Tutorial 1. Handling protein glycosylation using Coot and Refmac

1. Introduction

In Refmac, recognition of sugar residues and covalent linkages that they make to protein (e.g. ASN) and to each other is done automatically. However some manual work is involved at the stage of model building. This is not always a trivial task and the simple example selected for t is tutorial does not reflect all possible complications. The example structure has only one protein molecule in the AU, the protein is small and has only one glycosylation site, and all the sugar residues are present in a single conformation. High resolution data provide good elec ron density maps and make it easy to build the polysaccharide from single sugar residues.

2. Procedure

(1) The directory "jligand/1_glycan" contains the  tutorial data (files 4gos.mtz, model.pdb). Set up ccp4i project "1_glycan" with this directory as the project directory.

(2) Run Refmac5 with model.pdb and 4gos.mtz to generate map coefficients. Use default output file names.
        Input files: 4gos.mtz, model.pdb
        Output files: 4gos_refmac1.mtz, model_refmac1.pdb
Double click on the Refmac job in the ccp4i job list to open the job result page. Check refinement statistics. Open output files in Coot (Co t button in the section Output files)

(3) With output model and density from step (2) opened in Coot, add NAG residue into the difference map near ASN A112. Save the new model as model_refmac1_coot1.pdb in the project directory of the project "1_glycan".
        Input files: 4gos_refmac1.mtz, model_refmac1.pdb
        Output file: model_refmac1_coot1.pdb

(4) Refine model_refmac1_coot1.pdb against 4gos.mtz. Use default output file names.
        Input files: 4gos.mtz, model_refmac1_coot1.pdb
        Output files: 4gos_refmac2.mtz, model_refmac2.pdb

(5) The complete polysaccharide in this example contains three types of sugar residues: NAG, BMA, MAN. Reiterate steps (3,4) to build the whole polysaccharide inferring the type of the next added sugar residue from the features of the electron density maps.

  NAG (N-acetyl-D-glucosamine)                BMA (beta-D-mannose)                     MAN (alpha-D-mannose)

3. Controls

• If Refmac recognises the new link, it automatically adds a corresponding LINKR record to the output PDB-file.

• The following line in the Refmac log-file means that geometrical restraints associated with the covalent link are applied:
WARNING: New link was found

• No chiral volume outliers in the Refmac log-file mean that the type of link that has been applied is correct

• No strong "red" or "green" density on or near the added residue mean that the type of residue was determined correctly.

4. Notes

• Step (3), the incorporation of the first sugar monomer, is detailed in the next section.

• Step (3) includes manual fitting of the first sugar residue into its density.
Coot has an option of semi-automatic ligand fitting (Coot > Calculate > Other Modelling Tools > Find Ligands) and you can try it as well. Note, however, that the automatic procedure does not always work, especially when the electron density corresponds to a chain of several sugar monomers and you are looking for only one of them. Therefore we describe manual procedure which is guarantied to work (in our example) and which is worthy exercising in any case.

• To select the default output pdb-filename in CCP4i, place the cursor on the input pdb-filename and press Tab. Similarly select the default output mtz-filename.

• Good bookkeeping is quite important for multi-step procedures such as modelling polysaccharides. For this reason we pay attention to file names in this tutorial. For the same reason we do not run refinement directly from coot, but rather perform it via ccp4i interface, where bookkeeping is a bit better organised and previous work is easily traceable next time you open the interface. However, you may try and complete the whole exercise in a single session of Coot using Coot's own interface for Refmac.

• In the example used in this tutorial, the whole polysaccharide can be built in one go. So if you prefer, you can try and add all missing sugar monomers during a single coot session and then refine the complete structure at step (5). However, in more complicated cases the step-by-step procedure is advantageous as it helps avoid rebuilding the whole polysaccharide if something has gone wrong with one of the monomers (incorrect monomer type has been chosen, or incorrect link has been applied by Refmac because of inaccuracy in the initial fitting).

• At lower resolution and with longer sugar chains the chances are high that the sugar monomers remote from protein would have multiple conformations, but the density would not be good enough to model them correctly. In this case, modelling the density with water molecules (pseudo-waters) is more appropriate. Do not over-interpret the density!

5. Details of step (1), adding NAG linked to ASN

After step (2), the model with missing glycan moiety (model_refmac1.pdb) and corresponding 2–1 and 1–1 maps (4gos_refmac1.mtz) are opened in Coot from Refmac result page. Here is a detailed description on building the first sugar into the density.

• Select "Icons and text" in the toolbar at the right margin of the main Coot window.

• Go to residue A 112 ASN (coot > Draw > Go To Atom ... and select residue from the list). Note the green density, in which we will build the polysaccharide.

• Coot > File > Get monomer...; Enter NAG in the text box, press OK

• For easier positioning of the monomer, delete H-atoms from NAG: press "Delete" button in the toolbar, check "Hydrogens in Residue" checkbox in the "Delete window" and click on any atom of NAG molecule in the Coot main window.

• Open "Rotate translate" from the toolbar, click any atom of NAG molecule and position NAG so that it fits into the corresponding density reasonably well. In the correct orientation the O1 atom of NAG should be the closest to the ND2 of ASN A112.

• าReal space refine zoneำ; move individual atoms to their correct position using left mouse button with while keeping Ctrl button pressed

• Make sure that the O1 atom of NAG is in the correct position (very close to ND2 of ASN A112). Delete this atom, as it is not present in the bound NAG.

• Merge NAG into the original model as a residue A 201. This involves the following four steps.
– Coot main window > Edit > Merge Molecules*; check the box next to "NAG_from_dict" and press Merge button.
– Figure out what is the chain Id and residue number of NAG (in this case this should be B 1 for the first added monomer; chain ID will be incremented with each added monomer if you will try and add several monomers in a single Coot session) Then go to Coot main window > Edit > Renumber Residues* and select Chain ID B, residue numbers 1 to 1, Offset 200. Press Renumber button.
– Coot main window > Edit > Change Chain IDs*; select chain Id of the new NAG residue (B in our example), check "Use residue range" checkbox and define the residue range according to the residue number of NAG (from 201 to 201 in our example). Type in "A" in the text box "Chain ID". Press "Apply".
– Mid-mouse click on any atom in NAG to check that the procedure worked (the merged NAG residue should become NAG A201)

* In old version of Coot, this menu item is found in Coot main window > Calculate

• Perform the last check before saving the model and exiting Coot.– Open "Display Manager" window. There are two molecules, "model_refmac1.pdb" and "NAG_from_dict". The latter had been initially loaded from dictionary and fitted into the density. "Merge molecules" action has added a copy of this molecule to "model_refmac1.pdb".
– Using "Display" checkbox, show and hide these molecules in turn to make sure (a) that copy of "NAG_from_dict" is indeed present in "model_refmac1.pdb" as residue NAG A201 and (b) that the copy is at the same position as the original.

• Save the molecule "model_refmac1.pdb". Set the filename for output coordinates to "model_refmac1_coot1.pdb". This is a good filename for further bookkeeping with ccp4i.
– Note "CCP4i Project Directory" menu in the window "Select Filename for Saved Coordinates". Select the project 4GOS and the output pdb-file will be saved in the project directory.

• Exit coot.