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Backbone Assignment

Backbone Assignment

Once you have Set up your NmrResidues and finished the Pick and Assign routine, you are ready to sequentially link your residues and then sequence-specifically assign them. This is done using the Backbone Assignment routine.


Open three spectrum display modules next to one another in your drop area: one containing your HSQC and two containing your 3D assignment spectra (e.g. HNCACB/CBCACONH and/or HNCA/HNCOCA). Open the Backbone Assignment module with BB or via Assign / Backbone Assignment. Select your NmrChain in the drop-down menu (probably the @- NmrChain). Then open the settings box by left-clicking on the gear icon. Here you can select your Match module which should be one of your 3D spectrum displays. A good use of space is to place the Backbone Assignment module underneath your HSQC spectrum display.

Now open the sequence graph with SG or via View / Sequence Graph. This module contains two sections. The lower part shows the protein sequence. The upper part shows the NmrResidues for the NmrChain selected in the drop-down menu. Underneath the NmrResidues you should see amino acid type predictions (these will be more accurate if you have assigned CB as well as CA NmrAtoms). If you have specified experiment types for your spectra, then you will see colour-coded lines that show which NmrAtoms are linked through peaks in the your various spectra. You don't strictly need to set experiment types in order to do the backbone assignment, but it can be a helpful way to check for mistakes. Go to Setting Experiment Types and Backbone Assignment: Finding and correcting mistakes for more information.

Linking in the i+1 direction

Begin your backbone assignment by double-clicking on an NmrResidue, e.g. @100, in the Backbone Assignment module. Your HSQC and one of the 3D spectrum displays will now navigate to this NmrResidue position and the NmrResidue Project ID (PID) will be shown in the strip header. The @100 CA and CB chemical shifts will be marked. In your match display you will see three strips with three possible matches (note that the match needs to be in the CBCACONH/HNCOCA type spectrum). The strip headers in the match display will show that you are matching in the i+1 direction, the NmrResidues of the possible match, and also a percentage showing how good Analysis rates the match (the higher the better). First of all you need to determine which is the correct match. Usually this will be the left-hand strip, but that may not always be the case if there are several good matches. Once you have decided on the correct match, left-click on the header of that strip and drag it onto the >>> on the right-hand side of the header on the other 3D spectrum display. This will create a sequential link between the two NmrResidues. Several changes will the occur across the modules. The main 3D spectrum display will add a strip with the new NmrResidue and draw marks at its CA and CB chemical shifts. The match module will automatically update with new matches for the new NmrResidue. The Backbone Assignment module will also change: a new NmrChain will have been created and the two linked NmrResidues added to it. Note that the NmrChain name starts with a # to indicate that the chain is ordered, i.e. the residues in it are sequentially linked.

You are now ready to identify the next i+1 match and drag that strip onto the the >>> to link the next residue. Continue in this way until you are no longer able to find a good match.

Linking in the i-1 direction

This works analogous to the i+1 matching. However, you need to start the assignment by double-clicking on an NmrResidue of the type @100-1, i.e. an NmrResidue which is defined as being the predecessor of another. The match lines will now be drawn for the CAi-1 and CBi-1 chemical shifts. When you have found a match, drag the strip header on to the <<< on the left-hand side of the header on your main 3D spectrum display. Note that this time the match needs to be in the HNCACB/HNCA type spectrum.

Making sequence specific assignments

Your Sequence Graph module will continue to update as you link your residues if you set the NmrChain to the one you are currently creating/elongating. Based on the amino acid type predictions of the NmrResidues, the program will look for possible matches between the chain of linked residues and the sequence. Possible matches are shown in orange on the sequence. Once you are sure that the match is unique and correct, you can make the sequence specific assignment. Simply drag one of the NmrResidues in the Sequence Graph onto the amino acid in the sequence that you want to assign it to (the amino acid will turn blue while you hover over it). Once you release the drag, the sequence-specific assignment will have been made and all the residues in the linked NmrChain will update to their new assigned names.