Assigning peaks to resonances and spin systems

Assigning New Resonances

Given that we now have some peaks and can move between the different spectra we will start some assignment of the peaks to resonances. Initially we will assign the peaks anonymously, that is to say we will link peaks to a resonance number, but not say which atoms the resonances comes from. Initially such an assignment is not very useful, but we can go on to link related peaks to the same resonance numbers. For example we can say that a whole column of peaks in the 3D TOCSY and NOESY and an HSQC peak are derived from the same amide resonances. When we specify which atoms the resonances derive from then all of these linked peaks will automatically be assigned to those atoms.

To start an assignment choose (pick) the isolated HSQC peak at the location 7.12, 119.51 (1H,15N) , and with the cursor near its centre, press 'a' (or click R: Assign: Assign HQSC...). Note it doesn't matter which peak you choose really, but we will be referring to this one later. The Assignment: Assignment Panel popup will appear containing two rows of tables, one for each of the HSQC dimensions. In the most left table of each row click the button [<New>], this will add the resonances [1] and [2] to the 1H and 15N dimensions of the peak. Now click [Set Same Spin System] at the bottom left of the popup. The resonance assignments will become {1}[1] and {1}[2]:

The {1} annotation signifies that the resonances are both in spin system number 1, which in this instance indicates that they both belong in the same amino acid residue.

Now mark the peak 'm' and navigate to the equivalent position in TOCSY and NOESY spectra in window2 via R: Navigate: 1H - 15N in window2:

Ensure the peaks along the marker in window2 are picked 'Shift' + 'Ctrl' + left click and drag). Note that if you cannot pick some 3D peaks they may not have a maximum within the selected depth range. If this the case you can adjust the depth position or width.

Now assign one of the 3D peaks at the marked amide location, e.g. this red crosspeak:

Press 'a' with the cursor over the peak and find the Assignment: Assignment Panel popup. You will see that the popup has now updated for the 3D peak and consequently there are three dimension rows. Because the peak position closely matches the chemical shift value for resonances [1] and [2] they appear in the right hand tables. We can link these existing resonances to the 3D peak by clicking on their rows in the right-hand table. When you do this the resonance annotation appears on the left hand side (and in the spectrum window) to indicate that the peak is now linked, i.e. assigned to the resonances:

Note that you can remove the resonance assignment in the Assignment: Assignment Panel popup by selecting a row from the left hand side and clicking [Clear contrib]. The use of the term "contrib" here reflects the fact that a given peak dimension could potentially have a contribution from multiple resonances. So for example you can could have an ambiguously assigned NOESY peak where two different pairs of close resonances contribute to the measured peak intensity.

With the 3D peak's amide dimensions fully linked we will quickly give the other 3D peaks at the same amide position the same assignment. Do this by zooming out in the window so that you can see all the peaks along the marker line, select all the peaks including the assigned one (left click & drag) then select R: Assign: Propagate assignments:

This will cause the resonances assignments {1}[1] & {1}[2] (displayed on the spectra as "{1}[1],-,[2]") to be spread appropriately across all the selected peaks.

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