Keys, Commands & Abbreviations

Mouse functions

Keyboard shortcuts

Document Abbreviations

Loading Spectra

Open an existing project

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.  When the Analysis menu bar has appeared select M:Project:Open Project.  Navigate to find and then select the CcpnDemo001 project (the directory initially will be in green and then when you have selected it in deep purple). Then click [Open]. To save space, if you wish you can minimise the Extend-NMR panel at this stage.

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.

Now save the new project by selecting M:Project:Save. The first time you save a project that has moved locations the SaveAs dialog is brought up.  At this point you can choose to change the project name and/or the location where the project directory will be saved.  For now just hit the "Save" button.

After this first save, M:Project:Save will just automatically save to the existing location.  M:Project:SaveAs will let you save your project with another name, or in another location, but will still have the directory name the same as the project name.

It is a good idea to save now and again in case something goes wrong (either with your work or with the code).

This project has two windows, "window1" and "window2".  The first is a two-dimensional window with an HSQC spectrum in it, and the second is a three-dimensional window with two spectra in it, an HNCA and an HNcoCA.

Opening Spectra from File

To open a new spectrum go to M:Experiment:Open Spectra. In the resultant Open Spectra popup navigate to the CcpnDemo001/spectra/ directory and select 182.spc.par (the other three files are the spectra that are already open).  You can open up more than one spectrum at one time, but there we will just open one.

If necessary enlarge the popup (click and drag the bottom edge down) so that you can see the entry in the bottom table. This table is a list of how the selected spectra will be placed into the CCPN data structure. Each file will be associated with an experiment and a spectrum that are in turn linked to a shift list. For the most part each experiment carries only one spectrum, as will be the case in this tutorial. However, it is possible to have one experiment with several spectra, e.g. when they are processed differently. All the spectra will use the same shift list, as the experimental conditions are the same, so leave that field alone.

Enter a name for the selected experiment by double clicking on the experiment name cell, "Expt_4", and entering the name and then pressing <Return> or clicking outside the box. Set the name of the experiment to "N-NOESY" or similar.  The spectrum name could also be altered, but this is not necessary here and the spectrum name being inherited from the spectrum file ("182.spc") is informative.

Note that the file format option at the top op the popup is set to "Azara", which is correct for the files we will open. If the spectra were stored in a different format (Bruker, Felix, NMRPipe, NMRView, UCSF, Varian or Factorised) this option would have to be altered. Finally click [Open Spectrum].

Now file and referencing verification dialogues will appear. Again, enlarge the popup so you can see the entire table. The {Verify Referencing} tab will be open - it shows the parameters you normally need to look at. The {Verify File Details} tab lets you edit where and how the file is stored, but this its not normally needed. Click [Commit] on the button at the top of the popup (from either tab) to continue. Note that you can change spectrum referencing and file setup at any time after loading via M:Experiment:Spectra.

Note that you may skip all the verification steps, if you know your parameters to be correct, by selecting the "Skip verification dialogs" option before you click [Open Spectra]. You can change almost all the spectrum referencing and file parameters at a later time should you need to make adjustments. The only thing that you cannot change after a spectrum is loaded are its primary axes; in terms of number and which isotopes they refer to.

Setting Experiment Types

After the verification there is one final popup where you specify the types of the experiment that was run. This information is very useful later on; for example Analysis uses the knowledge of which experimental dimensions correspond to single bond transfers to automatically remove impossible assignment options.

Set the synonym for the new experiment to "15N HSQC-NOESY". Note that for this experiment there are two different full types with the same synonym (double click the Full Type column). So the "15N HSQC-NOESY" could be either H_H[N].NOESY  or H[N]_H.NOESY, with the difference being that the NOESY transfer can come either  before or after the HSQC step. For our experiment the selection H_H[N].NOESY is correct. When the experiment type is set click [Done] at the top right or [Close - All Done] at the bottom. You may have to expand the popup to see the [Done] button. After a few moments you will hopefully see the newly loaded spectrum appear. You can now also close the Open Spectra popup.

Spectrum Windows and Peaks

Window Components and Views

Once the new spectrum has loaded, a new window, "window3" will automatically be created. This is a three-dimensional window with "1H" along the x and y axes and "15N" along the z axis.  A new window is created like this only if no existing window matches in isotopes.

Note that if you minimise or close 99a window you can get it back again by going to M:Windows:HN: window3 (or equivalent).

You can move around the spectra within the windows using several different inputs. To zoom in and out use the <PgUp> and <PgDn> keys, the middle mouse wheel (if you have one), or hold <Shift>, click the middle mouse button, and move the mouse up and down.  If you zoom out from your spectra so that you can see their edge you will notice a dotted line which denotes their border.

You cannot zoom out further than the maximum size allowed in each dimension.  To change this size open up M:Window:Axes and select the Axis Types tab (if it is not already selected).  Each Axis Type has a Region, which can be edited by double clicking on the relevant cell.

To pan around the spectra you can click and drag the scrollbars at the edges of the windows, use the arrow keys, or click and drag on the spectra with the middle mouse button.

Looking in "window2" move the extra scrollbar at the very bottom of the window. - Left click and drag. This scrollbar is present on the 3D window to change the depth or plane of the spectra being viewed. On a 4D window there would be yet another scrollbar. To change the thickness of the displayed planes click and drag the side of the depth slider with the middle mouse button.

Staying in window2, click on "Spectra" at the top of the window. Here you will see two coloured buttons, one for each of the 3D spectra. If you click the buttons you can independently toggle the contour displays for the two spectra on and off.

Setting Contours

To change contour settings click on the "Contours" button at the top of a window.  This lets you do simple adjustments.  The green arrows will raise or lower the contouring floor, while the +1/-1 will change the number of contours.  The "Pos/Neg" button allows you to swap between showing just positive or just negative or both contour levels. Note that using these contour options only changes the spectra that are visible at the time within the window. If you click on the "More.." button you will bring up a more detailed contour level setting dialog.

Peak Picking and Manipulation

Focusing on the HSQC spectrum the next task is to define some contour extrema as peaks. There are two common ways to pick peaks. One is to search for all extrema in a boxed region. Try this in the HSQC window ("window1") by holding down <Shift> and <Ctrl> whilst clicking with the left mouse button and dragging the box to define a rectangular pick region.

Note that when the peaks are picked the contours will carry diagonal crosses to denote the peak position.  Each peak will also have some annotation to the top right of the cross, which we discuss below.

Locate the contours near the point at 8.7 ppm on the 1H axis and 119.4 on the 15N axis.  These contours represent two overlapping peaks where the extrema search will only pick one of the two underlying signals. To define the second peak position hold down <Ctrl> and click on the location to pick the new peak.

Now we will select some of the peaks, e.g. artefacts or noise, for deletion. To delete peaks click with the left mouse button and drag a box over a region containing peak crosses (without holding down any keys). When the mouse button is released you will see that the peaks in the defined region are highlighted with a border around the cross. To select just a single peak click near its centre with the left mouse button. To delete the selected peaks press <Del> or select R:Peak:Delete selected.

If you have peaks selected and then select a different set of peaks you will see that the selection is completely substituted for a new one. You can add to an existing peak selection by holding <Shift> while you choose. Note that you can select peaks in several different spectra, and from different windows, in this manner.

Markers and Navigation

Pick and select an isolated peak in the HSQC spectrum. Put a mark through it by holding the cursor over the peak centre and pressing <m>. The lines produced are a multi-dimensional marker at the peak position and will be visible at the equivalent 1H-15N location in the 3D window. To go to this equivalent position in window2, with the cursor over the marked peak select R:Navigate:1H - 15N in window2. Note that there are also navigation options for window3. There is only one navigation option for window2 because it is an HCN window and there is only one way for the HN axes from window1 to map to it. However window3 has HHN axes and thus has two navigation options, where the second option would take you to the 1H position on the vertical axis, rather than the horizontal axis, which represents the amide proton.

Multi-dimensional marks, vertical ruler lines and horizontal ruler lines can be added to any window location, not just on peaks, using the <m>, <v> and <h> keys respectively. To increase the number of marks and rulers that can be displayed at one time select M:Window:Marks and Rulers or R:Markers:Options. Note that you can clear all marks and rulers with the <n> key.

Strips and Strip Navigation

Now we will start to manipulate "strips" which are sub-divisions of a window that are connected (in terms of their view) in one of the screen dimensions, but independent in the other dimensions. Go to window2, select the "Strips" option at the top and click [+], this will add a vertical division to the window. Click and drag with the middle mouse button to move the spectra - you will see that the vertical axes of the strips are tied together, but the horizontal axis is independent. The depth dimensions are also independent, e.g. if you move the bottom most, 15N scrollbar. Which depth dimensions are moved depends upon which strip is active. The active strip is indicated by an asterisk "*" next to its strip number and is set either by double-clicking (left mouse) within a strip or by using the strip options at the top of the window; by clicking [1] or [2] or whatever. The active strip is also the one that will be removed when the [-] button is clicked. You can rearrange the strip order, moving the selected strip with the green arrows, and swap between vertical and horizontal strips with the toggle button. To remove all the strips press the vertical and/or horizontal buttons to the right of "Clear:".

Manually locating strips at interesting positions can be tedious, but there are various options to build strips from pre-defined locations, for example using peaks. To make strips using peak locations select three (picked) peaks in the HSQC spectrum and select (using the right mouse button) R:Strip:Peak Location Strips:1H-15N in window2 (the same option is found in R:Navigate). The result is three strips located at the amide positions corresponding to the HSQC peaks.