": select ".../MSP.pdb" and "Flexible?" check boxes and then press "Find Ligands" button
Fitted Ligands window: select "Fitted Ligand 0" and press OK to navigate to fitted ligand
Refine fitting
Append fitted ligand to the protein molecule
Edit (Calculate in old Coot) > Merge Molecules...; select "Fitted ligand 0"
Make sure that the ligand is appended
Open Display Manager and switch on and off the molecule model_refmac1.pdb

6. Dock PLP into electron density and save the structure with ligands

Load description and coordinates of PLP from the standard library,
Coot: File > Get Monomer...; enter 3-letter code PLP
Remove hydrogen atoms
Fit PLP into its density
Refine fitting
Delete O4A atom from PLP
Append fitted ligand to the molecule model_refmac1.pdb
Make sure that the ligand is appended
Take a note of PLP residue number and chain ID (should be "D 1")
Save molecule model_refmac1.pdb
select project 3_newlink and keep default filename model_refmac1-coot-0.pdb
Exit Coot

7. Refine structure with ligand and link

Edit the file linkrecord.pdb. This file contains one line, which is an instruction for Refmac to apply the link LYS-PLP to specified residues.
If you have followed instructions precisely, model_refmac1-coot-0.pdb will contain PLP as residue 1 in chain D ("PLP D 1"). Check if this is correct using a text editor or in 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.
Insert the LINK-record from linkrecord.pdb into model_refmac1-coot-0.pdb, just before CRYST1 record.
Run Refmac with the completed model and additional library refmac.cif.
In the line Libin specify additional library refmac.cif
Input files: 1ajs.mtz, model_refmac1-coot-0.pdb, refmac.cif
Output files: 1ajs_refmac2.mtz, model_refmac2.pdb
Note the decrease in R-free compared to the previous Refmac run
In Output Files section of Result viewer press Display to see the content of the output PDB-file. Check, if this file contains LINKR record (a few lines before CRYST1). If it does, then everything was done correctly.
Launch Coot to examine the electron density of the ligands