In the mean time CCPNmr Analysis is probably the best NMR assignment software available and I would strongly recommend using it. For details on how to download and install it, please see the CCPN website.
Currently there is no detailed manual for CCPNmr Analysis and while the Tutorial provided on the CCPN website makes sense to anyone already familiar with protein NMR assignment, others have found it rather cryptic. A wiki-style manual is currently being put together, but is still slightly patchy. In the mean time, here is a description of the basic functionalities required for protein NMR assignment.
When starting a new project begin with creating it by going to the Project pull-down menu, selecting New and giving the project a name. Analysis will create a file called ProjectName.xml and a directory called ProjectName in which it stores various other bits of data related to the project.
So as to be able to assign atom names to your resonances, you need to tell Analysis what molecules you are investigating. First of all you generate a Molecular System (as you may have more than one molecule in your NMR tube) to which you then add molecules (so-called Molecular Chains). The Molecular Chains are in turn generated from Molecule Templates. Inclusion of proteins/DNA/RNA is extremely straight forward and will be described here. Support for unusual amino acids and other molecules is in the process of being added. In the mean time if you contact the CCPN team they can probably help you include other molecules into your project.
To create a molecular system go the Molecules pull-down menu and select Molecular Systems. Click on Add Molecular System and give it a name.
To add molecules first edit the molecular templates by clicking Edit Molecular Templates or select Edit Molecular Templates from the Molecules pull-down menu. Click on Add a New Molecule and give it a name.
If you are adding a Protein (or DNA/RNA) click on Add Polymer Residues and type or copy/paste your sequence (making sure that the correct type of polymer is selected above). Select the cyclic polymer box if necessary. Click OK.
Back in the Molecular Systems pop-up you can now select your molecule template (next to arrow in centre panel) and click Make Chain from Template. Give it a chain code (usually this will simply be A, but if you have several molecules in your molecular system you should give them different chain codes, e.g. 'A', 'B' etc.). For proteins you will be asked whether the Phe/Tyr Hd1/2 and He1/2 atoms are equivalent due to rotation or not. In the vast majority of cases these aromatic rings have sufficient space to be able to rotate and the Hd1/2 and He1/2 atoms will thus end up with identical chemical shifts. There are some molecules where rotation is hindered. Unless you know this to be the case for your molecule, simply click Yes.
If your sample contains other NMR observable molecules, add these to your molecular system, too.
Go to the Experiment pull-down menu and choose Open Spectra. Make sure you select the correct data format at the top of the box - a large number are possible (Azara, Bruker, Felix, NMRPipe, NMRView, ucsf). Change the Experiment Name if you wish (you can always change it again at a later stage, if you want). In general, you should use a new experiment name for each spectrum you read into Analysis. It is in fact possible to read several spectra into one experiment, but this should only be done if the spectra are derived from the same data but processed differently. Change the spectrum name if desired. By default Analysis will read your spectrum into the first possible window. If you have a large number of windows or spectra you may want to change this to All or None instead (though the All option can slow you down if you have a lot of windows). In certain cases you may be using several shift lists - in that case make sure you have the correct one selected (but again this can also be changed at a later stage).
You will now see three pop-up boxes, one after the other. The first gives you information on the matrix size of you spectrum, simply click OK. The second shows you the referencing information. It can be worth checking that this looks reasonable and that the data was referenced correctly when processing it. When reading in Bruker data, for instance, the isotopes may not be defined correctly. Change things if necessary (though again this can be done at a later stage, too).
The third window asks you to select an Experiment Type. Analysis likes to know what type of experiment each spectrum is. It is then able to do intelligent things like automatically know that certain dimensions must belong to the same amino-acid etc. You can narrow down the experiment types on offer by selecting the Category (through-bond, through-space etc.). Then select the name either using the common names for them Type Synonym or the CCPN-defined naming system Full Type. Once you get your head round it, I find the latter easier than the former, as the common names are sometimes different to what I would use. The CCPN naming system essentially puts those atoms which are observed in capitals and those which are used for magnetisation transfer steps in the experiment in lower case. I think the transfers are generally via J-couplings except where stated otherwise (e.g NOESY, TOCSY).
Each window within Analysis has a defined dimensionality (i.e. it is either 2D, 3D, 4D etc.) and the isotopes for each axis are fixed. So you might have windows with (x,y,z)=(1H,15N) or (1H, 15N, 13C) or (1H, 13C, 15N) or (1H, 1H, 15N) etc. When you open your first spectrum, Analysis will create a window for it to be shown in. However, in some cases the window may not be the one you want: for instance an HNCO is usually opened in a (1H,15N,13C) window, but personally I prefer to look at it in a (1H,13C,15N) type of window. If you open further spectra and windows already exist with the correct number and types of axes, then no new window will be created - instead the spectrum is shown in those windows which are already present. When no window is present in which the spectrum will fit, a new one is created.
You can create your own windows by going to the Window pull-down menu and selecting New Window. Simply select what axes you would like and what isotopes they should be associated with. In the lower panel Analysis will show you if there are spectra which will fit into this window. If this is the case you have the option of selecting which (if any) spectra should be shown in this window (this is particularly useful when you have lots of spectra, as the program can get very slow if it tries to draw 20 spectra on top of one another in the new window).
If you want to duplicate a window, simply go to the Edit Window option in the Window pull-down menu. Select the window you want to duplicate and click Clone.
The Edit Window pop-up has a number of useful features:
Aspect Ratio: this will change the relative scaling of the x and y axes - change this to make your peaks look more round or elongated.
It is possible to change the background colour. By default the background colour is white and a light background is said to be better for the eyes, but many people prefer a dark background or are used to this from other programs.
If you have spectra with two axes belonging to the same isotope (e.g. (1H,1H)), then by default these have different Panel Types (e.g. H1 and H2). If you set them both to be H1, then your mouse will become symmetric across the diagonal and show two crossing-points rather than just one. This can be very useful for spectra such as 2D H-H NOESYs or solid-state carbon-carbon correlation spectra.
You can also select to see a 1D trace of the spectrum as you move across it with the mouse - displayed either on the mouse (X/Y cross-hair trace) or along the side of the spectrum (Side Trace). You can switch these settings on and off in the Edit Window pop-up, but in practice it is easier to do this via the mouse under the Right Click / Window options. To change the scale of the 1D traces use the Home and End keys.
There are a number of ways in which you can change the appearance of your spectra. A number of things can be changed under Edit Spectrum in the Experiments menu, such as the colour of your positive and negative contours. You can also order you spectra which will define the order in which they are drawn on top of each other and the order in which their buttons appear in the Spectrum Toolbar. You can also give your spectrum a short-cut.
Analysis has some extremely nice ways of moving around your spectra using the mouse and keyboard.
Zoom:
(a) roll your mouse wheel
(b) press mouse wheel/middle button + Shift, then move the mouse
Moving around the spectrum:
(a) press mouse wheel/middle button and move the mouse
(b) use the scroll bars
Moving through z-planes:
(a) roll the mouse wheel while holding down Ctrl
(b) use the scroll bar
To go from a place in one spectrum to an identical place in another:
Right click the mouse and go to Navigate, then select the relevant window (and whether you would like identical axes to be swapped or not)
To increase/decrease the z-plane thickness:
make the z-plane scroll bar thicker or thinner by pressing the middle mouse button on the edge of it and dragging.
| Left-click + Shift (+drag) | add to the selection |
| Left-click + Shift + Ctrl + drag | pick peaks |
| Middle-click + drag | move the spectrum around |
| Middle-click + Shift + drag up/down | zoom in/out |
| Middle-click + Ctrl + drag | zoom in on the dragged region |
| Middle-click + drag the edge of the z-scroll bar | in/decrease the plane thickness |
| Right-click | menu with various options |
| Mouse-wheel | zoom in or out |
| Mouse-wheel + Ctrl | move through z-planes |
Analysis can draw marks and rulers. The difference is that rulers are only drawn in one dimension (horizontal or vertical) where as marks are drawn in all dimensions. When you draw a mark or ruler in one window, it is automatically also drawn in all other windows. This is a very useful way of checking whether peaks are really at the same height or not. To draw a ruler press h or v for horizontal and vertical rulers, respectively, and it will be drawn at the current mouse position. To draw a mark, press m. n will remove all marks and rulers. You can also draw marks and rulers using the Marker menu in your Right Click Mouse menu. This also allows you to remove only marks or only rulers rather than all at the same time. By default only one mark and ruler can be drawn at any one time (i.e. if you draw a second, the first goes away). To increase the number you can draw, go to the Options menu and select Marks and Rulers. Here you can also select the colour of your marks and rulers. Marks have the added feature that if you draw them near to a peak, they will automatically position themselves on the peak and be black. Otherwise they will go at the precise mouse position and be the colour selected in the Marks and Rulers pop-up.
You can pick peaks by positioning the mouse where you would like to have one and then right-click the mouse and select Add Peak from the Peak menu. Alternatively, a better way is to drag the mouse over a region while holding down Shift and Crtl - this will place peaks at any maximum in that region. It is worth being aware of the Peak Find Parameters pop-up in the Crosspeaks menu. Here you can define a number of parameters which relate to peak picking. By default only positive peaks are picked, but here you can change this to negative only or positive and negative. Other parameters (which I hardly ever change, but you might find useful) include whether peaks are picked on, above or below the contour level, the picking of diagonal peaks and the method by which the volume of a peak is calculated.
The volume of a peak is calculated at the time when it is picked which means that you do not need to perform an additional peak integration step if you want to get a peak volumes.
Analysis is based round the concept of a Resonance - it makes sense, but is difficult to explain. But I will try. Each resonance is associated with one particular atom in your molecule. In theory you might think that it is associated with the particular chemical shift of that atom, but the reason for having the concept of a resonance, is that the chemical shift itself is dependent on many outside influences, such as temperature, pH, salt, ligand etc. This means that any one atom is not associated with any one particular chemical shift. Thus the resonance is there to hold it all together. The reason why you can't simply use the atom as your central object is that to start with you obviously don't know which peak corresponds to which atom.
When you pick a peak in Analysis the first thing you have to do to it, is create a new resonance for each dimension. At a later stage you may then add further attributes such as the atom type (Cα or Cβ), or the full assignment, i.e. exactly which atom in the molecule it corresponds to (Asp10Cα or Trp54N). Resonances are numbered from one upwards and are always shown in square brackets in the peak labels and can be looked at and manipulated using the Browse Resonances pop-up which can be called up from the Assignment pull-down menu. The Browse Resonances pop-up is also useful for navigation purposes, as you can mark selected resonances and go to their position within a specified window.
A spin system basically contains all resonances which belong to one amino acid in a protein (or one nucleotide in DNA/RNA or one sugar ring in a carbohydrate). When two resonances are known to be in the same spin system they can be added to a new spin system. Spin systems are initially numbered one upwards and their numbers are shown in curly brackets in the peak labels. Spin systems can be manipulated in the Spin Systems pop-up which is accessed via the Assignment menu. It is possible to associate a spin system with a particular amino acid type and also to merge them (e.g. if resonances originally associated with separate spin systems later turn out to belong to the same one).
Peaks can be assigned by using the assignment pop-up. Access this by holding the mouse over a peak and pressing a or doing right-click / Assignment / Assign Peak. Alternatively it can be called directly from the Edit Peak List pop-up. The assignment pop-up will show a field for each dimension of the peak. On the left there is the information on isotope type and chemical shift and what assignments have been made for the peak. On the left hand side are possible assignment options. Note that more than one assignment can be made for a single peak (as it may be the result of two overlapping resonances).
Several options are available. Of particular note are the Double Tolerances and Correlated Dims options. By default each spectrum has certain tolerances for each dimension within which Analysis will look for assignment options. Sometimes it is useful to double this when doing assignments, if the peaks are slightly off in the different spectra. The tolerances can be changed for spectra by calling the Tolerances pop-up from within the Edit Spectrum pop-up.
The Correlated Dims option makes use of the fact that the experiment type is known and that for some experiments certain dimensions have to belong to the same spin system. For example for a peak in an HSQC spectrum the two dimensions (H and N) have to belong to the same spin system (residue). Thus, if Correlated Dims is selected, only those options are shown where both dimension fit to the same spin system - a useful way of reducing your assignment options quickly.
By default each spectrum has one peak list associated with it. But you can add extra (empty) ones, duplicate (clone) peak lists or read in a peak list from another programme (using the Format Converter). Only one peak list is active it any one time, so if you are working with several peak lists, then make sure the correct one is active. Under Current Peak Lists in the Crosspeaks menu you can see and manage all your peak lists.
To actually look at a particular list and the peaks within it, go to Edit Peak Lists in the Crosspeaks menu. A useful feature within this pop-up is the ability to choose a window from the top right hand drop-down menu and then go to Find Peak. That window will then centre on the selected peak and be marked with a thick black mark.
Strips are particularly useful when doing assignments. Basically it is a way of seeing several parts of a 3D spectrum next to one another at differing z-planes (see Visualising 3D Spectra for more information on strips). To manage your strips click Strips in your Spectrum Window Toolbar. + will add strips. The strips are numbered (top right hand corner) and the z-plane is displayed in the bottom left hand corner of each strip. There is a button for each strip and the active one is coloured green. - will remove the active strip and Clear will remove all strips. Left and Right will move the active strip one place to the left or right; in this way you can change the order of your strips. Note that if you navigate from another window into a window containing strips, you will navigate to the active strip.
All tables in Analysis have some basic elements in common. Cells which can be edited by the user have a small E in the bottom left hand corner. Columns can be sorted by clicking on the column header. A small arrow then indicates the direction of sorting. Repeated clicking reverses the order. In any table it is possible to right-click the mouse and then select Filter so as to filter for certain strings (generally or in particular columns). It is also possible to right-click the mouse and select Export so as to export any combination of columns to a tab- or comma-separated file.
There are numerous keyboard shortcuts. You can see what these are and also change them if you go to Macros and then Organise Macros. Here is a list of the default settings:
Spectrum Manipulations
| Page Up | Zoom out |
| Page Down | Zoom in |
| Up | Move spectrum up within the window |
| Down | Move spectrum down within the window |
| Left | Move spectrum left within the window |
| Right | Move spectrum right within the window |
| Home | Zoom the slice range down |
| End | Zoom the slice range up |
| c | Centre the window where the mouse is |
| j | Scroll left orthogonally |
| k | Scroll right orthogonally |
| i | Increase the number of contours |
| o | Decrease the number of contours |
| e | Raise the countour level |
| r | Lower the contour level |
Marks and Rulers
| h | Create a horizontal ruler |
| v | Create a vertical ruler |
| m | Create a mark |
| n | Clear all marks and rulers |
Pop-Ups
| a | Bring up the Assignment pop-up |
| b | Bring up the Browse Atoms pop-up |
| u | Bring up the right-click Mouse Menu |
| s | Show the selected peaks in a pop-up table |
Peaks
| p | Move selected peak |
| P | Automatially centre the peaks on the closest maxima/minima |
| q | Move peak label |
| w | Automatically set the peak label positions such that they do not overlap |
| W | Reset the peak labels to their original positions |
| l | Unite peak positions |
| s | Show the selected peaks in a pop-up table |
Other
| S | Save project |
