Efficient resonance assignment
Initialise root resonances
The linking together of related peaks in different spectra by assigning them to common (anonymous) resonances is something that can be partially automated to speed up the assignment process. We can use the HSQC positions to define unique amide locations and pick and assign related spectra base upon these "root" locations. The first step in this automation is to define new amide resonance and spin system identifiers for all the peaks within the HSQC spectrum. Initially ensure you have most of the HSQC peaks picked:
Then select M: Assignment: Initialise Root Resonances. In the Assignment: Initialise Root Resonances popup, there is a table called 'Amide Sidechain Peaks' with a few rows filled. Some of the peaks which you picked in the HSQC will be from NH2 groups of amine side chains, and you need to handle those before you can initialise the peak list. Clearly the NH2 groups give two peaks, one for each hydrogen but both have the same 15N resonance (and thus 15N chemical shift). If such pairs of peaks were processed in the same manner as the backbone amide peaks they would become linked to two different pairs of resonances in two spin systems, when in reality they should carry the same 15N resonance and be in only one (side chain) spin system. Click on a row of the table to view the peaks, first making sure you set it to follow the right window (here window 1 or 3). If you think this looks like side chain NH peaks, double click the 'Confirmed' column so it changes to "Yes". When you are happy with all of them, click [Initialise Peak List!]:
This command calls the resonance and spin system assignment routines, that we used to assign the first peak, on all of the HSQC peaks. Note that the routine knows which spectrum to work with because we set the experiment type of the HSQC correctly as H[N]. You will now see that all of the peaks carry assignments of the form {x}[y],[z]. If you look at the NH2 peaks that you confirmed, you will see that both peaks belong to the same spin system and that the 15N dimension is assigned to the same Resonance.
Linking root resonances to 3D peaks
The next part of the assignment process is to link resonances, as we did before, from the HSQC to the corresponding trains of peaks in the 3D experiments. From the menu select M: Assignment: Pick & Assign from Roots. In the {Windows & Spectra} tab in the Pick & Assign from Roots popup that appears ensure that window1 is selected in the "Root Window:" pulldown menu, select window2 in the pulldown menu under 'Target Windows' and click [Add Target Window:]:
Now take a quick look at the {Tolerances} tab, check if the parameters look OK, and go on to the {Link Peaks} tab:
You will notice that the peaks from the HSQC are listed in the table. If you click on one of the rows, window1 will centre on that peak and the location of window2 will move to the same amide frequencies. Rearrange the positions of window1 and window2 so that you can clearly see both and the popup. Select a row corresponding to an HSQC peak that is not overlapped and click [Pick & Assign Root Resonances]. You will see that peaks are picked in the 3D spectra, in the box defined by the tolerances, and assigned to the amide resonances from the HSQC. You can go on to further amide positions by clicking [Next Root]. With appropriately set tolerances you may also click [Pick All & Assign Root Resonances] to process all of the amide positions - you can still use the 'Next Root' function (etc.) to loop through the peaks afterwards. Note that closely overlapping amide resonances would still have to be checked or linked by hand.
In the {Windows & Spectra} tab, on the row corresponding to the TOCSY, double click on the |Assign Non-root dim?| column. Then after having linked one of the amide spin systems to the 3D experiments, click [Assign Non-root Resonances]. You will see that the F2 dimension of the TOCSY peaks are now each assigned to a different resonance (which we could then go on to assign to the appropriate NOESY peak).
It is important when picking peaks and assigning resonances in this automated manner that noise and artifact peaks are not picked. Of course any offending peaks can be deleted afterwards, but most can be avoided by setting the picking tolerances to appropriately small values and setting the contour levels so that the noise is not visible. By default the peaks are picked only above the visible contour base level.
Now that we have defined and linked many resonances, look in the main resonance table at M: Resonance: Resonances. You will see that all of the resonances are listed here and many operations can be performed on them. The important thing to note here is that the chemical shift of each resonance is automatically calculated from the positions of the peaks to which it is assigned. Note that a resonance assigned to only one peak will have no deviation in its shift, but those assigned to several 3D peaks will deviate as the amide peak position varies slightly. By default the chemical shift values are the average of the assigned peak positions, where each spectrum is weighted equally. However, different dimensions of different spectra can carry different shift weightings, set at M: Experiment: Spectra in the {Tolerances} tab:
The value of a shift may be influenced more by the more precise experiments.