For this tutorial you will need the example spectra and project files from two archives.

The first part uses this CCPN project (94MB total size): CcpnDemo002.tgz
The second part uses this CCPN project (266 MB total size): CcpnDemo003.tgz

Keys, Commands & Abbreviations

Mouse functions

Mouse Button + Keys	Function
Left	Select peak
Left + Shift	Select peaks in region (additive)
Left + Control	Pick peak
Left + Shift + Control	Pick peaks in region
Middle	Drag canvas
Middle + Shift	Zoom (with up/down)
Right	Options menu
Mouse wheel	Zoom

Keyboard shortcuts

Key	Function
Cursor (Arrow)	Pan in window
PgUp	Zoom Out
PgDn	Zoom In
Del	Delete Selected peaks
F1-F12	Toggle spectrum
a	Assign peak
h	Add horizontal ruler
m	Mark cursor position
n	Remove marks & rulers
p	Move selected peak
v	Add vertical ruler

Document Abbreviations

Abbreviation	Meaning
M:n1	A top-level menu named "n1", accessed from the main Analysis menu
M:n1:n2	A sub-menu item "n2" accessed from top-level menu "n1"
R:n3	A menu item "n3" accessed from a spectrum window with a right mouse click
<Return>	The Return key
{title}	A tab named 'title'
[command]	A button named "command"
text[]	A text entry box named "text"
->	OS command line prompt
>>>	Python shell line prompt

Assigning Peaks to Resonances and Spin Systems

Start the Extend-NMR GUI

If the Extend-NMR GUI and CcpNmr Analysis are not already open, start Extend-NMR on the command line by typing:

-> extendNmr

(This assumes that the bin/ directory is on your path, otherwise you will need to type the full path or be in the bin/ directory.) Once the program has started select M:CcpNmr:Analysis.

Open an existing project

In the Analysis menu bar select M:Project:Open Project.  Select [Yes] if Analysis asks whether it can close the current project and [No] if it asks to save the current project. Navigate to find and select the CcpnDemo002 project (the directory initially will be in green and then when you have selected it in deep purple). Then click [Open].

You might get a warning that various files have moved location.  You might also get a dialog with a list of spectra paths (because those also have moved location).  If the paths are all in grey then just click the "All Done!" button at the bottom.  If any path is in red then Analysis cannot find the corresponding spectrum data file, so either you need to tell Analysis where it is (by double clicking the path cell and navigating to the correct location) or accept that that particular spectrum will not have its contours displayed.

This project has three windows, "window1", "window2" and "window3".  The first is a two-dimensional window with an HSQC spectrum in it, and the other two are three-dimensional windows, one with an HNCA and an HNcoCA, and the other with an HSQC-NOESY.  The HSQC has been peak picked but not the other spectra.

Efficient Resonance Assignment

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. Of course you can also do this manually if you wish. We can use the HSQC positions to define unique amide locations and pick and assign related spectra based 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.  Select M:Assignment:Initialise Root Resonances.

When you see the 'Initialise Root Resonances' popup, there is a table called 'Amide Sidechain Peaks' with a few rows filled. Some of the peaks 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"). If you think this looks like side chain NH peaks, double click the 'Confirmed' column so it changes to 'Yes'. Hint: there are five NH2 side chains in this protein. When you are happy with all of them, click 'Initialise Peak List!' at the top.

This command calls the anonymous resonance and spin system assignment routines on all of the HSQC peaks. Note that the routine knows which spectrum to work with because the experiment type of the HSQC is set 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.

The next part of the assignment process is to link resonances 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 Pick & Assign from Roots popup that appears ensure that window1 is selected in the Root Window pulldown menu and select window2 in the pulldown menu in the Target Windows section, and click [Add Target Window:]. Repeat this again to add window3 to the target list: select window3 from the pulldown list and [Add Target Window] again. Now take a quick look at the {Tolerances} tab and set the "Root 1H Dim1" tolerance to 0.03 ppm and the "Root 15N Dim 2" to 0.2 ppm. Check if the other 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 and window3 will move to the same amide frequencies. Rearrange the positions of the windows so that you can clearly see all of them, 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.

It is important when picking peaks and assigning resonances in this automated manner that noise and artefact 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.

Sequential Protein Backbone Assignments

We will now look at the sequentially assigning protein backbone spin systems using triple-resonance experiments. There are two basic, although linked, parts to the process. The first is the linking of sequential spin systems (collections of resonances that relate to one residue) on the basis of matching peak positions. The second is the matching of runs of unassigned spin systems to residues within a sequence.

Open an existing project

We will now leave the current CCPN project behind and load a new one; this differs from the old one in that it has two more spectra; HNcoCaCB and HNCACB, and that all the peaks have been picked and linked to the amide 'root; resonances as described above (i.e. using the Assignment:Pick & Assign from Roots option). In the Analysis menu bar select M:Project:Open Project. Select to close the exiting project, but there is no need to save. Navigate to find and select the CcpnDemo003 project, then click [Open].

Spectrum Setup

First make sure that window2 and window4 are visible and arranged such that window2 is tall and narrow and on the left of a tall and wide window4 (both windows should be as tall as possible). Both window2 and window4 have HCN axis and can display the triple resonance spectra. Note that to make a new window you could use either use  M:Window:New window or clone an existing window via R:Window:Clone. Using the "Spectra" tab in the windows ensure that the HNcoCA and HNcoCACB are the only spectra turned on in the narrow window and that all four triple resonance spectra are turned on in the wide window.

Select M:Assignment:Protein Sequence Assignment. You should start out in the 'Window & Spectra' tab. Select window2 as the 13C Window in the Query section and window4 as 13C Window in the Match section. Make sure that the "Use" column is set to "Yes" for the query HNcoCA and HNcoCACB spectra in the top table. For the bottom table set the "Use" column to "Yes" for only the HNCA and HNCACB spectra (i.e. not the through-carbonyl experiments). This setup means that we are going to compare specified 13C peak positions in the HNcoCA and HNcoCACB experiments with potentially matching peak positions in the HNCA and HNCACB experiments.

The rationale here is that the through-carbonyl experiment's peaks have the carbon shift of the preceding alpha and beta carbons along the polypeptide chain at a given amide location, where the HNCA & HNCACB have both the intra-residue and preceding alpha & beta carbon peaks for each amide. Thus we can potentially use both spectra to say two amide spin systems are sequentially connected by saying that an inter-residue peak of the HNcoCA or HNcoCACB derives from the same resonance as an intra-residue peak of the HNCA or HNCACB.

Note that this system can readily use other backbone experiments like HNcaCO, HNCO, HAcacoNH, HAcaNH  etc. with the same approach and that this tutorial only uses the alpha & beta carbon experiments for simplicity.

Linking Sequential Spin Systems

Now go to the {Spin System Table} tab and click on the row that corresponds to spin system 8. You will see that the two triple resonance windows move to new locations. The location of window2 is at spin system {8} and split into two regions one for the CA and the other for the CB peak. The other, window4, has moved to the position of any peaks that match the carbon frequencies of the query peaks. In this case there are three potential matches, but the middle strip that corresponds to spin system {71} is the only one that matches both the CA and CB positions well. Note that if the "Filter 13C By Inter/Intra Type" option in the {Options} tab is set to off then spin system {8} matches even more strips. Because we already have a good match we don't really need this option on, but it is useful if the previous-residue and same-residue peaks overlap significantly in the match strips. In the case of spin system {8} matching {71} the CA position does not intersect the purple HNcoCA position, it clearly matches the separate orange HNCA peak (and similarly for the CB position).

In the Spin System Table click to highlight the row corresponding to spin system {71} in the Match Peak Positions panel, and then click [Set Seq Link]. You will now see that the tables of the popup update to show that {71} is set as "i-1" of {8}. Also note that in the spectrum window the peak annotations of the aligned CA & CB peaks have changed to illustrate that they are both assigned to the same 13C resonances. Now click [Goto i-1], which will repeat the carbon shift matching, but this time from a sequence position one earlier in the sequence.  Repeat the above procedure for spin system {71}: select the best match and link. For this exercise go on to sequentially link six spin systems in total. If all goes according to plan, the order of spin systems (going i-1each time) will be {8} -  {71} -  {42} -  {37} -  {48} - {41}.

Assigning Segments to the Sequence

You will see that as you select the various spin systems in the main table, in the lower right Residue Types table there is a display of the probable types of amino acid residue. This prediction is based upon how well the shifts within the spin system match the chemical shifts in the RefDB database. As spin systems are connected sequentially, amino acid type predictions are made for the whole sequentially connected section. In the lower left hand table the connected spin systems, given their probable amino acid types, are matched to the protein sequence. Here the highest scoring positions of residue type match for various five residue sections are listed. You will see that the unassigned residue positions are coloured grey, and the one assigned regions become blue.

Click in the Spin Systems Table on the row for spin system {37}, which is in the middle of our linked region, and you will see that the residue type predictions are strong at and either side of this position. The highest scoring option in the Sequence Locations table (hopefully with a score of about 66.7) should correspond to the region from residue 17 Gly to 21 Leu, with the other sequence locations having lesser scores. Simply select the row for this highest-scoring location and click [Assign Selected] and then [OK] to confirm the assignment. You will see that all the residues in the section (by virtue of their links for the most part) become assigned to the selected section, and that the colors in the 'Sequence Locations' table change.

Select the option M:Molecule: Atom Browser.  Make sure that the elements [N] and [H] are displayed (click the button to get the green hydrogen assignment options) and look at the amide atom for 19 Thr. You will see that not only is 19 Thr assigned (i.e. the atom option goes dark green), but the atoms in the residues which we just connected sequentially are also assigned. Go through the spectra and the Protein Sequence Assignment (with 'Goto i+1')  to verify that all the connected spin systems have been assigned. Note that in this instance it was possible to assign the resonances to unique atoms, as well as assign the backbone spin systems to the sequence, because the resonances had their atom type set previously.

Automatic Protein Backbone Assignment

The semi-automated sequence assignment mechanism described above is supplemented in CcpNmr Analysis by automatic assignment routines. At the moment only one called "Nexus" is publicly available, but MARS and other routines will be incorporated in the future. The automated assignment routines are great for saving time, but they won't always work for all parts of proteins, especially where peaks are missing or severely overlapped. In such instances you can run the automation to assign the easy parts and then fill in the rest more carefully, where possible, using the more manual routines.

Fortunately the data in the example CCPN project being used here is pretty good, and the peaks positions have been checked and have undergone a degree of curation. Accordingly, we can assign most of pure protein sequences automatically with a couple of clicks. To do this open M:Assignment:Automated Seq. Assignment and in the resulting popup you will see that the four triple-resonance spectra are already selected; changing the "Use" column entry to "No" would mean the spectrum was not used for the assignment. All of these default settings are correct, so move on to the {Spin Systems} tab. In the spin systems table you will be presented with all of the resonances, available in the chosen spectra, that are linked to a backbone amide as either same-residue (Intra Residue) or previous-residue (Inter Residue). Initially the table is quite bare, with only a few resonances (and their chemical shift values) displayed for the few residues that we have already assigned. To fill in the rest of the table click [Find Resonances From Peaks], selecting [OK] at the confirmation dialog. This function will then automatically extract and label the CA and CB chemical shifts from the same- and previous-residue peaks. This is done by considering the overlap of the through-carbonyl spectra with other spectra, together with the peak intensities and relative chemical shifts that are expected to be visible (in an experiment of that type).

With the resonances populated in the spin systems table, move on to select the {Automation} tab. Here, set "Use existing assignments?" to on and accept the other default settings and click [Run Nexus]. Once the procedure has finished all of its iterations you will be shown a graph of the assignment scores for each residue; where blue means good, yellow dubious and red bad. Moving to the {Predictions} tab you will hopefully see large regions of good (blue) prediction. Before a spin system (and thus all of its contained resonances) can be assigned to the predicted residue, the prediction must be confirmed. To confirm the prediction either select a series of good rows (use <Crtl> + click in the table) and click [Confirm Selected] or set all blue regions en masse by clicking [Conform Above Score Threshold]. With your choices conformed click [Commit Assignments] and confirm [OK] to actually do the full assignment; which will affect the peak labels.

Look at the peaks in window1 to see the result of the assignment, and admire your handywork.