Tutorial 3. Using a new ligand and link descriptions

1. Introduction

This tutorial uses a 1.6 Å 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