Assigning resonances to atoms

Once we possess sufficient information, the resonances to which peaks are linked may be assigned to specific atoms. For the purposes of this tutorial we will cheat with prior knowledge and link spin system number 1 "{1}" to Leucine 21. Find the HSQC peak that we first assigned by going to M: Peaks: Peak List and {Peak Table} and select the HQSC from the "Peak List:" pulldown menu. Click on the row of the {1}[1] {1}[2] peak:

and click [Find Peak]. (If it is difficult to find then click on the |Assign F1| column heading which will sort that column.)  In reality we could choose any peak that we have assigned to the spin system and connect its resonances to 21 Leu Hn and 21 Leu N.

With the cursor over the centre of the peak press 'a', (or select R: Assign: Assign HSQC:5.spc1:6 from the right mouse menu). Once the popup appears select the first hydrogen resonance (top-left table) and then click on [Assign {1}[1]]. This will bring up the Molecule: Atom Browser popup:

Make sure the correct Chain is selected in the "Chain:" pulldown menu, and make sure that the hydrogen and nitrogen atoms are displayed (click [H] and [N]), then scroll down to click on H in the 21 Leu column:

Return to the Assignment: Assignment Panel popup and you will see that the resonance is now annotated as 21LeuH and that the 15N resonance has been assigned (note that we use the IUPAC convention of naming amide hydrogens in proteins as "H" rather than "Hn"). The 15N resonance was assigned because it is directly bound to the 1H resonance - Analysis knows this because we have set the experiment type to H[N]. Return to the Molecule: Atom Browser popup (M: Molecule: Atom Browser) and you will note that the atom sets you have chosen in the browser will change colour to reflect their assigned status.

Now that the Resonances are connected to atoms you will see that the peak annotation has changed to "21LeuH,N", not only for the peak we directly assigned, but also for all of the 3D peaks, that are linked to the same resonances. If you look in M: Resonance: Spin systems, {Assignments}, you will also see that the spin system has been unambiguously assigned to the leucine residue by virtue of our choice of atoms:

During such atomic assignments it may be useful to have representative chemical shift values for the possible atomic assignments. These can be accessed at any time at M: Resonance: Reference Chemical Shifts by selecting the appropriate "Ccp Code:" and "Atom Type:" (heavy atoms or hydrogens).

Connection of residues

Next we will make an inter-residue connection. In window2 mark 'm' the NOESY 21 Leu derived peak just below the diagonal at 7.4 ppm:

Returning to window1 you will note that two vertical lines are present on the HSQC, one through the 21 Leu spin system and the other at the amide H shift of a nearby residue. Looking at the HSQC along the line at 7.4 ppm we see that two peaks are intersected, although neither is dead centre:

(the marked NOESY peak is distorted by the diagonal peak). Cheating again, the most above peak of those two at 111 ppm in the 15N dimension is the residue N-terminal to 21 Leu.

We will bring the 3D TOCSY and NOESY peaks of this neighbouring spin system up in a new window strip:

In the HSQC window, select both the 21 Leu HSQC peak and the peak at 7.36, 111.0 ppm by (left click and drag with 'Shift') so that they are highlighted by surrounding squares. Now in the right mouse menu select R: Strip: Peak Location Strips:1H - 15N in window2 (Alternatively we could have manually added a new strip to window2 and, after having activated the destination strip, used the same navigation function as before in the second HSQC peak).

In the new strip of window2 you will see the neighbouring spin system with its return amide-amide NOESY peaks:

The spin system in the second strip is 20 Gln. So as before, pick the appropriate peaks in the spin system, assign one of them to the resonances in the HSQC spectrum, then propagate the assignments to the remainders.

Finally we will assign the connecting amide-amide crosspeaks. In the 21 Leu strip click R: Assign: Assign peak over the marked peak just below the diagonal.

In the Assignment: Assignment Panel popup you should select the 20 Gln H resonance for the second hydrogen dimension. Now assign the reciprocal amide-amide NOESY peak in second strip. With the assignment complete, select M: Chart: Assignment Graph and then in the {Options} tab click on the button named after your 3D NOESY experiment. Returning to the {Graph} tab you will then see the connection you have just made to the spin systems in the graphical display:

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