Assign user interface


Project menu items

The first step in NMR spectrum assignment is to set up the database. The Assign module organizes an NMR research project in the FELIX database management system into an entity. To start an assignment project, therefore, you must first define this entity.

Note: None of the Assign menu items work until a project is opened.


Assign/Project

Using the set of Project menu items you can build a new project, open an existing one, show it, delete it, add new experiments, read the library, or redefine the molecule whenever necessary (for example, when assignment and refinement is done in an iterative fashion, new assignments are made after a refinement step, so the redefinition of molecule coordinates is important).

The project entity is built using the Assign/Project (<Alt>-ap) menu item and contains information about the molecule or complex under investigation and the NMR spectra (preferences, file names, etc.). The project entity contains several other entities, such as patterns and frequencies, which can be defined in the building stage.

For Assign to function properly, you should specify the name of the molecule or complexes (MSI .car or XPlor .pdb format). This file is read in and used throughout the whole module.

If the project already exists (that is, if it was built in a previous session) this menu item will open it. If FELIX fails to find the name of the project on its own, you need to enter the name in the control panel.

Note: Even though nothing prevents you from defining multiple projects in a database, it is a strongly recommended that you open a separate database for each project.

Opening the project opens the Experiments table, which enables you to quickly switch between spectra (see the "The Experiments table-Assign/Experiment" section below).

If you have already opened the project, using this menu item again shows the entity in a spreadsheet table.


The Experiments table-Assign/Experiment

Once the project is built, you can specify the spectra that were measured on the molecular system under investigation. You can also redefine the plotting or other attributes of the experiment, and you can delete an experiment from the database. The first time you use this menu item, the Experiments table is created and displayed. From that point on, you must use the table to add, delete, change, or switch between experiments.

Experiment/Select

Accessing 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).

Experiment/Add

To add an experiment to the project, select the Experiments/Add (<Alt>-xa) menu item from the Experiments table. During the experiment setup, you must specify your preferred plotting parameters, which are saved in the project entity.

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 order of measurement, the folding, the type of experiment (J or NOE), the number of J steps for J-type experiments, mixing time for NOE, and several other items, such as the peak and volume entities.

You can define up to 12 spectra in one project.

Experiment/Change Attributes

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.

Note: If you redefine the plotting parameters outside this menu item, the effect of that redefinition remains as long as you do not chose a different experiment or exit the program.

Experiment/Delete

You can delete an experiment from the project by using the Experiment/Delete (<Alt>-xd) menu item from the Experiments table. This menu item is useful if you work with more than the currently available 12 spectra in your project. (For example, after you are done with spin-system assignment using a couple of spectra, you can delete them and add new ones for which you want to make peak assignments-typically these are NOE-type spectra.)


Assign/Define Library

FELIX also provides you with a library, pd.rdb, which was compiled using data from the literature. You can (and really should) read this library using the Assign/Define Library (<Alt>-al) menu item. This library is an ASCII file and contains information concerning the frequent residue types that occur in biopolymers. Currently it contains information about standard amino acid residues and deoxynucleotides. If you want to work with ribonucleic acids you should read in the rna.pdb file. You can edit these library files, add new residue types, or create a whole new library, but be sure to maintain the library syntax. Also, it is important that you maintain the biosym.alias file if you add new residue types.Please see Chapter 3., Tasks for more information.

The following is an example item from the library:

! ASN
!
! H - N H2 Od
! | | |
! H - Ca - Cb - Cg - Nd - H2
! |
! O = C
!
RESIDU ASN N
RESATM H HN 1 8.16 0.62
RESATM H HA 2 4.74 0.30
RESATM H HB1 3 2.74 0.32
RESATM H HB2 3 2.88 0.27
RESATM H HD21 7 7.75 0.32
RESATM H HD22 7 6.99 0.55
RESATM N N 8 123.0 5.0
RESATM C CA 10 53.0 2.0
RESATM C C 9 150.0 50.0
RESATM O O 15 0.0 999.0
RESATM C CB 11 38.0 3.0
RESATM C CG 12 100.0 999.0
RESATM N ND2 16 125.0 50.0
RESATM O OD1 17 100.0 999.0
! NH AH B1 B2 D1 D2 N A C O Cb Cg Nd Od
CONECT 0
CONECT 3 0
CONECT 4 3 0
CONECT 4 3 2 0
CONECT -6 -5 -4 -4 0
CONECT -6 -5 -4 -4 2 0
CONECT 1 2 3 3 5 5 0
CONECT 2 1 2 2 4 4 1 0
CONECT 3 2 3 3 5 5 2 1 0
CONECT 4 3 4 4 6 6 3 2 1 0
CONECT 3 2 1 1 3 3 2 1 2 3 0
CONECT 4 3 2 2 2 2 3 2 3 4 1 0
CONECT 5 4 3 3 1 1 4 3 4 5 2 1 0
CONECT 5 4 3 3 1 1 4 3 4 -5 2 1 2 0
ENDRES


Assign/Read Coordinates

Although it is necessary to read in a molecule at the stage of building the project, you still may want to reread the molecule file. The most obvious reason would be that the starting model was just a linear chain, and at one point you may have a refined model, so you must reread the molecule into FELIX. To do this, you can use the Assign/Read Coordinates (<Alt>-ao) menu item, since the number of atoms was not changed.


Manual spin-system picking-the Assign/Frequency Clipboard menu items

The Assign/Frequency Clipboard pullright (<Alt>-af) of menu items deals with the 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 (which then can be promoted to patterns) or for manipulating a collection of frequencies copied from patterns or prototype patterns. You can also display it as lines through 2D plots, and tiling, or strip plots can be spawned from it.


Assign/Frequency Clipboard/Zero Clipboard

Before you start picking a spin system manually, you must first make sure that there are no frequencies in the clipboard, using the Assign/Frequency Clipboard/Zero Clipboard menu item (<Alt>-afz). This allows you to delete all frequencies from the list.

Note: This action occurs immediately, without asking for confirmation.


Assign/Frequency Clipboard/Add One

The Assign/Frequency Clipboard/Add One menu item (<Alt>-afa) allows you to add a frequency to the frequency clipboard by picking a chemical shift from a plot of a spectrum.


Assign/Frequency Clipboard/Delete One

The Assign/Frequency Clipboard/Delete One menu item (<Alt>-afd) allows you to delete frequencies from the frequency clipboard using the control panel.


Assign/Frequency Clipboard/Swap Two

The Assign/Frequency Clipboard/Swap Two menu item (<Alt-aft) allows you to exchange the order of two frequencies in the frequency clipboard.


Assign/Frequency Clipboard/Remove Duplicates

The Assign/Frequency Clipboard/Remove Duplicates menu item (<Alt>-afm) allows you to purge frequencies from the frequency clipboard that are considered duplicate entries, based on a chemical shift tolerance that you define.


Assign/Frequency Clipboard/Compare Frequencies

The Assign/Frequency Clipboard/Compare Frequencies menu item (<Alt>-afc) allows you to compare the current collection of frequencies (frequency clipboard) with each of the patterns or prototype patterns. This allows you to see how many fuzzy similarities there are with each of them. Therefore you must specify the target to compare against (patterns or prototype patterns). If patterns are used, a comparison should be based on general shifts or on spectrum-specific shifts. If the fuzzy similarity exceeds the Min Similarity parameter, you are notified. For comparison, the Tolerance should also be used. This menu item is useful when trying to find out if the frequencies in the frequency clipboard constitute a novel pattern.


Assign/Frequency Clipboard/Copy Clipboard To Pattern

Once a frequency clipboard is compiled and sorted, you can copy it to a pattern using the Assign/Frequency Clipboard/Copy Clipboard To Pattern menu item (<Alt>-afl).

You can choose to append to frequencies at the end of the pattern, overwrite (when each frequency in the pattern is overwritten by a frequency from the list), or substitute (when each frequency in the pattern is replaced by the one from the list that lies closest to it). Also, you can select new, which creates new pattern at the same time.


Assign/Frequency Clipboard/Copy Clipboard To Proto

With this menu item (<Alt>-afo) you can copy a clipboard to a new prototype pattern.


Assign/Frequency Clipboard/Copy Pattern To Clipboard

The Assign/Frequency Clipboard/Copy Pattern To Clipboard menu item (<Alt>-afn) performs the opposite action of the Copy Clipboard To Pattern: you can copy frequencies from a pattern to the frequency clipboard and manipulate them through the frequency clipboard's menu items.


Assign/Frequency Clipboard/Copy Proto To Clipboard

The Assign/Frequency Clipboard/Copy Proto To Clipboard menu item (<Alt>-afp) allows you to choose a prototype pattern as a source of frequencies for a frequency clipboard.


Assign/Frequency Clipboard/View Clipboard

With this menu item (<Alt>-afv) you can list the actual contents of the frequency clipboard in the output window.


Assign/Frequency Clipboard/Sort Clipboard

The Assign/Frequency Clipboard/Sort Clipboard menu item (<Alt>-afs) allows you to sort the frequencies in the clipboard in ascending or descending order.


Assign/Frequency Clipboard/Tile Clipboard

The Assign/Frequency Clipboard/Tile Clipboard menu item (<Alt>-afi) allows you to spawn a tile plot from the frequencies in the clipboard.


Assign/Frequency Clipboard/Strip Plot Clipboard

The Assign/Frequency Clipboard/Strip Plot Clipboard menu item (<Alt>-afr) allows you to spawn a strip plot from the frequencies in the clipboard.


Assign/Frequency Clipboard/Draw Clipboard

The Assign/Frequency Clipboard/Draw Clipboard menu item (<Alt>-afw) allows you to draw straight lines on the plot along the frequencies in the clipboard.


Prototype Pattern menu items

The menu items in the third subsection of the Assign 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 algorithms.


Assign/Collect Prototype Patterns

The Assign/Collect Prototype Patterns (<Alt>-ac) menu item contains options used in automated spin-system detection. In Assign, several choices are available: those based on systematic search of 2D spectra (i.e., COSY-type only, TOCSY-type only, TOCSY and NOESY type spectra, or the COSY, TOCSY, and NOESY spectra-based method); those using optimization to find spin systems (in TOCSY or TOCSY and COSY spectra); and those utilizing systematic searching in 3D homonuclear spectra, 3D double or triple resonance spectra, or 4D triple resonance spectra.

Note that 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 double-resonance menu items use the information of a 15N-separated TOCSY spectrum (15N HSQC-TOCSY, 15N HMQC-TOCSY, or 15N TOCSY-HSQC), with or without information from a 2D 15N-HSQC spectrum.

The methods available for spin-system detection in 3D or 4D heteronuclear triple-resonance spectra are contained in Triple Resonance options. Currently you can use the following types of spectra:

The algorithms used by the options in the Assign/Collect Prototype Patterns menu item are described in Chapter 2., Assign building blocks. What follows is a short description of the controls within each of these menu items.

Assign/Collect Prototype Patterns options

You can choose between two different automatic seed/expansion areas, depending on the molecule-one that is suitable for peptides/proteins and one that is suitable for nucleic acids. Here we describe these two different options together:

For the Seed/Expansion Area, you can select Use Defaults to use the default values (which you can review later by clicking the More button), or you can enter them in the next control panel by selecting the Review option. Also, there are two interactive options (which require a seed spectrum to be currently plotted)-you can start spin-system detection from a peak (Select via Peak) or by dragging an area with the cursor (Drag with Cursor). If you choose to enter the seed area you should enter it in ppm. This area is used to collect the seed peaks. Assign attempts to disentangle the spin systems from these peaks. Assign uses the Frequency Collapse Tolerance value (in ppm) to decide if a candidate frequency already belongs to the prototype pattern. This tolerance should reflect the chemical shift variations expected among different spectra.

If you choose at this point to start the control panel's action (by clicking OK), the spin-system detection uses default values for the remaining settings. If you want to review and possibly change the defaults, click the More button. The new control panel contains these controls:

a.   The Remove Intraproto Frequencies control indicates whether peaks that belong to already existing prototype patterns from an earlier run should be removed from consideration. This can help to avoid duplicate entries if several prototype pattern-detection runs are applied and the protos entity is not purged between runs.

b.   The Number of Frequencies in Proto Min and Max controls set the minimum and maximum numbers of frequencies a prototype pattern can contain. For proteins, a good starting point are the values 3 and 8, respectively. This option can also be used to selectively detect spin systems (e.g., glycines or AMX spin systems). For this, Min should be set to the number of expected frequencies minus one (to account for coinciding frequencies); and Max should be set to the number of expected frequencies plus one.

c.   The Number of Iterations control governs the number of extension loops that can be run for each seed peak.

d.   You can use the Frequencies per Iteration control to specify how many candidate frequencies to add to the prototype pattern in each extension loop (usually one or two).

The next set of controls to set are the tuning parameters: the number of contacts required for a candidate frequency to be eligible or membership in a developing prototype pattern. For a prototype pattern containing 2, 3, 4, or more frequencies, you should enter at least how many COSY, COSY, and TOCSY, and COSY and TOCSY and NOESY contacts should have a new frequency to be considered as a candidate. All the values should be 0 and any number entered for N frequencies should be the required number for N-1 frequencies.

The last controls to set are the ppm filter definitions. You may impose filtering conditions to select out any unwanted prototype pattern, based on the number of frequencies in certain chemical-shift ranges. For proteins you might specify, for example, that between 6 and 12 ppm there should be only one frequency, and between 3 and 5.5 ppm there should be at least one and at most three frequencies.

You can remove peaks that are already part of earlier detected spin systems from consideration by setting the Remove first control to True. You can specify the number of standard deviations to be used in chemical-shift comparison by setting the Max Std Dev control. Output Level can be set to Quiet, Low, Medium, or High. Finally, you must define the inter-spectrum chemical-shift tolerance between TOCSY and COSY spectra and between TOCSY and HSQC spectra.

With the Assign/Collect Prototype Patterns/3D Homonuclear menu item, you should first set the experiment you want to use, then, similar to the method used in the 2D Systematic Search menu item, you must specify the seed area and the expansion area in ppm (e.g., Seed area W3 Low and High). Then you should define which two frequencies from each seed peak to use as the two first frequencies of a new prototype pattern (Use seed peak W3 True/False, W2 True/False, and W1 True/False). You must also define what dimension of a candidate peak is to be used as a new frequency in the spin system (Use exp. peak W3 True/False, W2 True/False, and W1 True/False). The remaining controls are set like those discussed under the 2D Systematic Search menu item.

Under this option you can choose from three different methods:

The options for Triple Resonance are:

If you would like to use a different combination of spectra, you can use the Assign/Collect Prototype Patterns/User Settable menu item. You have to define a primary spectrum (e.g., an HSQC or an HNCO) and secondary spectra (e.g., an HSQC-TOCSY or a pair of HNCACB and CBCA(CO)NH). You also have to specify in which direction the search should proceed and what the expected atom types found along each dimension are.

Besides the built-in methods for spin-system collection, you can use virtually any combination of spectra. The method is a semiautomatic detection-that is, you need to click a position (or peak) in a primary spectrum (such as HSQC or HNCO) and then, according to the setup procedure, a spin system gets collected and stored, if there are correlated peaks in the spectra you defined. To achieve this, you must first set up your experiments and define how the connection between the peaks is expected to be, using the Assign/Collect Prototype Patterns/Semiautomated Setup menu item. Then you can use the Assign/Collect Prototype Patterns/Semiautomated Collect menu item (<Ctrl-=) to collect a new spin system. You can step through your spectra and do all the spin-system semi-automated detection until you use the Assign/Collect Prototype Patterns/Semiautomated Unset menu item.

The Assign/Collect Prototype Patterns/Extend Prototype Patterns menu items helps to collect new frequencies (that is, extend spin systems) into already existing prototype patterns. For example, if you detect the sextets (HN,i-Ni-Ca,i-Ca,i-Ca,i-1-Ca,i-1) on a pair of experiments, then you can use an HAHB(CO)NH spectrum to extend the spin systems to include the Ha,i-1 and Hb,i-1 frequencies. This is mainly useful when the backbone spins of a protein are collected via a triple-resonance method, and the sidechain resonances with a triple-resonance or double-resonance method.


Assign/Edit Prototype Pattern

This menu item provides you with several options:


Assign/Promote Prototype Patterns

After prototype patterns are collected, you can use the Assign/Promote Prototype Patterns menu items to copy all of them to patterns or you can copy patterns back to prototype patterns.

Copy Prototype Patterns to Spin Systems (Patterns)

With this menu item you can promote all spin systems (prototype patterns) detected with any of the automated collection methods to patterns. For homonuclear and double-resonance spin systems, FELIX will just copy over the frequencies and create new patterns. For triple-resonance experiments, FELIX will search through the list for potentially sequential spin systems using the tolerance (which you can set) and store that information in patterns as neighbors. Also, if you request it, this function can search for potential profiles and store them. Finally, FELIX can compare each potential pattern against all existing ones to discover if the new candidate pattern is unique. If it finds that the new pattern is not unique, it asks you to allow for deletion. This deletion option slows the speed of the function's execution, but allows you to avoid duplication of the patterns. In certain cases, proline spin systems can be detected as part of a protopattern showing up only as neighbors (i-1). This can occur if protopattern detection was done using CBCA(CO)NH and CBCANH spectra.

Copy Spin Systems (Patterns) to Prototype Patterns

By using this menu item you can copy final spin systems (patterns) back to prototype patterns.


Assign/Zoom Prototype Pattern

The Assign/Zoom Prototype Pattern (<Alt>-an) menu item is useful for 3D and 4D spectra when you want to visually inspect the spin systems resulting from automated detection. 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 also specify the required ranges, you can bring up the region by clicking OK.

Protopatterns table

The majority of the visualization menu items useful for analyzing prototype patterns are accessible through the prototype pattern table. The table is usually opened by using the automated collection menu items, but you can open it with the Edit/Prototype Patterns (<Alt>-ep) menu item. Before you can execute any menu items in the table, you need to select (click) a row or select multiple rows, depending on the action you want to perform. The menu items accessible through the table interface are briefly described below.

The Action/Zoom (<Alt>-az) menu item from the Protopatterns table is useful for 3D and 4D spectra when you want to visually inspect the spin systems resulting from automated detection. You can select a prototype pattern by clicking its number (highlighting the row) for which prototype pattern's frequencies should define the region. Then you need to either use the Action/Zoom menu item or click the Zoom icon in the Protopatterns table. To set the preferences for this menu item, you need to use the Preferences/Zoom menu item in the Protopatterns table.

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 either click 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 need to select the Action/Tile Plot menu item or use the Tile Plot icon in the Protopatterns table. The tile plot preferences can be set with the Preference/Tile Plot menu item in the Protopatterns table. To turn off the tile plot you can 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 needed row in the table. Then select the Action/Strip Plot menu item or use the Strip Plot icon in the Protopatterns table. The strip plot preferences can be set in the Preference/Strip Plot menu item in the Protopatterns table. To turn off the strip plot you can use the View/Plottype/Tile/Strip Plot menu item.

This menu item (<Alt>-an) allows you to define a strip plot from a 3D/4D spectrum, where the individual strips originate from potentially different planes of different spin systems. You first need to select the needed prototype patterns by clicking the rows in the table (after the first click use <Shift>-click). Then you need to click the Action/ND Strip Plot menu item or use the ND Strip Plot icon in the Protopatterns table. The strip plot preferences can be set in the Preference/ND Strip Plot menu item in the Protopatterns table. To turn off the tile plot you can use the View/Plottype/Tile/Strip Plot menu item.

This menu item (<Alt>-nf) in the Protopatterns table allows you to graphically add a frequency to the selected prototype pattern.

The ProtoPattern/Promote One Proto to Spin System menu item (<Alt>-np) in the Protopatterns table copies over the frequencies from the selected prototype pattern to a new pattern (spin system).

Note: If the selected prototype pattern is a triple-resonance spin system with frequencies from the neighboring spin system, this information is lost during the promotion-that is, this information is not transferred to neighbor probability, which is different from the way the Assign/Promote Prototype Patterns menu item works.  

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 result is listed in the text windowoutput window.

     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


Pattern (Spin Systems) menu items

The next section in the Assign pulldown contains menu items dealing with patterns or full spin systems-that is, those which have neighbor probabilities, residue type probabilities, and assignments.


Assign/Spin System

The Assign/Spin System menu (<Alt>-as) contains menu items for creating, deleting, and editing patterns.

Creating and Deleting Spin Systems

The Assign/Spin System/Add One menu item (<Alt>-aso) allows you to create a new empty pattern and add it to the database. You may define the name, color, comment, and root frequency, and the unique identification number is defined by the program.

The Assign/Spin System/Delete One menu item (<Alt>-asl) allows you to delete patterns from the database by clicking the unique number of the pattern to be deleted.

The Assign/Spin System/Clear All menu item (<Alt>-asc) allows you to delete all patterns from the database.

Note: Accidental use of this menu item can destroy several weeks worth of work.

Editing Spin Systems

After a pattern is established, you may edit it. New frequencies can be added to or deleted from a pattern. Attributes or shifts can also be edited.

This menu item (<Alt>-asa) is similar to the one in the Frequency Clipboard, but it allows you to directly add frequencies to the patterns.

This menu item (<Alt>-asm) allows you to type in a new frequency which is then appended to a pattern.

The Delete Frequency menu item (<Alt>-asd) allows you to delete frequencies from the patterns.

The Assign/Spin System/Edit Attributes menu item (<Alt>-asi) allows you to change the name, color, comment, and root frequency of the pattern.

The Assign/Spin System/Auto Root menu item (<Alt>-asr) allows you to copy the generic shift of the first frequency of the pattern to its root frequency attribute. This frequency is then used in the homonuclear neighbor detection routines of Assign/Neighbor menu items.

The Copy Generic Shift To Specific menu item (<Alt>-asg) allows you to copy the generic shifts to spectrum-specific shifts for frequencies in all patterns or in a specific pattern. You must specify the experiment for which specific shifts are to be redefined. The proper spectrum-specific shifts of assigned frequencies in patterns are crucial to the success of automated peak assignment.

The Copy Specific Shift To Generic menu item (<Alt>-ass) allows you to copy the spectrum-specific shifts of frequencies of one or all patterns to generic shifts. You must specify which experiment's spectrum-specific shifts to copy.

This menu item (<Alt>-asu) automatically goes through the specified spectrum and tries to update the spectrum-specific shifts for patterns using possibly intrapattern peaks within specified tolerances.

The Edit Frequency Chemical Shift menu item (<Alt>-ase) allows you to change the chemical shifts of frequencies of pattern. You can edit the generic shift or the spectrum-specific shift. If you select the latter, you automatically select the experiment which will undergo a shift change.

The Assign/Spin System/Graphical Edit Frequency menu item (<Alt>-asf) allows you to change the chemical shifts of frequencies of a pattern in a graphical way. You are asked which pattern's chemical shifts to edit, then lines are drawn across the spectrum at these frequencies. You must select the frequency to be edited with the crosshair cursor, then position the crosshair at the new frequency location and click. You can continue to edit the frequencies in that pattern until you want to exit the menu item (by pressing <Esc>).

The Tile+Show+Edit Frequencies menu item (<Alt>-ast) is similar to the Graphical Edit Frequency menu item, but after asking which pattern is to be edited, it tiles the pattern, shows the frequencies, and then allows you to edit. Press <Esc> to exit this menu item.

The Rename Spin Systems menu item (<Alt>-asn) allows you to change the default names of all or particular patterns to the residue they belong to. If you assign all frequencies in a pattern (for example, pa2) to a specific residue (for example, ALA_32), then this action updates the name of the pattern and, in subsequent actions (for example, tiling and listing), the name of that pattern shows as that residue.


Spin System Extension-Assign/Extend Spin Systems

Three automated methods for adding frequencies to spin systems (patterns) are accessible through the Assign/Extend Spin Systems (<Alt>-ad) menu item:

Extend via HCCH-TOCSY

Using this method on the current 3D HCCH-TOCSY experiment allows you to automatically add new frequencies to the patterns starting from Ca, Ha frequency pairs.

Extend via H(CCH)(CO)NH or C(CH)(CO)NH

Using this method on the current 3D H(CCH)-TOCSY-(CO)NH or 3D C(CH)-TOCSY-(CO)NH experiment allows you to automatically add new frequencies to the patterns.

Note: This option works only if the neighbor relationships between the spin systems are unambiguous (the probabilities are set to 1), since it adds aliphatic Ha or Ca frequencies not to the ith pattern but to the i-1 neighbor.

Extend via HBHA(CO)NH

Using this method on the current 3D HBHA(CO)NH experiment allows you to automatically add new frequencies to the patterns.

Note: This option works only if the neighbor relationships between the spin systems are unambiguous (the probabilities are set to 1), since it adds aliphatic Ha and Hb frequencies not to the ith pattern but to the i-1 neighbor.


Residue Type Probability-Assign/Residue Type

The items in the Assign/Residue Type menu (<Alt>-ay) deal with the residue type probabilities of patterns.

Assign/Residue Type/Score Residue Type

The Assign/Residue Type/Score Residue Type menu item (<Alt>-ays) allows you to automatically match patterns and residue types.

Two algorithms are available for scoring. One is adapted for proton or all-atom scoring. Here, for each atom in the library, Assign tries to find a matching frequency in the pattern. The best-matching frequency may lie no more than a user-defined standard deviation away from the expected value. If a required number of atoms can be matched, a score is computed and stored in the database. The scores are then stored in the database as probabilities that one particular pattern belongs to a certain residue type.

The other method uses the Ca and/or the Cb chemical shift distribution to score patterns. This option can be triggered by setting the Scoring method to CACB only.

These probabilities can then be used by the Assign/Sequential menu items to make sequence-specific assignments.

Assign/Residue Type/Match Residue Type

The Assign/Residue Type/Match Residue Type menu item (<Alt>-aym) allows you to compare a pattern with a residue type defined in the library.

For each atom in the residue and for each frequency in the pattern, a result is printed: [ d = (actual shift - expectation value)/standard deviation]; therefore the lower the value of d, the higher the probability that the particular frequency belongs to the atom.

Assign/Residue Type/Set Residue Type

The Assign/Residue Type/Set Residue Type menu item (<Alt>-ayt) allows you to manually set the probability (or "score") that a particular pattern belongs to a certain residue type. You can then list it using the Assign/Residue Type/Show Residue Type menu item.

Assign/Residue Type/Show Residue Type

The Assign/Residue Type/Show Residue Type menu item (<Alt>-ayw) allows you to list the score you set with the Assign/Residue Type/Set Residue Type menu item or calculated with Assign/Residue Type/Score Residue Type menu item.


Neighbor Probability-Assign/Neighbor

The Assign/Neighbor pullright menu (<Alt>-an) contains a set of menu items that deal with the neighbors of patterns (the patterns belonging to consecutive residues in the sequence).

The first part of the pullright contains menu items for automatically detecting potential neighbor patterns in a 2D NOESY spectrum (Find Neighbor Via 2D NOE), in a homonuclear 3D [J,NOE], [NOE,J] or [NOE,NOE] spectrum (Find Neighbor Via 3D NOE), in a 15N-separated NOE spectrum (Find Neighbor Via 3D N-15 NOE), or in triple-resonance experiments (Find Neighbor Via 3D/4D). Please see Chapter 2., Assign for a description of the algorithms for the Find Neighbor menu items.

Assign/Neighbor/Find Neighbor Via 2D NOE

In the Assign/Neighbor/Find Neighbor Via 2D NOE (<Alt>-an2) menu item the following controls can be set:

Assign/Neighbor/Find Neighbor Via 3D NOE

In the Assign/Neighbor/Find Neighbor Via 3D NOE (<Alt>-an3) menu item, you should specify the Range of freq for the pattern in ppm. Only the NOESY peaks that lie in this range are used. Normally for proteins this would be the Ha, Hb region, but you may also include the HN region.

Assign/Neighbor/Find Neighbor Via 3D N-15 NOE

In the Assign/Neighbor/Find Neighbor Via 3D N-15 NOE (<Alt>-ann) menu item, you can use the default parameters or specify the maximum number of frequencies of the particular patterns to use in the detection algorithm (Number of Frequencies to use in Pattern). You should then specify the atom type and ranges in ppm to use for the First Type (usually the HN frequencies) and for the Second Type (usually 15N).

Then you should specify the dimension to use for candidate search (Candidate (NOE) Dimension) and the region in ppm. Only the NOESY peaks that lie in this range are used. Normally for proteins this would be the Ha, Hb region, but you may also include the HN region.

Several additional parameters can be modified:

Assign/Neighbor/Find Neighbor Via 3D/4D

This menu item (<Alt>-an4) allows you to find neighbors for existing patterns in a triple-resonance 3D or 4D experiment, such as HN(CO)CA.

Assign/Neighbor/List Neighbors

The Assign/Neighbor/List Neighbors menu item (<Alt>-anl) lists the neighbor relation between patterns set in manual or automated routines.

Assign/Neighbor/Show Neighbors Via Strip Plot

This menu item (<Alt>-anr) allows you to view possible neighbors found in automated routines by spawning strip plots on a 3D spectrum (e.g., HSQC-NOESY or CBCANH).

Assign/Neighbor/Set Neighbors Manual

The Assign/Neighbor/Set Neighbors Manual menu item (<Alt>-ans) allows you to set one pattern as another's neighbor (i  -  i  + 1 connectivity or i  -  i  -  1 connectivity) with a certain probability.

Assign/Neighbor/Delete Neighbors

The Assign/Neighbor/Delete Neighbors menu item (<Alt>-and) deletes a previously set neighbor relation.


Search for Sequential Assignment -Assign/Sequential

The Assign/Sequential (<Alt>-aq) pullright contains two menu items to make automated sequence-specific assignments and several menu items to review them.

Assign/Sequential/Systematic Search

The Assign/Sequential/Systematic Search (<Alt>-aqs) menu item allows you to find potential matchings of the collection of patterns onto the sequence using a systematic search. The patterns should have neighbors and residue type assignments. The molecule and the library should also be defined.

Note: This option works only on unbranched biomacromolecules.

Assign/Sequential/Simulated Annealing

The Assign/Sequential/Simulated Annealing (<Alt>-aqa) menu item finds optional matchings of the collection of patterns to the sequence, using the simulated annealing method. The patterns should have neighbors and residue-type assignments. The molecule and the library should also be defined.

The next three settings are used to fine-tune the simulated annealing algorithm: you can change the Temperature factor to a lower number if most of the sequence is well defined or set the number of iterations in Iteration factor. The Sequential/individual factor setting can be used to change the weight of the neighbor information vs. spin-system probability scores.

Finally, select the appropriate Output level.

Assign/Sequential/Show Suggested Via Strip

This menu item (<Alt>-aqr) allows you to show the stored results of an Assign Sequential/Systematic Search run as strip plots on a 3D spectrum.

Assign/Sequential/Compose Stretch with Strip Plot

This menu item composes a "stretch" of spin systems, starting with a specified pattern, using only neighbor probabilities. The stretch is then stored in the stretch entity. The action also creates a strip plot for the current 3D triple resonance matrix.

Assign/Sequential/Suggest Assignment For Strip

This menu item suggests a sequence-specific assignment for a specified stretch, using the residue-type probabilities.


Spin System Assignment-Assign/Assign Spin System

The last step in assigning spin systems is to name the resonances within them. The menu items to do this are in the Assign/Assign Spin System (<Alt>-aa) pullright.

Assign/Assign Spin System/Residue Type

The Assign/Assign Spin System/Residue Type (<Alt>-aar) menu item allows you to assign all frequencies in a particular pattern to a residue type (but not number) in the sequence (i.e., a non-sequence-specific assignment). A typical assignment is that each frequency has a name like 1:VAL_*:*, where wildcards (*) mean that the exact residue number and the atom name is not defined yet.

Assign/Assign Spin System/Sequence Specific

The Assign/Assign Spin System/Sequence Specific (<Alt>-aas) menu item allows you to assign all frequencies in a particular pattern to a residue type and number in the sequence (i.e., a sequence-specific assignment) but no atom name is specified. A typical assignment is that each frequency has a name like 1:VAL_27:*, where the wildcard (*) mean that the exact atom name is not defined yet. Meanwhile, you can request that the program make suggestions based on matching the chemical shifts of the frequencies against the libraries and naming the frequencies with atom names (Assign Frequencies should be set to Yes). In this case the frequencies in the pattern will have names like 1:VAL_27:HN.

Assign/Assign Spin System/Frequency

The Assign/Assign Spin System/Frequency (<Alt>-aaf) menu item allows you to manually assign the frequencies of the patterns.

First, you select the frequency of the assignment to be built. You can do this by clicking the Frequency control panel and then clicking SELECT. Next you select a residue type from the Residues list and click FILTER. The program then fills in the #s list with valid residue numbers and the Nuclei list with all protons (or carbons or nitrogens, depending on the type of frequency) of that particular residue type. If you now click one number from the #s list and one nucleus from the Protons list, clicking the BUILD button fills in the atom specification for that particular frequency.

If the atom specification is acceptable, click ADD or Return to transfer it to the Possible Assignment list and store it in the database. Alternatively, you may directly type in the name of the frequency in the Atom Spec box. Up to six possible assignments can be stored with each frequency in the database. Unique or most-probable assignments can be specified in the database by selecting them from the list and clicking SPECIFY, in which case this assignment shows up in the top line under the Assignment header. You also can despecify-that is, make all assignments equally possible-by clicking SPECIFY without highlighting any assignment. You can delete any assignment by clicking DELETE.

Assign/Assign Spin System/Unassign Spin System

The Assign/Assign Spin System/Unassign Spin System (<Alt>-aau) menu item allows you to unassign all frequencies in a pattern.


Assign/Chemical Shift Index (CSI)

If the resonance assignments are done for all the patterns, then you can derive some secondary structural information-this method is called the "Chemical Shift Index."

Assign/Chemical Shift Index/HA Chemical Shift Index

Calculate the chemical shift indices based on assigned Ha chemical shifts in current patterns (<Alt>-aih).

Assign/Chemical Shift Index/CA Chemical Shift Index

Calculate and tabulate the CSI, based on assigned Ca shifts in current patterns (<Alt>-aia).

Assign/Chemical Shift Index/CB Chemical Shift Index

Calculate and tabulate the CSI, based on assigned Cb shifts in current patterns (<Alt>-aib).

Assign/Chemical Shift Index/C Chemical Shift Index

Calculate and tabulate the CSI based on assigned Có shifts in current patterns (<Alt>-aic).

Assign/Chemical Shift Index/Consensus CSI

Calculate a consensus CSI table, if the Ha, Ca, Cb and Có CSI's are already calculated and stored (<Alt>-aio).

Assign/Chemical Shift Index/Dihedral Restraint

Based on a consensus CSI table, this menu item calculates dihedral angle restraints for Y and F for consensus secondary structural regions (<Alt>-aid).


Reporting Spin Systems-Assign/Report Spin System

After the assignments are done, you can create reports of particular spin systems with the Assign/Report Spin System (<Alt>-ar) menu item, which either lists information to the output window or stores the frequencies, frequency names, neighbors, and generic and spectrum-specific shifts in a file.


Assign/Zoom Spin System

The Assign/Zoom Spin System menu item (<Alt>-az) is useful for 3D and 4D spectra when you want to visually inspect the final spin systems or patterns. You can define which pattern's frequencies should define the region. If you select a pattern name from the Source list and then click the Show button, the Frequencies box is filled in with the frequencies of that pattern. If you then select the desired Orientation and specify which frequency should be at which dimension, also specifying the required ranges, the region can be brought up by clicking OK.


Spin System Table-Edit/Spin Systems

Most of the visualization menu items useful for analyzing patterns (spin systems) are accessible through the spin-system table. The table is opened by using the Edit/Spin Systems (<Alt>-es) menu item. Before you can execute any menu items in the table, you need to select (click) a row or multiple rows, depending what you want to do.

Action/Zoom

The Action/Zoom (<Alt>-az) menu item from the Spinsystems table is useful for 3D and 4D spectra when you want to visually inspect the frequencies in spin systems. You can select a pattern by clicking its number (highlighting the row) for which pattern's frequencies should define the region. Then you need to use the Action/Zoom menu item or click the Zoom icon in the Spinsystems table. To set the preferences for this menu item, use the Preferences/Zoom menu item in the Spinsystems table.

Action/Draw

You can visualize a pattern by drawing straight lines on the actual spectrum along the frequencies of particular spin systems. First you need to select one or two patterns from the table and then click the Action/Draw (<Alt>-ad) menu item or click the Draw icon in the Spinsystems table. By using the View/Draw Frequencies or the Clear Frequencies item in the right-mouse menu, you can turn these lines off. To set the colors, you can use the Preference/Draw menu item from the Spinsystems table.

Action/Tile Plot

The Action/Tile Plot (<Alt>-at) menu item is used to define a tile from any pair of patterns. You first need to select two patterns by clicking two rows in the table. Then you need to select the Action/Tile Plot menu item or use the Tile Plot icon in the Spinsystems table. Tile plot preferences can be with in the Preference/Tile Plot menu item in the Spinsystems table. To turn off the tile plot you can click the View/Plottype/Tile/Strip Plot toggle.

Action/Strip Plot

The Action/Strip Plot (<Alt>-as) menu item in the Spinsystems table is used to define a strip plot from a specific pattern. First you need to select a pattern by clicking the needed row in the table. Then you select the Action/Strip Plot menu item or use the Strip Plot icon on the Spinsystems table. Strip plot preferences can be set with the Preference/Strip Plot menu item in the Spinsystems table. To turn off the strip plot you can click the View/Plottype/Tile/Strip Plot toggle.

Action/ND Strip Plot

This menu item (<Alt>-an) allows you to define a strip plot from a 3D/4D spectrum, where the individual strips originate from potentially different planes of different spin systems. You first need to select the needed patterns by clicking the rows in the table (after the first click, use <Shift>-click). Then you need to select the Action/ND Strip Plot menu item or use the ND Strip Plot icon on the Spinsystems table. Strip plot preferences can be set with the Preference/ND Strip Plot menu item in the Spinsystems table. To turn off the tile plot you can click the View/Plottype/Tile/Strip Plot toggle.

Spinsystem/Add Frequency via Cursor

This menu item (<Alt>-sf) in the Spinsystem table allows you to graphically add a frequency to the selected pattern.

Spinsystem/List Residue Type

The Spinsystem/List Residue Type menu item (<Alt>-st) in the Spinsystem table allows you to list the "score" that you set with the Assign/Residue Type/Set Residue Type menu item or that you calculated via the Assign/Residue Type/Score Residue Type menu item for the previously highlighted pattern.

Spinsystem/List i+1 Neighbors

The Spinsystem/List i+1 Neighbors menu item (<Alt>-sl) in the Spinsystem table lists the i+1 neighbor patterns from the database for the current (highlighted) spin system.

Spinsystem/Show i+1 Neighbors Via Strip Plot

This menu item (<Alt>-sn) allows you to view possible i+1 neighbors for the current spin system in the database by spawning strip plots on a 3D spectrum (e.g., HSQC-NOESY or CBCANH).

Spinsystem/List i-1 Neighbors

The Spinsystem/List i-1 Neighbors menu item (<Alt>-s-) in the Spinsystem table lists the i-1 neighbor patterns from the database for the current (highlighted) spin system.

Spinsystem/Show i-1 Neighbors Via Strip Plot

This menu item (<Alt>-as) allows you to view possible i-1 neighbors for the current spin system in the database by spawning strip plots on a 3D spectrum (e.g., HSQC-NOESY or CBCANH).

Spinsystem/Delete Neighbor

The Spinsystem/Delete Neighbor menu item (<Alt>-sd) in the Spinsystem table allows you to interactively delete neighbors that were shown as strip plots for a particular spin system. You need to click a particular strip to delete the spin system defining that strip as a possible neighbor.

Spinsystem/Perpendicular Strips

The Spinsystem/Perpendicular Strips menu item (<Alt>-sp) in the Spinsystem table allows you to interactively define a set of strip plots that are perpendicular to the currently viewed plot of a 3D double- or triple-resonance spectrum. For example, first display an HN-N plane of an HNCA on CBCANH or 15N NOESY spectrum at a spin system's intraresidue or interresidue 13C or HA frequency. Then select the Spinsystem/Perpendicular Strips menu item in the Spinsystems table, click the same peak (the target spin system), then click the peaks that are in the same plane (the possible neighbor spin systems) and from which strip plots will be spawned. (If FELIX cannot find a peak in the spin system table, you should go back and manually add that spin system to the spin system table using the frequency clipboard.) When there are no more candidate peaks left in the plane, press <Esc>. FELIX then draws the strip plots, target spin system first.

The menu item works properly if the ND Strip preference is set (e.g., which is the NH axis and which is the 15N axis, etc.).

To set then the neighbor relations you can use the Spinsystem/Set i+1 Neighbor or Spinsystem/Set i-1 Neighbor menu item in the Spinsystems table. You need to set the appropriate neighbor relationship (i.e., i+1 or i-1) depending on whether the spectrum is double or triple resonance and whether the target spin system's peak was selected as an intraresidue or interresidue peak.

Spinsystem/Set i+1 Neighbor, Spinsystem/Set i-1 Neighbor

The Spinsystem/Set i+1 Neighbor (<Alt>-se) and Spinsystem/Set i-1 Neighbor (<Alt>-si) menu items in the Spinsystem table allow you to visually set neighbors for a spin system if an ND strip plot is displayed.

Select the Spinsystem/Set i+1 Neighbor or Spinsystem/Set i-1 Neighbor menu item from the Spinsystems table then use the crosshair cursor to select first the target spin system's strip and then the neighbor spin system's strip. If Set i+1 Neighbor is selected, the neighbor spin system is marked as the i+1 neighbor of the target spin system. If Set i-1 Neighbor is selected, the target spin system is marked as the i-1 neighbor of the neighbor spin system.

Spinsystem/Assign

The Spinsystem/Assign menu item (<Alt>-sa) from the Spinsystem table is similar to the The Assign/Assign Spin System/Sequence Specific menu item.

Spinsystem/Report

After the assignments are done you can list the frequencies, frequency names, neighbors, and generic and spectrum-specific shifts for the current spin system in the output window, using this menu item (<Alt>-sr) from the Spinsystem table.


Stretches of Spin Systems-Edit/Stretches

A set of menu items dealing with stretches of spin systems is accessible within the Stretches table. These stretches of spin systems can be composed with either the Assign/Sequential/Systematic Search menu item or the Assign/Sequential/Compose Stretch menu item with the Strip Plot controls. The table is automatically opened by these menu items when it is created, or alternatively, you can open the table with the Edit/Stretches (<Alt>-ec) menu item. Before you can execute any menu item in the table, you must first select a stretch by highlighting (clicking) its row.

Action/ND Strip Plot

The Action/ND Strip Plot (<Alt>-an) menu item in the Stretches table allows you to define a strip plot from a 3D or 4D spectrum, where the individual strips originate from potentially different planes of the spin systems of the stretch. You first must select the stretch by clicking its row in the table. You then select the Action/ND Strip Plot menu item or use the ND Strip Plot icon on the Stretches table. You can set strip plot preferences using the Preferences/ND Strip Plot menu item in the Stretches table. To turn off the strip plot, click the View/Plottype/Tile/Strip Plot toggle.

Stretch/New

The Stretch/New (<Alt>-sn) menu item in the Stretches table allows you to define a new stretch. You must select a pattern from the control panel to add a new stretch to the table. You can then manually add other spin systems.

Stretch/Compose Stretch with Strip Plot

The Stretch/Compose Stretch with Strip Plot (<Alt>-sc) menu item in the Stretches table is the same as the Assign/Sequential/Compose Stretch with Strip Plot menu item. This menu item composes the stretch of spin systems, starting from a specified pattern, and using only neighbor probabilities. The stretch is then stored in the stretch entity and displayed in the table. The menu item also creates a strip plot for the current 3D matrix.

Stretch/Add Pattern

The Stretch/Add Pattern (<Alt>-sa) menu item in the Stretches table allows you to manually add a spin system to the current (active) stretch, using the control panel.

Stretch/Swap Pattern

The Stretch/Swap Pattern (<Alt>-sp) menu item in the Stretches table allows you to swap out a pattern from the current stretch. You must first click the pattern in the strip plot with the large crosshair cursor and then select a new pattern from the control panel. The menu item then swaps the two patterns in the entity and in the strip plot.

Stretch/Delete Pattern

The Stretch/Delete Pattern (<Alt>-sd) menu item in the Stretches table allows you to interactively delete a pattern by selecting the strip plot of the current stretch with the large crosshair. The pattern is also deleted from the table.

Stretch/Suggest Assignment for Stretch

The Stretch/Suggest Assignment for Stretch (<Alt>-ss) menu item in the Stretches table suggests a sequence-specific assignment for the current stretch of spin systems using the previously defined residue-type probabilities.

Stretch/Assign One Stretch

The Stretch/Suggest Assignment for Stretch (<Alt>-ss) menu item in the Stretches table performs a sequence-specific resonance assignment for the patterns in the current stretch, using either suggested assignments (from the Assign/Sequential/Systematic Search menu item or from the Stretch/Suggest Assignment menu item in the Stretch table) or assignments you specify.


Assign Residue Table-Edit/Residues

After you have made sequence-specific assignments for some spin systems, you can look at the assignments not only in the Spinsystem table, but also in the Residues table. This table is activated through the Edit/Residues (<Alt>-er) menu item.

File/Save As

The File /Save As (<Alt>-fa) menu item saves a report about the current resonance assignments for the given residue to a .txt file.

Action/Zoom

The Action/Zoom (<Alt>-az) menu item in the Residues table is useful for 3D and 4D spectra when you want to visually inspect the frequencies in spin systems assigned to specific residues. You can select a residue that is assigned to a pattern by clicking its number (which highlights the row), which should the define the region for the pattern's frequencies. You then use the Action/Zoom menu item or the Zoom icon in the Residues table. To set the preferences for this action, use the Preferences/Zoom menu item in the Residues table.

Action/Draw

You can visualize an assigned residue by drawing straight lines on the actual spectrum along the frequencies of the particular spin system to which it is assigned. First you select one or two residues from the table, then select the Action/Draw (<Alt>-ad) menu item or click the Draw icon in the Residues table.

Action/Tile Plot

The Action/Tile Plot (<Alt>-at) menu item is used to define a tile from any pair of assigned residues. You first select two patterns by clicking two rows of residues in the table, then select the Action/Tile Plot menu item or click the Tile Plot icon in the Residues table. You can set tile plot preferences with the Preferences/Tile Plot menu item in the Residues table. To turn off the tile plot, click the View/Plottype/Tile/Strip Plot toggle.

Action/Strip Plot

The Action/Strip Plot (<Alt>-as) menu item in the Residues table is used to define a strip plot from a specific assigned pattern. First you select a pattern by clicking the needed row of residues in the table. You then select the Action/Strip Plot menu item or click the Strip Plot icon in the Residues table. Strip plot preferences can be set with the Preferences/Strip Plot menu item in the Residues table. To turn off the strip plot, click the View/Plottype/Tile/Strip Plot toggle.

Action/ND Strip Plot

The Action/ND Strip Plot (<Alt>-an) menu item allows you to define a strip plot from a 3D/4D spectrum, where the individual strips originate from potentially different planes of different spin systems. You first select the needed patterns by clicking the rows of the residues in the table (<Shift>-click to select more than one row). You then select the Action/ND Strip Plot menu item or click the ND Strip Plot icon in the Residues table. You can set strip plot preferences with the Preferences/ND Strip Plot menu item in the Residues table. To turn off the tile plot, click the View/Plot type/Tile/Strip Plot toggle.

Action/Show Residue

The Action/Show Residue (<Alt>-ar) menu item in the Residues table can display one or more residues in the molecule by blanking the other residues. You can only use this menu item if you have activated the 3D molecule display through the Model/Draw Molecule menu item.

Residue/Assign

The Residue/Assign (<Alt>-ra) menu item in the Residues table can be used to assign the frequencies for a selected pattern to the atoms of the current residue.

Residue/Report

The Residue/Report (<Alt>-rr) menu item prints a report in the output window about the current resonance assignments for the residue.


Peak Assignment menu items


Assign/List Peak

With the Assign/List Peak (<Alt>-at) menu item you can query the database for a particular peak's positions, assignment status, and membership in spin systems.


Assign/Peak Assign

The Assign/Peak Assign (<Alt>-ak) pullright. contains several menu items for changing the assignments of a peak. A peak is considered to be assigned if the frequencies defining it are assigned and if this assignment is transferred to the peak itself. Therefore, in an Assign strategy, the frequencies in the patterns are the primary targets of the assignment. Once the frequencies are assigned, you can transfer this information to peaks automatically (Assign/Peak Assign Peak/Autoassign Peaks) or manually (Assign/Peak Assign/Manual Assign Singly or Assign/Peak Assign/Manual Assign Multiply).

Assign/Peak Assign/Manual Assign Singly

The Assign/Peak Assign/Manual Assign Singly (<Alt>-aks) menu item is used to manually assign peaks. If you assign peaks manually, FELIX first checks to see whether the frequencies defining that peak are already assigned in patterns. If the frequencies are assigned, they show up as possible peak assignments. You can then select from the listed names for a peak or create a different assignment.

First you select the Dimension for which the assignment is to be built by choosing from the Dimension popup. Then you select a residue type from the Residues list and click the FILTER button. The program then fills in the #s list with valid residue numbers, and the Nuclei list with, for example, all protons of that particular residue type. Clicking one number from the #s list and one nucleus from the Nuclei list, then clicking the BUILD ASN button fills in the atom specification for that particular frequency. To accept these specifications, press <Enter>, which transfers this atom specification to the peak and also stores it in the database. You can also select a frequency assignment from the Use Frequency Assignments list and make it the actual peak assignment by pressing <Enter>. You may delete any peak assignments by clicking the DELETE ASN button or just clear an erroneous one from the box with the CLEAR ASN button. You can use several utilities in the Show or the Restraints pulldown. To use the Restraints/NOE_DIST or NOE_VOL options you must have scalar peaks defined and also must have measured the peak volumes. With these options you can define or redefine restraints peak by peak. With the Show utilities you can: measure distances between the constituent atoms of possible frequency assignments in the current model, see the NOE buildup (if you measured one previously), see unassigned atoms that are within 7 ┼ of the atoms to which this peak can possibly be assigned, and draw the frequencies on the peak box to help to distinguish visually between the possibilities. Finally, if you are working with a 2D NOE spectrum you can jump to the symmetric peak and assign it, verify the assignments, or identify overlap problems.

Assign/Peak Assign/Manual Assign Singly 3D

The Assign/Peak Assign/Manual Assign Singly 3D (<Alt>-akd) menu item is used to manually assign peaks in a 3D HSQC-NOESY type experiment. You must first select and display a 3D HSQC-NOESY type experiment before selecting this menu item. This menu item changes the cursor to one that you can use to select the peak you want to assign. FELIX then checks the cross-peak table for the HSQC-NOESY experiment to see if assignments already exist. If they do, they are displayed along with the chemical shift and tolerance for each dimension.

The control panel that appears contains two list boxes. The list box on the left displays matching assignment pairs for the HSQC dimension. To be listed, both assignments must be from the same residue and the frequencies for each assignment must both be within the given tolerance for that dimension, as shown in the control panel. Along with the assignments, the difference in chemical shift between the selected cross-peak and the assigned frequency is show for both HSQC dimensions. Often, the correct assignment is the only one listed. If multiple possible assignments are listed, the correct one can usually be selected based on the chemical shift deltas. If no possible assignments are listed or if you want to display more possibilities, then you can increase the tolerance for the corresponding dimensions and click the Refresh button. All assignment pairs matching the new tolerances are then displayed.

The list box on the right shows the possible assignments in the NOESY dimension. To generate this list, FELIX goes through all assigned frequencies and displays those that are within the specified tolerance for the NOESY dimension. The distance to this proton is displayed, along with the difference in the chemical shift between the selected cross-peak and the assigned frequency. This list is sorted by distance. After you select a new HSQC assignment pair in the left list box or change the NOESY dimension tolerance, update the list by clicking the Refresh button.

After you are satisfied that you have selected the correct assignments, you can transfer the assignments to the HSQC-NOESY cross-peak table by clicking the Assign button in the Store Assignment rectangle. You can then click NEW PEAK to select a new peak for assignment and start the process over, or click Cancel to exit the control panel.

Assign/Peak Assign/Manual Assign Multiply

In FELIX it is possible to have peaks with multiple competing assignments stored. You can use automated assignment to store that information, or you can use the Assign/Peak Assign/Manual Assign Multiply (<Alt>-akm) menu item to store, alter, or delete such information. If a peak has multiple possible assignments stored, then in the control panel this will show up. Then you can delete from those multiple assignments, add new ones, or promote one particular multiple assignment to a single assignment. You can use utilities from the Show or the Restraints pulldown. To use the Restraints/NOE_DIST_OVLP or NOE_VOL_OVLP options, you must define scalar peaks and measure the peak volumes. With these menu items you can define or redefine restraints peak by peak. From the Show pulldown you can: measure distances between the constituent atoms of possible frequency assignments in the current model, see the NOE buildup (if you measured one previously), see unassigned atoms that are within 7 ┼ of the atoms to which this peak can possibly be assigned, and draw the frequencies on the peak box to help to distinguish visually between the possibilities. Finally, if you are working with a 2D NOE spectrum, you can jump to the symmetric peak and assign it, verify the assignments, or identify overlap problems.

Assign/Peak Assign/Autoassign Peaks

The Assign/Peak Assign/Autoassign Peaks (<Alt>-aka) menu item is used to transfer frequency assignments to peak assignments. Therefore it is crucial that you be finished with frequency assignments before you use this menu item. You should also set and adjust the spectrum-specific shifts for the spectrum to which peaks are to be assigned, if necessary. Since the algorithm uses the molecule's structural information to decide whether the two atoms that the NOE transfer are assigned to are within the specified cutoff, the results depend on the actual structure: fewer peak assignments are made for a linear-chain protein than for a partially or fully folded one. The algorithm is described in Chapter 2., Assign building blocks You can specify the cutoff distance for NOEs (Rejection Cutoff) and the Peak Entity to assign. You can also specify whether to strictly enforce this distance cutoff (Strictly Enforce Dist). With this setting, when there is only one assignment possible for a given peak, then the assignment is retained, although the distance between the two atoms is more than the rejection cutoff. This option can be useful if all the resonances are assigned in a molecule and the structure is unfolded. You can choose to let the program multiply assign peaks; if so, it is possible to define a second cutoff distance (Unambiguous Cutoff), which is used to distinguish between multiple possible assignments. You can choose not to assign peaks that are already fully assigned (Skip Fully Assigned) or multiply assigned (Skip Multiply Assigned). Finally, you can select the Output Level.

Assign/Peak Assign/Reassign Peaks

The Assign/Peak Assign/Reassign Peaks (<Alt>-akr) menu item reassigns certain peaks based on a list. You would typically use this menu item after hotspots were found in generated structures. Hotspots can result from misassignment or overlapped peaks. For misassignment, you use a list to reassign (or unassign) those peaks. You can use a simple ASCII file that just has the two (or three or four, depending on dimensionality) names in a row separated by a blank, or you can create a list from within Insight II using the following procedure: first load the restraints on all the refined molecules into Insight II using the NMR_Refine/Restraints/Read molname* command. Then execute the NMR_Refine/Distance/List command. The provided numvioltofelix script redirects the resulting output to another file. This is the file you can use in the Filename parameter. Then you proceed similarly to the previously described Manual Assign Singly menu item, but instead of selecting the peaks by cursor, the peaks are automatically centered in your current frame after selecting them from the table and the corresponding control panel is opened.

Assign/Peak Assign/Check Consistency

The Assign/Peak Assign/Check Consistency (<Alt>-akc) menu item checks the consistency between frequency assignments and peak names. In FELIX, each peak has a stored pointer to a frequency and a name for each dimension. If after assigning a peak to a frequency, you reassign that frequency, there can be an inconsistency-that is, the peak name will be different from the frequency name (that it points to). This can happen if, for example. you assign a frequency first to an HB1 and assign the peak accordingly, and then reassign that frequency to HB2. The peak name is still HB1, but the frequency to which it points is named HB2. To correct this kind of discrepancy you need to use the Check Consistency menu item. In the control panel, the Peak Entity defaults to the current experiment's peak entity. You can change this setting.

Assign/Peak Assign/Unassign Peaks

The Assign/Peak Assign/Unassign Peaks (<Alt>-aku) menu item serves to unassign all peaks in the currently active experiment.

Note: There is no way to cancel this menu item.


Pattern/List Membership

You can use the Pattern/List Membership (<Alt>-nl) menu item to find out which pattern a particular peak belongs to (i.e., which is the closest pattern within the spectrum-specific tolerance).


Pattern/List Frequency

This menu item allows you to determine which pattern(s) a particular cursor position can belong to (<Alt>-ny).