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 should select your Match module which should be one of your 3D spectrum displays and will be the display in which possible matches are shown. Then select your Search module a different 3D spectrum display which will display the peaks on which your search is based. By default, the program will look for CA and CB matches, but you can also add C (carbonyl) matching or remove the CB matching depending on the spectra you are using. A good use of space is to place the Backbone Assignment module underneath your HSQC spectrum display. We also suggest that you set the aspect ratio of your two 3D Spectrum Displays to be the same (e.g by clicking on Fixed in both Spectrum Displays) or by adjusting them manually and then clicking Locked on both to keep them that way.
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). You will probably also see colour-coded lines that show which NmrAtoms are linked through peaks in the your various spectra. You don't strictly need the Sequence Graph module to be open while you link your residues, but it will provide you with some helpful information.
Begin your backbone assignment by double-clicking on an NmrResidue, e.g. @100, in the Backbone Assignment module. Your HSQC and your 3D Search 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 Spectrum Display, you will see three strips with three possible matches (note that the match needs to be in the CO-based spectrum, e.g. HNcoCA/HNcoCACB/CBCAcoNH/HNCO). 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. You increase the number of matches shown in the Settings panel of the Backbone Assignment module.
Once you have decided on the correct match, left-click on the header of that strip and drag it onto the right-hand orange arrow in the header on the Search 3D Spectrum Display. This will create a sequential link between the two NmrResidues. Several changes will then occur across the modules. The Search 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. (It can be helpful to order the NmrResidue table of an ordered NmrChain by Index which will put them in N- to C-terminal sequence order.) Finally, the Sequence Graph should also update to show you the new NmrChain and the two sequentially linked NmrResidues.
You are now ready to identify the next i+1 match and drag that strip onto the right-hand orange arrow to link the next residue. Continue in this way until you are no longer able to find a good match.
Once you have linked at least three NmrResidues in a row, the Sequence Graph module will start to make suggestions for a possible sequence-specific assignment: in the lower section, it will colour possible matching residues in orange. As you increase the length of your sequentially linked NmrChain, this will hopefully narrow down to a single possible stretch. This information may be helpful if you need to discriminate between two possible good matches; if you can see that the next residue is likely to a Serine and one good match shows the next residue to have chemical shifts consistent with this and the other doesn't, then you will know which one to choose.
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 left-hand orange arrow in your Serach 3D Spectrum Display. Note that this time the match needs to be in the HNCACB/HNCA/HNcaCO type spectrum.
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.
It is worth being aware that the Backbone Assignment and Sequence Graph modules both contain a Link to current NmrChain option in their settings. If this setting is selected in both modules, then they should each update to display the NmrChain selected by the other module. If you do not wish these modules to be linked in this way, simply deselect these settings.