Here are some hints and tips on using CCPNmr Analysis with solid-state MAS NMR data. There are various features which are particularly useful for solid-state MAS NMR spectroscopists, but which users may not be aware of. So here is a collection of some of the features which I have found especially useful.
From version 2.2.1 onwards several additional features have been added to the program which are specially designed for solid-state MAS NMR spectroscopists. These include an update of the labelling feature and experiment types as well as some side-band identification features and double quantum axes. An article on this will hopefully be published soon.
When reading in a new spectrum CCPNmr Analysis will always ask you to specify the Experiment Type. The solid-state experiment types have been overhauled for version 2.2.1 and most solid-state experiment types are now included in the CCPNmr Analysis software. Note that the experiment types do not correlate with pulse sequences, since some pulse sequences will give near identical spectra (e.g. PDSD, DARR and RFDR) and in other cases a simple change of the mixing time will substantially alter the peaks observed (e.g. DARR at different mixing times). Instead the Experiment Type should be chosen in accordance with the kind of spectrum observed, e.g. CC (one-bond), CC (relayed) or CC (through-space). A full table of all solid-state experiment types is available on the CCPNMR Wiki site. Additional experiments can be added by users or the CCPN team can add them.
The advantage of setting your Experiment Types when you read in your spectra is that the program can then help you to filter assignments, for instance.
A major advantage of CCPNmr Analysis is the fact that it is very easy to overlay spectra. In fact, by default, Anaylsis will overlay all the spectra it can (see Organising Windows and Spectra on how to manage your windows and spectra effectively). Using the Spectra toolbar at the top of each spectrum window it is very easy to switch different spectra on and off. The fact that it is so easy to overlay spectra in both 2D and 3D is, to my mind, a major advantage of CCPNmr Analysis over Sparky.
By default your mouse will form a single crosshair with one vertical and one horizontal line. However, when you have two axes belonging to the same atom type (e.g. in a carbon-carbon correlation spectrum) it is really useful to have a double crosshair (with two vertical and two horizontal lines) which will trace equivalent points on either side of the diagonal. In order to set a double crosshair mouse, go to Windows in the main menu and select Window. When you click on a window in the upper part of this pop-up, you will see the axes displayed in the lower part. One of the columns is headed Panel Type. By default these are all set to be different, so for a 13C-13C window, they will be called C1 and C2. If you set the panel types to be the same, then you will obtain a double crosshair mouse. I find it useful to go through all windows and make sure that all panel types are C1, N1 and H1 and then you will always see a double crosshair mouse in diagonal spectra and you will always see equivalent mouse lines in all other windows, too.
CCPNmr does not give you one window per spectrum. Instead it gives you windows with set x-y or x-y-z axes (e.g. 13C-13C or 13C-15N-13C or 13C-13C-15N). All spectra which contain those axes are then displayed in that window. Using the Spectra tab at the top of each window you can switch individual spectra on and off. However, you are not limited to the number or type of windows that you have. You can use this in order to organise, group and optimally display your spectra.
For instance, I will often often create a second 13C-13C window for my carbon-carbon correlation spectra. The first I will set such that I can see the aliphatic region and the second so that I can see the carbonyl region. In order to duplicate a window go to Window in the Windows menu, select the window you want to duplicate and click on Clone.
In some cases you may find that Analysis is overlaying spectra which you don't really want to overlay. For instance, you might like to display your NCACX spectra in a 13C-13C-15N window. Because the x and y dimensions are both 13C, Analysis will also place all your carbon-carbon correlation spectra into this window. I find this unhelpful and prefer not to have all my carbon-carbon correlation spectra visible in the Spectra toolbar of my NCACX window: it unnecessarily clutters the toolbar and makes it is hard to find the NCACX spectra. You can remove the carbon-carbon correlation spectra from the window and from the toolbar by going to Window in the Windows menu and then selecting the Spectra & Peak List Mappings tab. Right at the top you can select the relevant window. In the top part of the pop-up you will then see all the spectra which can be mapped into that window (and in the lower part the corresponding peak lists). Simply make sure that the columns Spectrum In Toolbar, Pos. Contours Visible, Neg. Contours Visible and Slice Visible are all set to No for those spectra which you do not want to see in that window.
For 3D and higher dimensionality spectra it is useful to view different planes of your spectrum. When you read in a 3D spectrum, Analysis will simply create a default window, e.g. a 13C-13C-15N window for an NCACX. You can create a second window with 13C-15N-13C axes in order to see the 13C-15N plane rather than the 13C-13C plane of the spectrum. Go to New Window in the Windows menu. Give your new window a name and at the top select the axes you want. If you want to exclude certain spectra from the toolbar from the start, then make sure that these spectra have a No in the In Toolbar? column. If there are a lot of spectra which are mapped to the window and they are all visible, it can take a long time for Analysis to draw all the contours. So it is often better to select all spectra and click on Selected Not Visible (once the window has been created you can switch the spectra back on using the Spectra toolbar). Finish by clicking Create Window!.
Strips are commonly used to visualise small sections of 3D spectra. Using the Strips button at the top of a window you can access the Strips toolbar. This allows you to add or remove strips (blue + and -), change which strip is active (green number buttons) or move the active strip relative to the others (green arrows). By default the strips are vertical. However, for some solid-state MAS spectra, such as NCACX or NCOCX spectra is easier to use horizontal strips. A little switch button in the toolbar allows you to switch between using horizontal and vertical strips.
In addition to strips you can also insert Separators. Right-click the mouse and go to Strip and Add horizontal/vertical separator (you may need to go to Switch to horizontal/vertical first to switch between the horizontal and vertical functions). While strips each have their own x,y and z chemical shift values, a separator generates strips which share the same x or y chemical shift value and the same z chemical shift value. This allows you, for example, to view the aliphatic and carbonyl regions of a spectrum in the same window without the intervening aromatic region. In a 3D the z-dimension would nonetheless remain correlated. Separators can fulfil a similar purpose as the Synchronize Spectra function in Saprky.
There are a couple of ways in which you can make strip plots fairly quickly. One option is to generate your strips using the Resonances pop-up. First of all, filter the table for the resonances of interest by clicking on the ? in the Assign Name column header. In the filtering pop-up select Regular Expression and enter N|Ca or N|C$ if you want to generate NCa or NCO strips, respectively, and then click Filter Include. (Or change the filtering expression for other types of strip - note that | signifies and, $ signifies end of text.) Now select the pairs of resonances from the residues you would like to generate strips for (use the Ctrl key to select several non-consecutive rows in the table; it will also help to sort the table by Residue or Spin System #). Finally select which window you want the strips to appear in using the drop-down menu above the table and then click Strip Selected to generate the strips. Your strips will now be created in the chosen window. Obviously you have to make sure that the isotopes match the isotope axes in your chosen window.
An alternative is to generate your strips using the Peak Table of the spectrum of interest (via the Peak / Peak Lists menu). You can now select one peak from each strip that you want to have created (use the Ctrl key to select several non-consecutive rows in the table), choose the window in which you want the strips to be displayed using the drop-down menu in the top right-hand corner and then click Strip Selected. A series of strips (one for each peak selected) will now be created in that window.
You can print your strips using the Window / Print Window pop-up. Set your Options as desired and ensure the correct window, spectra and peak lists are selected. In the Region tab you can either set your regions by Min/Max (useful for setting the x dimension of horizontal strips, for example) or by Centre/Width. The Centre/Width option will allow you to set the strip width to be whatever you would like, regardless of the width currently shown in the window.
Manipulating strips in graphics software packages
If you generate a series of NCa and NCO strips you can interleave them quite quickly in a program such as Inkscape (free from www.inkscape.org), Adobe Illustrator or Corel Draw. Simply start by ungrouping everything until you can see that the strips have been separated from one another. Then select the area of one strip at a time and group that selection again. Now you can move the strips around and easily reorganise them or interleave strips from different spectra.
The only thing you have to watch out for is that each strip is surrounded by four lines to create the box around it. If you select the top strip when everything is ungrouped you will also have selected the top line of the second strip. Therefore, after selecting the area of the top strip, click once on the bottom line while holding down the Shift key (to remove one line from the selection) before you group your selection again. If you now move the top strip, you should see that the second strip still has a box surrounding it. This may sound a bit strange, but should become fairly obvious when you try it out.
When Analysis makes assignment suggestions it looks for possible assignments within certain tolerance limits. By default these are set quite low, at values which are suitable for solution NMR. For solid-state MAS spectra it is often useful to use rather larger tolerances. You can set these tolerances by going to the Experiment menu and selecting Spectra. Then go to the Tolerances tab. The top part of the pop-up shows the Assignment Tolerances for the selected spectrum and you can edit them. Below you can see an overview of the tolerances in every spectrum. On the whole you will want to increase all tolerances in all spectra. The easiest way to do this is to set the tolerances for the first spectrum at the top (I usually use 0.5 ppm for all 13C and 15N dimensions). In the lower panel you can then select all spectra (make sure that you select the first sepctrum last, so that it is a darker shade of pink) and then click Propagate Assignment Tolerances. The tolerances from the first spectrum will now to copied to all the other spectra. Note that you can temporarily double the tolerances which you have set by clicking on Double Tolerances in the Assignment Panel.
It is possible to read in 1D spectra into Analysis. As with higher dimensional spectra, it is possible to overlay these and the mouse crosshair is automatically relayed to other windows for easy comparison.
When looking at assignment possibilities in the Assignment Panel there are a variety of useful options available. Intra-residue is a useful option for spectra recorded with short mixing times where you would on the whole only expect to see intra-residue cross-peaks. Double Tolerances allows you temporarily to double the tolerances set for that spectrum. In this way you can have a slightly lower value as your standard and not be swamped with assignment possibilities all the time. But when there is a peak which is slightly out for some reason you can still find the correct assignment option and make the assignment. Correlated Dims uses the Experiment Type selected previously to reduce your assignment options. For example, in an NCACX experiment the N and Cα must always belong to the same residue. The is encoded in the Experiment Type and so when you select Correlated Dims for an NCACX the assignment possibilities will be reduced to those where the N and Cα belong the same residue (unless there are no such pairs within the tolerances used). The CX dimension will not be affected since this is a through-space transfer and could go to any residue. Only if you select the Intra-residue option as well, will you only be given fully intra-residue assignment options. By selecting a Labelling Scheme you can filter the assignment possibilities based on the way in which your molecule is labelled. See below for more details.
The Assignment Panel not only allows you to make firm assignments, but also so-called Tentative Assignments by clicking on Tentative Assign. The assignment will be made, but there will be a ? after it to indicate that it is tentative. This allows you to make a provisional assignment and in a sense mark an assignment as less sure than a proper assignment. It is also possible to make several tentative assignments to one resonance. You may, for instance, face a situation where you have narrowed something down to two possibilities, but cannot distinguish between these yet. In this case you can make two tentative assignments - that way your peaks will be marked accordingly, and you can easily eliminate one assignment at a later stage and make a proper assignment. I find this helpful in order to minimise the amount of loose paper notes I have to keep - instead the information on provisional assignments is saved safely within the project.
From version 2.2.1 onwards it is possible to add the spinning speed at which an experiment was recorded by going to the Experiments popup in the Experiment menu and then going to the Experiment Details tab. Analysis will then automatically add dashed side-band diagonals to your spectrum where relevant. You can also use V, H and M to draw vertical rulers, horizontal rulers or marks which are repeated at side-band intervals. This is a convenient way to identify whether for instance a peak in the aromatic region of the spectrum is in fact a side-band peak from the carbonyls or not.
If you have identified a peak as being a side-band peak you may want to mark it in some so that you don't forget that it is a side-band peak. There are two possibilities of how you can do this. Either, right-click, go to Peak and then Set merit and 0.0. Alternatively, right-click, go to Peak and then Set details - you can then type something like side-band into the comment box. Now go to the Peak menu and select Draw Parameters. Make sure that Merit Symbol or Details are selected, depending on which one you opted to go with. If you are using the Merit Symbol, then go to the Merit Sympbols tab and enter a symbol such as * or ! into the Poor merit box and click on Set Symbols. Now your side-band peak should be marked either with a symbol or a comment to remind you that it is not a real peak.
It is likely that a rather more sophisticated system of properly identifying side-band peaks will be introduced some time in the future. But in the mean time this is a reasonably good fix.
Some 13C-13C correlation spectra can be recorded with a double quantum chemical shift axis in the indirect dimension (e.g. INADEQUATE, POST-C7 etc.). The observed chemical shift is then the sum of two individual chemical shifts and pairs of peaks are observed at positions δ1,(δ1+δ2) and δ2,(δ1+δ2). When reading in such a spectrum you should select the C[CC(DQ)] Experiment Type. A modified diagonal is now drawn at along (δ, 2δ). The mouse will have crosshairs at both δ1 and δ2 in the direct dimension and at (δ1+δ2) in the indirect dimension. Mouse crosshairs will also be replicated at the two single quantum chemical shifts in all the other windows for easy comparison with other spectra (it may be necessary to make sure that the Panel Type in the Window, Edit Windows popup for the single quantum dimension is the same as that for your other single quantum windows).
When making assignments for such a DQ spectrum, the chemical shift sum in the indirect dimension is automatically split into its two constituent single quantum chemical shifts. Assignment options for these are presented and be selected as normal.
Several labelling schemes have been incorporated into Analysis. Along with the standard 15N, 13C and 15N,13C,2H schemes, this also includes the 1,3-13C-glycerol and 2-13C-glycerol labelling schemes which are particularly useful for solid-state MAS NMR spectroscopists. You can use the labelling schemes to filter assignment options, for example. Simply select a Labelling Scheme from the list in the Assignment Panel and the assignment possibilities will be limited to those which are in agreement with the labelling scheme. For the 2Glycerol labelling scheme, for example, it will take into account that you cannot see any intra-residue Cα-CO peaks, but it will allow inter-residue Cα-CO assignments. Whenever you can select a labelling scheme for filtering you are also given the option of selecting the Min Isotope Fraction. This is the minimum fraction which must be labelled at any site for it to be classed as being labelled. This is important for the glycerol labelling schemes: some sites are fully labelled or fully unlabelled, but many are partly labelled, because there are a variety of isotopomers. The minimum degree of labelling necessary at a site for you to see peaks depends on various factors including your protein and your signal-to-noise ratio. For this reason you can always adjust the Min Isotope Fraction yourself. Note that the glycerol labelling schemes include the various isotopomers at 1:1 ratios since this ratio appears to vary somewhat from protein to protein.
Labelling schemes can also be used when creating distance restraints.
From version 2.2.1 onwards you can also associate an experiment with a particular labelling scheme so that the labelling scheme is taken into consideration automatically and you don't have select the labelling scheme by hand each time. Go to the Molecules menu and select the Isotope Labelling popup. In the top half of the popup select New Sample (for the correct molecule). You can now add a labelling scheme for this sample in the lower half of the popup by selecting New Pattern from Scheme (making sure that the correct labelling scheme is selected in the drop-down menu on the right). You will see that the Labelling Pattern in the top half has now been updated. By double clicking in the Experiments column you can select the experiments which are associated with this sample. One of the advantages of using this feature is that you can create a sample which contains two labelling schemes, e.g. 50% U-15N and 50% U-13C labelling. Analysis will then automatically know that any 15N-13C cross peaks are intermolecular cross peaks and distance restraints will be generated accordingly.
You can add or edit labelling schemes by going to Molecule and selecting Isotopomer Schemes (version 2.1) or Reference Isotope Schemes (version 2.2 onwards). Either click New to create a new labelling scheme, or select a scheme you wish to alter and click Copy. In the Isotopomers tab you can specify each isotopomer and the way in which the atoms in that isotopomer are labelled. In the top half of the pop-up you will see all the isotopomers and you can add or delete them using the buttons below the list. Add Default Abundance Set will add a set of 20 amino acids using the Default Abundance. By default this is the natural abundance. If you wish, you can change the Default Abundance using the Set Default Abundances button above the list of isotopomers. This is useful if all amino acids have the same labelling. In the lower part of the pop-up you will see the labelling at each atomic site for the isotopomer selected above. Here you can change the weighting of different isotopes to create the correct degree of labelling if this is specific to certain sites. Unlabelled sites should on the whole be set to natural abundance levels rather than 0.
To create distance restraints, go to the Structure menu and select Make Distance Restraints. In the Settings tab you can set the main options for the kind of restraints you want to generate. At the top you select the Peak List on which you want to base your restraints. Under Restraint Distance Params you can set whether you want to use a Distance Function or Distance Bins. If you select the latter, you get a list of bins below (you may have to increase the size of the pop-up window to see it) which you can edit to suit your requirements. You can generate two different types of restraints: Assigned Restraints are made from assigned peaks only and if the assignment is unambiguous, then the restraints will be, too. Shift Match Restraints match the peak chemical shifts with your Shift List and generate ambiguous restraints. You can check the shift matching with Test Shift Match - this allows you to check for errenous peaks in advance. The ability to select a Labelling Scheme is particularly useful if you intend to make Shift Match Restraints for a sample with glycerol-based labelling. The Residue Ranges, Shift Match Tolerances and Chem Shift Ranges tabs allow you to select further options.
