The following flow diagram shows the different steps that need to be taken during the assignment process.
1. Find a peak in the HSQC to be used as a starting point
Begin by selecting a peak in the HSQC which looks nice and is not overlapped.
2. Navigate to this NH position in the CBCANH
Navigate from here to your CBCANH by right clicking the mouse and selecting Navigate and 1H – 15N in windowX (where windowX is the window in which you have the CBCANH). The strip should contain two Cα peaks and two Cβ peaks.
3. Identify the own and previous Cα/Cβ pairs by comparison with the CBCA(CO)NH
If you overlay the CBCA(CO)NH, then one of the Cα and one of the Cβ peaks (generally the weaker ones) should be present in the CBCA(CO)NH. These are the Cα and Cβ of the previous residue, the other two are the Cα and Cβ of the same residue as the NH group. The two carbons belonging to the same residue as the NH group can be assigned.
4. Assign the own Cα/Cβ
The assignment is done using the assignment panel (press a when the mouse is over the peak to be assigned). Select New for the carbon dimension. This will create a new carbon resonance and assign it to this peak. Since it belongs to the same spin system as the N and H resonances, click on Set Same Spin System, so as to add the new carbon resonance the NH spin system. Now assign the atom type (Cα or Cβ) by clicking Set Atom Type. This will bring up the Browse Atoms panel which will initially not show any atoms at all. Click on C to toggle the carbon atoms on and select any Ca (or Cb) to set the atom type.
5. Select the own Cα/Cβ and search for matching peak pairs in the CBCA(CO)NH
Now select your assigned Cα and Cβ peaks (drag the mouse over them together or individually while holding down Shift) and to make things easier, place a mark through each peak (hold the mouse of the peak and press m). Then hold the mouse over one of the two peaks, right click and select Peak, Match Peaks, In CBCACONH and F3 (make sure this is the carbon dimension!). Analysis will now look for strips in the CBCA(CO)NH spectrum where there are peaks that match these two carbon dimensions. In this way you will find the NH which follows your current one in the sequence, since the CBCA(CO)NH peaks are of the type Hi-Ni-Cαi-1/Cβi-1. Be aware that at the peak matching feature will only offer those spectra which are visible in your query window as possible options for matching! So if the spectrum in which you want to search for matches is not given as an option, make sure that it is ‘switched on’ and visible in the current window from where you are doing your matching.
Analysis will then bring up a new panel in which the Options are presented. The case below two matches were found which are ranked according to a scoring function. The top match has matches to both peaks, the lower one only to one peak. Thus the top match looks as though it is probably the correct one, though this should always be checked visually in the spectra as well (a peak found by analysis may for instance be noise and not a real signal, or if two peaks are overlapped the peak may not be placed correctly).
6. Display matching peaks in strips and select best match
To visually inspect your results, you can select as many of the possible matches as you like. Display Groups in Strips will then bring up these strips in whichever target window you have selected.
Once you have brought up your possible matches for visual inspection you may be lucky and find that there is quite definitely one match which is much better than the others.
7a. Good match: Assign the CBCA(CO)NH Cα/Cβ and set the NH as the sequential spin system
In this case you can now go ahead and assign the carbon dimensions in the matching strip of the CBCA(CO)NNH. Simply bring up the Assignment Panel for each peak (press m while the mouse is on the peak) and select the relevant carbon resonance.
To set the sequential spin system, select either peak, right click the mouse and go to Assign, Set Sequential Spin Systems, F1 0, F2 0, F3 -1. Analysis now knows that the spin system assigned to the F3 dimension of this peak belongs to the residue i-1 relative to the spin system assigned to the F1 and F2 dimensions.
Sometimes you will find that there are two very good matches and it is not possible to tell which is correct. In this case simply make a note of the spin system involved and come back to this later when you have an idea of the position in the sequence and the amino acid types that are involved.
7b. No good match
If you are unlucky and the spectra are not very good, you may find that none of the possible matches found by Analysis match very well. Alternatively, the following amino acid may be a proline – because it has no H attached to its N, proline does not give any signals in CBCA(CO)NH spectra and there is no possible match to find. If cannot find a good match, simply make a note of the spin system you have investigated and try your luck with another by starting at the beginning again.