The first step in an NMR spectrum assignment is to set up the database. The Analytical module organizes an NMR research project in the FELIX database management system into an entity. To start an analytical assignment project, therefore, you must first define this entity.
With the Project menu items you can build a new project, open an existing one, show it, delete it, add new experiments, or define the molecule whenever necessary.
The project entity is built using the Analytical/Project (<Alt>-lp) menu item and contains the NMR spectra (preferences, file names, etc.) and may contain information about the molecule. The project entity contains several other entities, such as patterns and frequencies, which can be defined in the building stage.
You do not need to define a molecule when starting the Analytical module - you can read a molecule in at any time (ISIS/Draw .mol, Accelrys .car or XPlor .pdb format).
If the project already exists (that is, if it was built in a previous session) Analytical/Project opens it; if FELIX fails to find the name of the project on its own, you need to enter the name in the control panel.
Opening an existing project brings up the Project table, which shows the contents of the entity in a spreadsheet.
Once the project is built, you can specify the spectra that were measured on the molecular system. You can also redefine the plotting or other attributes of the experiment and can delete an experiment from the database. The first time you use this menu item the Experiments table is created. From that point on, you use the table to add, delete, change, or switch between experiments.
Selecting the Experiment/Select menu item (<Alt>-xs) from the Experiments table quickly retrieves spectra from a project entity, so that you can display contour or density plots from related spectra with the same limits. When you switch from one spectrum to another, all parameters are automatically reloaded. This makes it unnecessary to manually switch entities (that is, the peak entity is reloaded after changing spectra).
To add an experiment to the project, select the Experiments/Add (<Alt>-xa) menu item from the Experiments table. During experiment setup, you must specify your preferred plotting parameters, which are saved in the project entity.
You can add the currently displayed 1D or 2D spectrum to the project or open a saved spectrum and add it to the project.
To insure proper functioning, the spectra included must be referenced in ppm. You can also specify various parameters of the spectrum, such as temperature, pH, solvent, the measured nuclei, the tolerance for frequency collapsing, the type of experiment (J or NOE), the number of J steps for J-type experiments, mixing time for NOE, and peak entity.
You can define up to twelve spectra in one project.
With the Experiment/Change Attributes (<Alt>-xc) menu item from the Experiments table, you can redefine plotting or other parameters of an experiment at any time.
You can delete an experiment from the project entity using the Experiment/Delete (<Alt>-xd) menu item from the Experiments table. This is useful if you work with more than the currently available twelve spectra in your project.
Although it is not necessary to have a molecule in order to properly use most of the menu items in the Analytical module, you still may want to read the molecule file. The most obvious reason is that you can do the peak assignment through the molecule display in FELIX. To read in a molecule, use the Analytical/Read Coordinates (<Alt>-lo) menu item.
After the molecule is read, it is displayed in interactive mode. You can rotate, translate, zoom, or reset the display. If the display is planar (i.e., all z coordinates are zero) you can rotate the molecule only in the screen plane (around the z axis). You can do this by moving the cursor while pressing the <Shift> key and left mouse button.
The Analytical/Label menu item (<Alt>-ll) is used to show an atom on the molecule by entering the atom name in the control panel. If the atom is found, its name is displayed on the molecule; otherwise a message is shown in the text window that the atom was not found. If Label in the control panel is set to Off you can clear labels; if the Atom name is set to wildcard (*) then all labels are cleared.
With the Model/Color (<Alt>-lr) menu item you can change the coloring scheme of the molecule. For example, you can change the color of all protons or all amide protons to yellow.
The Analytical/Frequency Clipboard (<Alt>-lf) menu items deal with a clipboard of frequencies (or frequency clipboard). A clipboard is a data structure that allows you to store and manipulate an array of chemical shifts and associated nuclei. This clipboard is mainly used for picking spin systems manually or manipulating a collection of frequencies copied from prototype patterns. You can also display it as lines through 2D plots; and tiling, or strip plots can be spawned from it.
Before you start picking a spin system manually, you must first make sure that there are no frequencies in the clipboard, using the Analytical/Frequency Clipboard/Zero Clipboard menu item (<Alt>-lfz>). This allows you to delete all frequencies from the list.
The Analytical/Frequency Clipboard/Add One menu item (<Alt>-lfa) allows you to add a frequency to the frequency clipboard by picking a chemical shift from a plot of a spectrum.
The Analytical/Frequency Clipboard/Delete One menu item (<Alt>-lfd) allows you to delete frequencies from the frequency clipboard using a list.
The Analytical/Frequency Clipboard/Swap Two menu item (<Alt>-lft) allows you to exchange the order of two frequencies in the frequency clipboard.
The Analytical/Frequency Clipboard/Remove Duplicates menu item (<Alt>-lfm) allows you to purge frequencies from the frequency clipboard that are considered duplicate entries, based on a chemical shift tolerance that you define.
The Analytical/Frequency Clipboard/Compare Frequencies menu item (<Alt>-lfc>) allows you to compare the current collection of frequencies with each prototype pattern. This allows you to see how many fuzzy similarities there are. You need to specify the target to compare against (prototype patterns). If the fuzzy similarity exceeds the Min Similarity parameter, you are notified. Use the Tolerance menu item for comparison. This function is useful when trying to find out if the frequencies in the frequency clipboard constitute a novel spin system.
With this menu item (<Alt>-lfo), you can copy a clipboard to a new prototype pattern.
The Analytical/Frequency Clipboard/Copy Proto To Clipboard menu item (<Alt>-lfp) allows you to choose a prototype pattern as a source of frequencies for a frequency clipboard.
With this menu item (<Alt>-lfv) you can list the actual contents of the frequency clipboard in the text window.
The Analytical/Frequency Clipboard/Sort Clipboard menu item (<Alt>-lfs) allows you to sort the frequencies in the clipboard in ascending or descending order.
The Analytical/Frequency Clipboard/Tile Clipboard menu item (<Alt>-lfi) allows you to spawn a tile plot from the frequencies in the actual clipboard.
The Analytical/Frequency Clipboard/Strip Plot Clipboard menu item (<Alt>-lfr) allows you to spawn a strip plot from the frequencies in the actual clipboard.
The Analytical/Frequency Clipboard/Draw Clipboard menu item (<Alt>-lfw) allows you to draw straight lines on the actual plot along the frequencies from the clipboard.
The Prototype Pattern subsection of the Analytical pulldown contains menu items relating to the prototype pattern entity. The prototype patterns are the generally rough spin systems that are the results of automated spin-system detection.
The Analytical/Collect Prototype Patterns (<Alt>-lc) menu item contains options used in automated spin-system detection. In the Analytical module, you can systematically search 2D spectra (i.e., COSY-type only, TOCSY-type only, HMQC and COSY, HMQC and TOCSY, HMQC and COSY and TOCSY, HMQC and COSY and HMBC, or the HMQC and TOCSY and HMBC spectra-based method).
The TOCSY type can refer to any homonuclear 2D experiment in which magnetization is transferred through J coupling, and the correlation extends over more than three bonds (e.g., RELAY or DOUBLE_RELAY experiments).
The algorithm used by this option is based on intra- and inter-spectral chemical shift comparison. What follows is a short description of the options in this menu item's control panel.
You can choose among seven common combinations of 2D experiments as the Method to search prototype patterns.
Analytical uses the Interspectral Tolerance for H (in ppm) to decide if a candidate 1H frequency already belongs to the prototype pattern. This tolerance should reflect the 1H chemical shift variations expected among different spectra.
Analytical uses the Interspectral Tolerance for C (in ppm) to decide if a candidate 13C frequency already belongs to the prototype pattern. This tolerance should reflect the 13C chemical shift variations expected among different spectra.
Allows you to graphically edit the frequencies of a prototype pattern.
Allows you to edit the prototype pattern, delete frequencies, assign spin types, and perform other manipulations.
Deletes one prototype pattern.
Deletes all prototype patterns.
The Analytical/Zoom Prototype Pattern (<Alt>-ln) menu item is useful when you want to visually inspect the spin systems resulting from an automated detection run. Using this menu item, you can define which prototype pattern's frequencies should define the region. Selecting a prototype pattern number from the Source list and then clicking the Show button fills in the Frequencies list with the frequencies of that prototype pattern. If you then select the desired Orientation, specifying which frequency should be at which dimension, then specify the required ranges, you can bring up the region by selecting OK.
Most of the visualization menu items useful for analyzing prototype patterns are accessible through the prototype pattern table. The table is usually opened using the automated collection menu items but you can open it using the Edit/Prototype Patterns (<Alt>-ep) menu item. Before you can execute any menu items you need to select one or more rows in the table, depending on what you want to do.
You can visualize a prototype pattern by drawing straight lines on the actual spectrum along the frequencies of a particular prototype pattern. First you need to select two protos from the table and then select the Action/Draw (<Alt>-ad) menu item or click the Draw icon in the Protopatterns table. Using the View/Draw Frequencies or the Clear Frequencies menu item in the right-mouse menu, you can then turn these lines off. To set the colors you can use the Preference/Draw menu item in the Protopatterns table.
The Action/Tile Plot (<Alt>-at) menu item is used to define a tile from any pair of prototype patterns. You first need to select two prototype patterns by clicking two rows in the table. Then you select the Action/Tile Plot menu item or click the Tile Plot icon on the Protopatterns table icon bar. Tile-plot preferences can be set with the Preference/Tile Plot menu item in the Protopatterns table. To turn off the tile plot, use the View/Plottype/Tile/Strip Plot menu item.
The Action/Strip Plot (<Alt>-as) menu item from the Protopatterns table is used to define a strip plot from a specific prototype pattern. First you need to select a prototype pattern by clicking the desired row in the table. Then you select the Action/Strip Plot menu item or click the Strip Plot icon on the Protopatterns table icon bar. Strip plot preferences can be set in the Preference/Strip Plot menu item in the Protopatterns table. Use the View/Plottype/Tile/Strip Plot menu item to turn off the strip plot.
The ProtoPattern/Add Frequency via Cursor menu item (<Alt>-nf) in the Protopatterns table allows you to graphically add a frequency to the selected prototype pattern.
The Protopattern/Delete Frequency via Cursor menu item (<Alt>-nd) in the Protopatterns table allows you to graphically delete a frequency from the selected and drawn prototype pattern.
The ProtoPattern/List Membership (<Alt>-nl) menu item in the Protopatterns table is used to find the particular peak to which a prototype pattern belongs, if any. The results are listed in the text window, for example:
Peak # D1 D2
------ -- --
53 9.3042 121.3826
Proto D1 Distance Proto D2 Distance
----- -- -------- ----- -- --------
5 9.31 0.580025e-02 5 121.425 0.0424
The ProtoPattern/Draw All Protopatterns menu item (<Alt>-na) in the Protopatterns table allows you to draw all prototype patterns in the currently displayed region.
With the Analytical/List Peak (<Alt>-lt) menu item you can query the database for a particular peak's positions, assignment status, and membership in protopatterns.