Analysis tutorial Data

For this tutorial you will need the example spectra and project files in this archive:

 analysisTutorialData.tar.gz

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	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
[command]	A button named "command"
text[]	A text entry box named "text"
->	OS command line prompt
>>>	Python shell line prompt

Loading Spectra

Start a new project

Start Analysis on the command line by typing:

-> Analysis

Once the program has started and the main Analysis menu bar has appeared select M:project:New. Into the query box that appears enter a name, without spaces, for the tutorial project, e.g. "TrialProject". Then click [OK] or press <Return>.

Now save the new project by selecting M:project:Save and click [Simple Save] at the bottom of the save project popup. At the subsequent prompts click [Yes], then [OK].

This popup window shows the directory into which the project files will be saved and gives the name of the main project XML file. This main project file gets its name from the name of the project which you just typed in. Alongside the main project XML file will be a project directory, which you were prompted about, also named after the project. It is in this directory that the majority of the project's data is stored.

Note that the next time you select M:project:Save the Save project popup window will not appear because the save already knows the location of the projects XML files. Close the Save project popup by clicking [Close].

Opening Spectra from File

To open some spectra from file go to M:Experiment:Open Spectra. In the resultant Open Spectrum popup select three spectrum parameter files to open. Do this by clicking on the line in top table corresponding to "5.spc.par", and then whilst holding down the <Shift> key click on the "7.spc.par" line. This will highlight three spectra to open.

Now enlarge the popup (click and drag the bottom edge down) so that you can see three entries in the bottom table. This table is a list of how the selected files 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 names for the three experiments by double clicking on the experiment name rows, e.g. "Expt_1", entering the name and then pressing <Return> or clicking outside the box. Set the names of the three experiments to "HSQC", "NOESY-HSQC" and "TOCSY-HSQC" respectively, or similar. The names of the spectra could also be altered, but this is not necessary here and the spectrum names being inherited from the spectrum file are 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 or UCSF) this option would have to be altered. Finally click [Open Spectra].

Now for each spectrum in turn file and referencing verification dialogues will appear. If you click [OK] on the first Verify Spectrum File Details popup you will get a warning about the data bit ordering of the file - just click [Yes] here. The next popup allows you to change the spectrum referencing, but all should be correct here so just click [OK]. Note that you can change spectrum referencing and file setup at any time after loading via M:Experiment:Edit Spectra. For the second and third spectra just toggle the "Is data big endian" option to on before pressing [OK] in the verification popups.

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].

Setting Experiment Types

After the verification there is one final popup where you specify the types of the experiments. 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 give impossible assignment options. For the HSQC spectrum set the category to "through-bond" and the synonym to "15N HSQC/HMQC" OR set the full type to "H[N]". Here the synonym is a human readable, but potentially ambiguous, name for the type of experiment whereas the full type is a precise, but often less readable, descriptor.

Set the synonym for the second experiment to "15N HSQC-NOESY" and the third experiment to "15N HSQC-TOCSY". Note that for these experiments there are two different full types with the same synonym (double click the Full Type column). For example the "15N HSQC-NOESY" can 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 experiments the defaults of H{N}_H.NOESY and H{N}_H.TOCSY are correct. When the experiment types are fully set click [Close].

Spectrum Windows and Peaks

Window Components and Views

Once the three spectra have loaded, two windows will automatically be created. One is a two-dimensional window "window1" containing the HSQC and the other "window2" is three-dimensional for the NOSY and TOCSY. Note that if you minimise a window you can get it back again by going to M:Window:window1 (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. 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. If you zoom out from your spectra so that you can see their edge you will notice a dotted line which denotes their border.

Note that you can change the way in which the arrow keys move the spectra but going to M:Other:General Options and selecting "Pan with keys track view". With this set ON the keys act as if to move the view, rather than the background.

Whilst 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. To move to a specific coordinate in depth dimension you can type a location into the "15N" box (and press <Return>) at the bottom left of the window. To centre the screen axes at a given position you can double click on the coordinates at the top bar of the window (to the right of "Strips") to get a dialog box.

Staying in window2, click on the blue triangle to the left of "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.

Window Options

There are various parameters that you can manipulate to change the way in which windows and spectra are displayed. Firstly we will make a new window which we can play with, whilst leaving the others alone. So go to M:Window:New Window. Select "1H" in the z1 pulldown menu to make a 3D window which is orthogonal to the current window. Note that such a window is capable of displaying all three spectra because the X-Y (screen) plane has 1H and 15N axes and this is valid for the HSQC as will as the 3D spectra. Toggle some of the spectra to visible by double clicking in the second column of the table and then click [Create].

We will manipulate this window via M:Window Edit Window. For the "window3" row of the to table try changing the background colour and the aspect ratio by double clicking on the appropriate columns. Also try adding '1D slices' to the axes with the "Slice visible" column in the lower table. Note that if you really don't like the scrollbars in the window you can turn them of here.

Slice panels can also be added to and removed from the windows via the mouse-menu (accessed with right click over the window). Select the options in M:Window. To change the intensity scale of the spectra in the slice panels use the <Home> and <End> keys

Setting Contours

Now we will investigate changing the contours. Firstly change the contour colours by going to M:Experiment:Edit Spectra and clicking in the "Pos. contour colours" column. To set the contour levels, within a spectrum window go to the top "Contours" option - click on the triangle to the left. Note that if you use the widget to the left you can raise and lower the contour levels for all visible spectra on-the-fly by licking on the "-" or "+" side as appropriate. To alter more of the contour settings now click on [Detailed Settings].

In the Edit Spectrum Contour Levels popup select an appropriate spectrum and set the levels as desired. You can quickly adjust the bottom most or "Base level" contour with the "/2" (divide by two) and "*2" (multiply by two) buttons. Note that any changes you make in the "Auto contour levels" panel will create the specified number of levels in a geometric series (e.g. 1, 2, 4, 8, ...), starting at the base level using the specified multiplier. If the "Add levels" option is on, the levels will be created by the addition of an increment, rather than by multiplication.

To add negative contours, set the "Negative" option on. If you wish to have a only one negative contour, for example to give an indication of truncation artefacts without negative contours generally, then select (with a mouse click and drag) the unwanted contour values in the "Manual Contour Levels" panel and press <Del> then click [Apply Manual]. When you are finished adjusting the contour levels click [Done] to close the popup.

Peak Picking and Manipulation

Focussing 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 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.

Locate the contours near the point at 8.7 ppm on the 1H axis and 118.4 on the 15N axis.  These contours represent two overlapping peaks where the extrema search will only pick one of the peaks. 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 M:Peak:Delete selected peaks.

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.

Next try moving a peak. For this select a single peak, move the mouse cursor to the desired position and press the <p> key. Alternatively you can hold the <p> while you move the mouse. If the selected peak is near, but not exactly at, an extremum you can shift it to the extremum with <P> (i.e. <Shift> + <p> )

Open a table containing a list of peaks at M:Crosspeaks:Edit Peak List. In the Peak List Editor popup select a spectrum for which you have picked peaks with the top left pulldown menu. Click on one of the peak rows so that it is highlighted in blue, then click on [Find Peak]. This will locate the peak and centre it within its window. With the table and the spectrum window both visible, select the peak in the contour window and move it to a different position. - Watch in the peak list table as the peaks ppm values are continuously updated. This is a general feature of tables in Analysis. The tables are updated when things change to reflect the immediate state of the underlying data. It is also possible to move a peak by editing its position value in the table. Do this by double clicking on a "Shift" column in the table for one of the peaks that you can see in the spectrum window. Edit the peak position for that dimension, but not so much that the peak would not be visible on screen, and press <Return> to see the peak move relative to the contours. 

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 M:Navigate:1H-1H in window 2. Note that there were two navigation options, where the second option would take you to the 1H position on the vertical axis, rather than the horizontal axis that 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:Other: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 on 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 set in 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. To remove all the strips with [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 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.

Assigning Peaks to Resonances and Spin Systems

Assigning New Resonances

Given that we now have some peaks and can move between the different spectra we will start some assignment of the peaks to resonances. Initially we will assign the peaks anonymously, that is to say we will link peaks to a resonance number, but not say which atoms the resonances comes from. Initially such an assignment is not very useful, but we can go on to link related peaks to the same resonance numbers. For example we can say that a whole column of peaks in the 3D TOCSY and NOESY and an HSQC peak are derived from the same amide resonances. When we specify which atoms the resonances derive from then all of these linked peaks will automatically be assigned.

To start an assignment choose the isolated HSQC peak at 7.1ppm (1H), 119.5ppm (15N), and with the cursor near its centre press <a> (or you can select R:Assign:Assign HQSC...). The Edit Assignment popup will appear containing two rows of tables, one for each of the HSQC dimensions. In the left most table of each row click the button [<New>], this will add the resonances [1] and [2] to the 1H and 15N dimensions of the peak. Now click [Set Same Spin System] at the bottom right of the popup. The resonance assignments will become {1}[1] and {1}[2] here the {1} annotation signifies that the resonances are both in spin system number 1, which in this instance indicates that they both belong in the same amino acid residue.

Now mark the peak (<m>) and navigate to the equivalent position in TOCSY and NOESY spectra in window2 via R:Navigate. Ensure the peaks along the marker in window2 are picked (<Shift> + <Ctrl> + left click and drag). Note that if you cannot pick some 3D peaks they may not have a maximum within the selected depth range. If this the case you can adjust the depth position or width,

Now assign one of the 3D peaks at the marked amide location: Press <a> with the cursor over peak the peak and find the Edit Assignment popup. You will see that the popup has now updated for the 3D peak and consequently there are three dimension rows. Because the peak position closely matches the chemical shift value for resonances 1 and 2 they appear in the right hand tables. We can link these existing resonances to the 3D peak by clicking on their rows in the right
hand table. When you do this the resonance annotation appears on the left hand side (and in the spectrum window) to indicate that the peak is now linked, i.e. assigned to the resonances. Note that you can remove the resonance assignment in the popup by selecting a row from the right hand side and clicking [Clear contrib].

With the 3D peaks amide dimensions fully linked we will quickly give the other 3D peaks at the same amide position the same assignment. Do this by zooming out in the window so that you can see all the peaks along the marker line, select all the peaks including the assigned one (left click & drag) then select R:Assign:Propagate assignments. This will cause the resonances assignments {1}[1] - {1}[2] to be spread appropriately across all the selected peaks.

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. The first step in this automation is to define new amide resonance and spin system identifiers for all the peaks within the HSQC spectrum. Initially ensure you have most of the HSQC peaks picked. Then select M:Macros:Initialise HSQC. This "macro" script calls the resonance and spin system assignment routines, that we used to assign the first peak, on all of the HSQC peaks. Note that the routine knows which spectrum to work with because we set the experiment type of the HSQC correctly as H[N]. You will now see that all of the peaks carry assignments of the form {x}[y] {x}[z].

Some of the peaks which you picked in the HSQC will be from NH2 groups of amine side chains. Clearly the NH2 groups give two peaks, one for each hydrogen and both have the same 15N resonance (and thus 15N chemical shift). When such pairs of peaks are processed by the initialisation macro they will be 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. Identify a pair of amine peaks (e.g. at 7.02, 110.2 ppm and 6.69, 110.2 ppm) and with the cursor over one of them open the Edit Assignment popup (<a>) and uncheck the Correlated Dims option. You will notice that although the peak is assigned to one 15N resonance (left hand side) there are two possibilities that are near identical in chemical shift (right hand side). We will now merge the other 15N resonance, which is assigned to the other amine peak to with the assigned resonance. To do this, select the row on the right hand table that corresponds to the unlinked resonance. The 15N dimension will now be assigned to two resonances, which in cases where resonances are overlapping would be valid, but here it is just a means to an end. Select one of the assigned resonances in the lower left table and click [Merge F2 Resonances] and then click [Yes]. Now we will merge the spin systems by selecting [Set Same Spin System] (you may have to do this on the other amine peak, sepending on which way the merge went) and then [OK] when prompted. Looking back at the peaks in the spectrum window you will see that they now relate to the same nitrogen and spin system.

The next part of the assignment process is to link resonances, as we did before, from the HSQC to the corresponding trains of peaks in the 3D experiments. From the menu select M:Assignment:Link Peak Lists. In the Link Peak Lists popup that appears in the ensure that window1 is selected in the Root Window pulldown menu and that window2 is selected in the pulldown menu to the right of [Add Window:].

Now  click [Add Window]. You will notice that the peaks from the HSQC are listed in the bottom table. If you click on one of the row in this table window1 will centre on that peak and the location of window2 will move to the same amide frequencies. Rearrange the positions of window1 and window2 so that you can clearly see both and the popup. Select a row corresponding to an HSQC peak that is not overlapped and click [Pick Target & Assign Root Resonancs]. 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 Targets & Assign Root Resonances] to process all of the amide positions. Note that closely overlapping amide resonances would still have to be linked by hand.

In the upper table, on the row corresponding to the TOCSY, double click on the "Assign Non-root dim?" column. Then after having linked one of the amide spin systems to the 3D experiments, click [Assign Target Non-root Resonances]. You will see that the F2 dimension of the TOCSY peaks are now each assigned to a different resonance (which we could then go on to assign to the appropriate NOESY peak).

It is important when picking peaks and assigning resonances in this automated manner that noise and artifact 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 contour base level.

Now that we have defined and linked many resonances, look in the main resonance table at M:Assignment:Resonances. You will see that all of the resonances are listed here and many functions can be applied to them. The important thing to note here is that the chemical shift of each resonance is automatically calculated from the positions of the peaks to which it is assigned. Note that a resonance assigned to only one peak will have no deviation in its shift, but those assigned to several 3D peaks will deviate as the amide peak position varies slightly. By default the chemical shift values are the average of the assigned peak positions, where each spectrum is weighted equally. However, different dimemsions of different spectra can carry different shift weightings (set at M:Experiment:Edit Spectra [Tolerances]) so that the value of a shift may be influenced more by the more precise experiments.

Defining Sequences and Molecular Systems

So far we have been linking related spectra with anonymous resonances. In the end we will assign these resonances to specific atoms, but first we must specify the molecules within our experimental sample that we may assign to. Creating a list of atoms to which we may assign has two distinct steps. The first is the specification of a molecule, usually with an amino acid sequence, that will act as a template. The second step is to build an assignable molecular system from the templates to give the atoms. The molecular system represents all the assignable molecules that may reside within the NMR sample, these include proteins, nucleic acids and small molecules. For this tutorial we make a mock molecular system with one protein chain and one small molecule. These two steps exist so that we may define a template sequence once, but have the potential to create several distinct polypeptide chains for assignment, for example if we have a homodimer.

Open M:Molecules:Molecular Systems and click [Add Mol System]. This will create the new blank molecular system to which we can add our protein chain (albeit only for part of  this tutorial). Click on the MS1 line to activate the lower section of the popup, then click [Edit Molecule Templates].
The popup which now appears allows you to create a molecule template which you can then use to create polymer chains within a molecular system. Such molecule templates determine the sequence information to make the residues of the chain. From here select [Add New Molecule]. You will then be prompted for the template name. Here you can either accept the default or enter a new name, then click [OK].

Now we will add a sequence to the new molecule template. So select the row in the top table containing the new molecule then click on [Add Polymer Residues] in the middle section. The next popup provides an input box for macromolecular sequences. First ensure [] Three Letter Codes, is selected, then cut and paste the following sequence (left mouse to select in the browser and middle mouse to paste) into the text window:

LYS ALA SER SER PRO SER SER LEU THR TYR LYS GLU MET ILE LEU LYS SER MET PRO GLN 
LEU ASN ASP GLY LYS GLY SER SER ARG ILE VAL LEU LYS LYS TYR VAL LYS ASP THR TYR   

The input sequence does not have to be perfectly formatted. You can click [Tidy] at any time to see how Analysis has interpreted the sequence. Also, you can switch between one- and three-letter codes after the sequences has been entered. If you look in the P:Ccp Codes pull-down menu you will see all of the residue codes that are currently available, many of which are modified amino acids.
Once the sequence has been entered, click [OK]. The molecular with all its associated atoms, bonds etc. will take a short time to build. When it is done the new molecule will appear in the Molecule Templates popup. Note that for new templates you can double click on the residues in the table to change them. Try this for a LYS to set its Descriptor to indicate a different protonation state.

Just for fun, we will now add a small molecule to our project. This could be part of the first molecule we made, i.e. linked to the protein in some way. However, here we will make a new molecule with [Add New Molecule] and after accepting the name click [Add Other Compound]. The Select Small Molecule popup will open. Here choose [DNA] at the top then in the right hand table select a compound, perhaps an unusual nucleotide, to display an idealised structure in the left hand panel. You can rotate the cartoon compound by clicking and dragging with the middle mouse button. Using <Shift> while dragging up and down with the middle mouse button will change the zoom level.
With any compound selected click [OK] to enter it into your second molecule template. Now [Close] the Molecule Templates popup. The templates with the sequence and compound you just entered will now be available form the Edit Molecular Systems popup.
Returning to the Edit Molecular Systems popup you will see the name of your newly created sequence template is displayed at the bottom of the central panel. Now click on [Make chain from template:] just to the left of the template name. You will be prompted about whether aromatic atom sets are equivalent due to rotation - just click [Yes]. Analysis will now build a polymer chain within the molecular system with a sequence derived from the template. If you click [Make chain from template:] again you will build a dimer of the same molecule with A and B chains.
To view the new polymer chain have a look at M:Molecules:Atom Browser. You can scroll through the sequence and by toggling the [H], [N] etc. you can display all of the assignable sets of atoms.

Assigning Resonances to Atoms

Once we posses sufficient information, the resonances to which peaks are linked may be assigned to specific atoms. For the purposes of this tutorial we will cheat with prior knowledge and link spin system number 1 "{1}" (HN at 7.1ppm 1H; 119.5ppm 15N) to Leucine 21. Find the HSQC peak that we first assigned by going to M:Peaks:Peak Lists:{Peak Table} and selecting the HQSC from the pulldown menu. Click on the row of the {1}[1] {1}[2] peak and click [Find Peak]. In reality we could choose any peak that we have assigned to the spin system and connect its resonances to 21 Leu Hn and 21 Leu N.

With the cursor over the centre of the peak press <a>, (or select R:Assign:Assign "spectrum name"  from the right mouse menu). Once the popup appears select the first hydrogen resonance (to left table) and then click on [Assign {1}[1]]. This will bring up the Atom Browser popup. Here, make sure that the hydrogen and nitrogen atoms are displayed (click [H] and [N]), then scroll down to click on Hn  in the 21 Leu column. Return to the Edit Assignment popup and you will see that the resonance is now annotated as 21LeuHn and that the 15N resonance has been assigned. The 15N resonance was assigned because it is directly bound to the 1H resonance - Analysis knows this because we have set the experiment type to H[N]. Return to the Atom Browser and you will note that the atom sets you have chosen in the browser will change colour to reflect their assigned status.
Now that the Resonances are connected to atoms you will see that the peak annotation has changed to 21LeuH,N , not only for the peak we directly assigned, but also for all of the 3D peaks, that are linked to the same resonances. If you look in M:Resonance:Spin Systems you will also see that the spin system has been unambiguously assigned to the leucine residue by virtue of our choice of atoms.

During such atomic assignments it is may be useful to have representative chemical shift values for the possible atomic assignments. These can be accessed at any time at M:Resonance:Ref Chemical Shifts  by selecting the appropriate residue code and atom types (heavy atoms or hydrogens).
Next we will make an inter-residue connection. In window2 mark (<m>) the NOESY 21 Leu derived peak just below the diagonal at 7.4 ppm. Returning to window1 you will note that two vertical lines are present on the HSQC, one through the 21 Leu spin system and the other at the Hn shift of a nearby residue. Looking at the HSQC along the line at 7.4 ppm we see that two peaks are intersected, although neither is dead centre (the marked NOESY peak is distorted by the diagonal peak). Cheating again, the peak at 111 ppm in the 15N dimension is the residue N-terminal to 21 Leu.

We will bring the 3D TOCSY and NOESY peaks of this neighbouring spin system up in a new window strip: In the 2D windows select both the 21 Leu HSQC peak and the peak at 7.36, 111.0 ppm by (left click and drag with <Shift>) so that they are highlighted by surrounding squares. Now in the right mouse menu select M:Strip:Peak Location Strips:1H - 15N in window2. Alternatively we could have manually added a new strip to window2 and, after having activated the destination strip, used the same navigation function as before in the second HSQC peak.

In the new strip of window2 you will see the neighboring spin system with its return Hn-Hn NOESY peaks. The spin system in this strip is 20 Gln. If you have not picked and assigned this part of the spectrum automatically, pick the appropriate peaks in the spin system, assign one of them to the resonances in the HSQC spectrum, then propagate the assignments to the remainder. Then assign one dimension of one peak to the H or the N of Gln 20.

Finally we will assign the connecting Hn-Hn crosspeaks. In the 21 Leu strip select R:Assign:Assign "Spectrum name" over the marked peak (just below the diagonal). In the assignment popup you should select the 20 Gln H resonance for the second hydrogen dimension. Now assign the reciprocal Hn-Hn NOESY peak in second strip. With the assignment complete, select M:Charts:Assignment Graph. Go to the {Options} tab and click on the button named after your 3D NOESY experiment. You will then see the connection you have just made to the spin systems in the graphical display. 

Loading Existing Projects and Save Options

Quit the current Analysis session, saving the extant project as you see fit.
Now at the Linux shell command line type:

-> ls 

And press <Return>. You will see that we have an XML file present in the current directory called TutorialProject.xml. This is the main file for the project that we are about to open. However, this file does not contain all of the project data. Indeed, the main XML file contains relatively little information. What it does do is specify the location of many other files that allows all of the information to be brought together when the project is opened and the data accessed. It is worth noting that these other XML files, which we shall see in a moment, are only loaded when required, when we access the data. In this way Analysis only loads only the files it needs. Conversely, when a project is saved, only the files corresponding to modified data packages will be written.

At the command line now type:

  -> tree TutorialProject/

This command will list all of the contents of the project directory. You will see that there are several XML files within the directory structure. Each file corresponds to a different type of data that may be loaded into the project. Thus the project XML file must always have its project directory present, with its other XML files.

Start Analysis with the existing project by typing:

  -> Analysis TutorialProject.xml

The project will take a moment to load, and when it is done several spectrum windows will appear on screen.

Initially open M:project:Save As and click on the "Urls", "Storages" and "Data locations" tabs to open three tables. The contents of these three tables illustrate the locations of the various data that are loaded as part of the project.

The first "Url" tables shows all of the directories where Analysis will look for its data. Note that some of these relate to the current project directory within the home directory; these are where all the user editable data are stored. Some of the data are stored in a directory that is part of the CCPN installation. These contain reference information that the user cannot change, for example the chemical element description.

The second, "Storages", table shows the location of the CCPN XML format data files. Each XML file represents a package (or sometimes part of a package) containing related data. For example NMR data is stored in the file ccp/nmr/Nmr.xml and the molecule template information is stored at ccp/molecule/Molecule.xml. These two locations are relative to the project directory because they use the project directory URL (number 2). The third table, "Data locations", shows where all of the spectrum data files are located. Again the file paths are relative to the URL used.

One very important point to note is that when a project is copied all of the absolute paths within the project must be updated accordingly. If the project files are copied via the operating system then the URL paths will not be updated and Analysis will attempt to load the data from its original locations. This can end up with two separate projects using the same XML files and thus serious problems.The recommended way of copying or moving a project is to use the Save As option, choosing a new project file and directory whilst ensuring that the "Correspondingly change related paths" is set on.

Working With Variable Chemical Shifts

Setting up and linking a new shift list

Here we are going to set up a new shift list for the second HSQC experiment present in the demonstration project. Given that the experiment is already loaded, all we need to do is go to the M:Experiment:Edit Experiments and in the Shift List column double click on the cell corresponding to HSQC_II. In the pulldown menu that appears simply select <New>. You will see that a second shift list is now listed. Now whenever an assignment is made in HSQC_II the shift remains separate from the other spectra. Indeed, any resonances that were already connected to HSQC_II before its shift list was changed will have its shift recalculated in two separate groups.

Assignment of HSQC_II, with its separately curated shifts proceeds pretty much as assignment normally does, with the only major difference being that for a new shift list (i.e. without any assignments under it) there will be no resonance possibilities that can appear when assigning a peak. - The only known shifts are distinctly different, representing different conditions.
To assign HSQC_II, even though we don't have shifts set, we can say that certain peaks in the two HSQC spectra are equivalent if we can see how they have moved under the different situations. Accordingly we can select a peak in the first HSQC and the corresponding peak in HSQC_II and propagate assignments. That is to say that the peak dimensions link to the same resonances (and hence atoms), even though things have moved. Try this for the peak at 7.2,119.0 ppm in HSQC and its equivalent at 7.3,120.0 ppm in HSQC_II. Use the mouse with a left click to ensure that these peaks (and only these peaks) are selected. Then in the right mouse menu select R:Assign:Propagate. Note that this function usually checks to ensure resonance positions are within tolerances, but here with the two shift lists it cannot so the connection is made anyway, which is exactly what we want.
You may like to repeat the above procedure for several HSQC peaks and their counterparts. With a few resonances represented in the second shift list have a look at the resonance browser M:Assignment:Resonances to see evidence of these links. In the resonance browser note that you can choose between the two different shift lists in a pulldown menu at the top, i.e set the ShiftList: pulldown to "ShiftList 2". Note that when the shift list changes the resonance positions shift and the number of peaks linked also changes. Apart from the resonances Analysis also provides direct access to the shift measurements. Looking in the menu at M:Measurements:Measurement Lists, you will see that there are tables of the shift values corresponding to each list. Note that other types of value like T1 rates, or Hetero-NOE values will also appear as measurements in these tables if they are calculated.

Copying peaks between related spectra

Propagating assignments between peaks by manually selecting them, although sometimes necessary, can be tedious if you have large number of peaks that are equivalent and have closely matching positions. In this instance Analysis provides a utility to compare all of the peak positions in two related spectra and transfer the resonance assignments efficiently, whilst still leaving sufficient scope for human intervention.
In this exercise we will transfer the alpha carbon assignments from the peaks in the HSQC experiment to the HSQC_II experiment. In the main menu we select M:Crosspeaks:Copy Peak Assignments. In the Copy Peak Assignments popup ensure that the HQSC experiment is set at the Source Peak List and that the HQSC_II is set as the Target Peak List.
In the top table you will see a list of the source peaks, together with an indication of how many destination peaks they match within the given tolerances. Note that when the Follow Peaks? option is set clicking on a source row will cause peak to be highlighted in a relevant window (window1). Also, selecting the source will display the potentially matching destination peaks in the lower table. Clicking on a target row will, as before, highlight the relevant peak.
This utility can be used in one of two ways. One can manually choose the correct destination for each source peak and [Assign Selected Target], or if you trust the completeness and accuracy of the destination one can automatically assign the closest destination peak to each source one with [Assign Singly Matched].

Linking Sequential Polypeptide Spin Systems

We will now look at the linking of sequential protein backbone spin systems using triple resonance experiments. Select M:Assignment:Link Seq Spin Systems. Now make sure that window2 is visible and expanded along one side of the screen. With the mouse over window2, select R:Window:Clone. This will produce an identical window containing the triple resonance backbone spectra. Now arrange window2 and the new window4 so that they are side by side, with window2 shrunk to about one quarter the width of window4 (both windows should be as tall as possible). Then switch off the HNCA spectrum in window2, via the "Spectrum" tab, so that only the purple HNcoCA is visible.

In the Link Sequential Spin Systems popup select window2 as the "Query" window and window4 as the "Matches" window. Now click on the [HNcoCA:111] button in the top panel and the [HNCA:110] button in the "Matches" panel. This setup means that we are going to compare specified 13C peak positions in the HNcoCA experiment with potentially matching peak positions in the HNCA experiment.

The rational here is that the HNcoCA experiment's peaks have the carbon shift of the preceding alpha carbon along the polypeptide chain at a given amide location, where the HNCA has both the intra-residue and preceding alpha 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 derives from the same resonance as an intra-residue peak of the HNCA. Note that you can readily use other backbone experiments like HNcoCACB, HNCO etc. within this same system and that this tutorial only used the alpha carbon experiments for simplicity.

In the "Spin Systems" panel click on the row that corresponds to spin system 90 You will see that the two triple resonance windows move to new locations. The location of window2 is at spin system {90} as found in the HNcoCA. The window with the HNCA, window4, has moved to the position of any peaks that match the alpha carbon frequency. In this case there is only one good match, which corresponds to spin system 89. Click to highlight the row corresponding to spin system {89} in the matched peaks panel, and then click [Set Seq Link]. You will now see that the tables of the popup update to show that {90} is set as "i+1" of {89}. Also note that in the spectrum window the peak annotations of the aligned  HNcoCA and HNCA peaks have changed to illustrate that they are both assigned to the same 13C resonance. Now click [Goto i-1], which will repeat the carbon shift matching, but this time from a sequence position one earlier in the sequence. This time spin system {89} matches two HNCA peaks, but we know that the spin system {88} peak is the correct one because the 76Lys peak is already assigned and because the 76Lys HNCA peak is not an intra-residue one, i.e. it is coincident with the 76Lys HNcoCA peak. [Set Seq Link] as {88}, then [Goto i-1]. Repeat the sequential linking procedure for {88} & {170}, {170} & {160}. Now we could then correctly link {160} to 7Lys, but instead we will simply assign the spin system {160} to the residue 8Ser.

With the cursor over the spin system {160} HNcoCA peak select <a> and then in the Edit Assignment popup click on the F1 assigned resonance row {160}H[81] at the top left, then click [ Assign {160}H[81] ] on the left of the bottom buttons. In the Browse Atoms popup that appears, ensure that the element [H] is displayed (click the button to get the green hydrogen assignment options) and select the Hn atom for 8 Ser. You will see that not only is 8SerHn assigned (i.e. the atom option goes dark green), but the atoms in the residues which we just connected sequentially are also assigned. Look through the spectra and the Link Sequential Spin Systems 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. For example, the amide proton in spin system {170} was set as type "H", thus when {160} was assigned to 8Ser, spin system {170} which we set as i+1 of {160} gets assigned to 9Tyr and Analysis knows that the proton must be 9TyrH.

Structures and NOEs

The final part of the exercise is to look at how we can use Analysis to generate distance restraints that may be used in a structure calculation and how we can pass intermediate structural information back into Analysis to help with NOE peak assignment.

Making Distance Constraint Lists
To make a list of distance constraints from the assigned peaks in an NOE peak list first go to M:Structure:Make Dist. Constraints. You will see that there are three panels. In the General options change the spectrum to N-NOESY:182. We can leave all of the other parameters alone for demonstration purposes. The Distance Params section would allow us to specify how the NOE peak intensities relate to the distance bounds of any generated distance constraints. The default method is to calculate a target distance as peak volume raised to the power of -1/6 multiplied by some scaling factor, such that the reference intensity (in this case defaults to the peak list's average volume) exactly corresponds to the reference distance (in this case 3.2 Angstroms). The upper and lower bounds of the distance constraint are calculated as fractional changes from the calculated target distance (default is 20% above and below) while observing absolute minimum and maximum values for the bounds (1.72 & 8.00 Angstroms respectively by default). The Residue Ranges section would allow you to make only constraints for specific assigned regions of your molecule.

To calculate constraints for the selected peak list simply press [Calculate constraints]. After a short pause you will see the Current Constraint Sets popup appear. This shows that you have one constraint set (a way of grouping related constraints and violations) containing one list of about 500 constraints. Click on the row of the constraint list in the central table and then click [View Constraints]. Note that you can also get to this point via M:Structure:Constraints.

The constraints popup will appear and in its table you will see the constraints listed, mostly as green coloured rows. Note that constraint number 3 has a second grey row. This indicates that constraint 3 is ambiguous and represents a possible connection between two different pairs of 1H resonances.

There is a second common way to generate distance constraints, which is to match the chemical shifts of resonances to NOE peak positions, thus generating potentially highly ambiguous distance constraints. Such constraints would typically be filtered to select only the correct contributing resonance pairs, by iterative structure generation and violation analysis in a program such as ARIA or CYANA.

To generate distance constraints by shift matching select M:Structure:Shift Match Dist. Constraints. The resulting popup has some similarities to the first constraint generating system, but there are two extra panels; to say which chemical shift ranges you would like to match to NOE peaks and what the tolerances for such shift to peak position match are. Typically you would set the tolerances based on the widths of your NOESY peaks and exclude a small chemical shift region around the water signal.

Select the N-NOESY spectrum as before and click [Calculate constraints], accepting the default parameters. Note that we are using the same constraint set as last time. You always have the option to put new constraints in a new constraint set, and indeed you should always use a new constraint set if any of the atomic assignments have changed. Each constraint set uses a frozen copy of the resonance-to-atom assignments at the time its restraints were created, so that you always have a record of what was really constrained, even if the resonances subsequently point to different atoms. By the same token, if you did not use a new constraint set after atomic assignments were changed the constraints would refer to the atoms from the old assignments not the new ones.

In the Constraints popup, which is hopefully still open (M:Structure:Constraints otherwise), select constraint list 2 from the
pulldown menu. You will now see that in contrast to the first list these constraints are highly ambiguous, with several possible resonance pairs for each constraint. If you click on the row of the first constraint "1:0" and then [Assign Peak], the Edit Assignment popup will appear to show the assignment possibilities and status for the peak that gave rise to this constraint. This llustrates that in the second dimension of this NOESY peak there are several resonances with shifts that closely match the peak position for that dimension. Note that the peak itself is not assigned to any of these, but that the correct contributing resonances may be assigned after a structural model had been calculated. Referring back to the Constraints popup, if you now click [View peaks] you will see that the NOE peak appear in a table from where you can then select the peak's row and click [Find Peak] to locate it in an appropriate window. Note that  a constraint may be linked to more than one peak, for example where there are symmetry related peaks that correspond to the same close resonance pair.

Loading Structures
Next we will load a protein structure, corresponding to the spectra that are present in this tutorial. This structure has been generated using the program ARIA, but in general such a structure could from an early calculation run or an homology model. To load the structure select M:Struture:Edit Structures, click on [Import] and select the file "G1.pdb" and finally [OK], when the structure and its coordinates appear in the Current Structures popup we know that all had loaded and we can click [Close].

Referring back to the Constraints popup, you are now able to select any constraint rows (using <Shift>/<Ctrl> keys) and then
click [View Selected On Structure] to illustrate graphically where on the loaded structure the constraints apply. The controls for the structural viewer are as follows:

• Rotate with middle-click & drag.
• Zoom with the mouse wheel, or middle-click, <Shift> & drag.
• Move with middle-click, <Ctrl> & drag.

The mouse right-click is reserved for a menu and the left-click will be used for selection (in the future).

The same sort of functionality is present in the Edit Assignment popup. - If you go back to the NOESY spectrum in Window 4, and assign a peak (by pressing <a> with the cursor over a peak), you can see assignment possibilities via the [Show On Structure] button. Also note that because we now have a structure the Edit Assignment popup will show distances between one peak assigned 1H resonance and the 1H possibilities in another peak dimension.

To make the whole process of comparing NOE peaks to a structural model efficient, Analysis has the M:Assigment:Link NOEs option. Select this from the menu and in the resulting popup set the NOE Peak List pulldown to N-NOESY, and set the Mark Toggle on (red). By clicking on the rows NOE peaks table you will see that several things happen automatically: The view of the selected windows (in this case Window 4) zooms and marks the selected peak; the graphical structure view highlights the possible atom connections that the peak could represent; and the lower table of the Link NOE Resonances popup shows the structurally possible, shift-matched resonance pairs ordered in terms of shift and geometric distance. In the lower table clicking on an assignment row and [Assign Selected] sets that assignment for that peak. Also, selecting [Predict Peaks] will use the entered structure and the known chemical shift values to predict the positions of peaks, near to the selected real peak, which correspond to close resonance pairs. These artificial peaks are labelled with the structural distance and are contained in an entirely separate list to the real peaks (so nothing is contaminated and they can be removed easily).

Calculating Structural Violations

Lastly, given the structure that we have loaded into Analysis and the distance constraints that we generated from the NOE peak list. We can investigate the compatibility between the structural model and the constraint distances to find violations.

Return to the Constraints popup (M:Structure:Constraints) and select constraint list 1 from the pulldown menu and click [Calculate Violations]. After a short pause you will see that several of the constraint rows turn red. These indicate that the constraint distances are inconsistent with (i.e. violate) the loaded structural model. The peak assignments from which this constraint list was generated obviously needs some work, so that hopefully a new and better structural model can be generated. Many of the violations are small,
but some are large, taking for example constraint 12 ("26SerH - 20LeuHba"), the violation is 1.6 Angstrom. If you select this row and click Assign Peak you will see that an assignment in the F2 dimension, to 22GluHba, is probably missing: the chemical shift "delta" is small and the distance given the structure is close. Clicking on the 22GluHba row in the Edit Assignment popup will ambiguously assign the peak. Hopefully when the next set of constraints is generated (and maybe a new structure calculated) the peak will represent a shared contribution from two close 1H pairs, and the 26SerH - 20LeuHba constraint will no longer appear too short.