Structure : Restraints and Violations

Display Structure Restraint and Restraint Violation Data

This popup window is used to manage and display structural restraints. Mostly such restraints are derived from NMR data and are applied during a structure calculation to restrict molecular conformations to those which are consistent with the observed data. Many kinds of NMR data can be used to generate restraints and the restraints themselves are of different types. This system will display tables for any kind or restraint described in the CCPN data model, but the most common kind of restraints are for distances and dihedral (torsion) angles.

This system is used for viewing and managing restraint information on a large scale, it is not used directly for the initial generation of the restraints themselves. Structural restraints may be imported into CCPN via the FormatConverter or they may be created with various various dedicated components available in CCPN software. For example distance restraints may be made via the Make Distance Restraints (using through space experiments like NOESY) and Make H Bond Restraints options. Dihedral angle restraints can be made from chemical shift information using DANGLE and from scalar couplings using the 3J H-Ha Coupling option.

Restraint Sets

The first “Restraint Sets” tabs lists all of the sets into which the restraint lists of the project are grouped into. The table indicates how many restraint and violation lists are in the set as well as an indication of the number of “fixed” resonance assignments that have been frozen in the set. Restraint sets may be deleted here, but are created elsewhere; when restraint lists are created they can be put into a new or existing set.

Within CCPN, restraints are stored in restraint lists which group together restraints of the same type (i.e. angle and distance restraints cannot be mixed in a list). Restraint lists are further grouped together into restraint sets. A restraint set often represents all of the restraint lists that will be used together at the same time during a structure calculation. However, this grouping is slightly more subtle because a restraint set has important consequences with regards to NMR resonance assignment. In essence, a restraint set takes a fixed snapshot of the resonance assignment status at the time the restraints were made. Consequently, if atomic assignments change after restraints are generated the restraints will remain linked to the original atoms. This is helpful to the user because it is always possible to know what was actually restrained in a structure calculation. The user should however be careful when putting new restraints into old restraint sets; if atom assignments have changed then even the new restraints (actually between NMR resonances) will still restrain the old atom assignments. Thus it is good practice to always make a new restraint set if assignments have changed.

Restraint Lists

The second “Restraint Lists” tab displays all of the restraint lists that are available in the selected restraint set. The restraint set may be selected in the pulldown menu above the tabs. Generally this table is used to give an overview of the restraint lists but several high-level functions are accessible via the buttons below the table. As described above, the actual restraint generation takes place elsewhere in dedicated systems.

Restraint lists my be deleted and merged/combined together into a single list (if they are of the same type). It is notable that restraint lists may also be split. Currently there are two means of doing this, the first is to [Split Ambig/Unambig] which separates a distance restraint list into two, where one list contains only restraints that are equivalent to a single assignment (only two linked atoms or prochiral sets) and the other list has restraints that represent multiple, ambiguous assignments. The [Split Violated] function is helpful during violation analyses, which aim to determine which restraints are incompatible (e.g. mistaken assignment, minor conformation or artifact) with a structural model. This will separate out a list of those restraints that are violated in a structure (beyond the restraint bounds) for further inspection.

Restraints

The third “Restraints” tab allows the user to view all of the individual restraints within a restraint list, selected via the left hand “Restraint List” pulldown. If any structure violation analyses have been performed on the list the user can select from the “Violation List” pulldown. Any restraints that were violated in the structural analysis will be coloured; red, orange or yellow depending on severity. Also where violations are recorded the columns for “Mean Viol”; average violation amount over a structure ensemble, and “Viol Fraction”; the proportion of an ensemble’s models that were violated, are filled.

Selecting from the “Structure” pulldown menu allows geometric information to be extracted from a structure ensemble, so that it may be compared with the values that are being restrained. Accordingly, if a structure violation analysis is performed using [Calculate Violations] then it is the selected structure that is compared with the restraints to find inconsistencies. Also, selecting as structure allows the “Struc Value” column to be filled with the value calculated from the resonance locations in the structure. Here the kind of value naturally differs according to which type of restraint list is displayed.

The “Value Method” pulldown menu is notable for distance restraints because it allows the user to switch between “NOE sum” and “Minimum” options. These are relevant because a restraint, which restrains pairs of resonances, may link groups that contain multiple atoms (e.g. a methyl group). With multiple atoms at the end of a distance restraint there is flexibility about how the distance is defined. The “Minimum” method to measuring distances simply records the shortest distance between any pair of atoms from either side of the restraint (e.g. the closes atoms in two methyls). The “NOE sum” method uses r^-6 distance summation to give a value that represents what the equivalent NOE estimated distance would be if a signal were recorded between the two atom groups. For example, if a restraint is to a methyl group all three methyl atoms will contribute to increase the intensity of an NOE signal, so the distance will appear to shorter than the single atom equivalent. Hence it is this shorter distance that will come from the NMR data and appear in the restraint, and thus the shorter distance that should be used in violation analyses. The get the “NOE sum” distance the pairs of atom distances are first converted into a kind of ‘NOE intensity’ (r^-6), these are added together to give the total ‘NOE intensity’, which is then converted back into a distance.

In the main restraints table the restrains are listed as coloured rows, but there will often be grey rows present. A grey row represents an alternative set of restrained resonances (e.g. a different pair of atoms) but relates to the same restrained value. The main coloured row for a restraint and the grey alternatives are both referred to as the “items” for the restraint. Hence at the bottom there is the [Delete Items] button to remove specific restraint alternatives. In this regard there is no difference between the first green item and the other possibilities; it is fairly arbitrary which item comes first. Naturally deleting all of the items (possibilities) in a restraint removes the whole restraint. Although the first item is often coloured green it may have other colours indicating a structural violation. A restraint with more than one item may reflect either a genuine signal overlap, e.g. an NOE peak is caused by two or more close atom pairs that have similar chemical shifts and cannot be separated, or ambiguous trial assignments that come from speculatively matching chemical shifts to spectrum peaks.

The various functions below the table allow restraints to be managed but the restrained resonances, and the parameter values they relate, to come from specialised restraint generation systems. In general the restraint information is derived from NMR experimentation and not adjusted by hand, Several functions allow the user to link between the restraints and any spectrum peaks that they are derived from. This can be done after selecting restraints with [Show Peaks] or after selecting peaks with [Show Restraints For Selected Peaks]. There are also functions to coordinate assignments between the restraints and peaks. How this is done in practice tends to vary with personal preference, but it is possible to both refresh a restraint according to the latest peak assignment with [Update Assignment From Peak] or refresh the peaks according to the restraints (e.g. after an ARIA or CYANA run that removes restraint ambiguity) with [Update Peak Assignments].

Violation Lists

The forth “Violation Lists” tab gives details of all of the violation analyses that have been recorded in the project. Strictly speaking, each violation list can contain results from several analyses but this is usually not the case in normal Analysis operation. The individual violation records are presented in the last “Violations” tab, and also affect the violation data presented in the “Restraints” table. Violation lists may be calculated, given a structure and a restraint list, inside Analysis using [Calculate Violations] in the “Restraints” tab, or they may be imported from external programs like ARIA.

A violation list is a grouping of violation entities, and these record an inconsistency between the restrained values of a restraint and what is actually observed in a structure (or ensemble). Essentially this means a structural value is outside the bonds of the restraint. Clicking on a violation list in the upper table will fill the lower table with an overview of the structure models that were used in the restraint violation analysis (usually just a single ensemble).

Violations

The last tab lists all of the violation items that were recorded in a selected violation list. This table will present some of the same information as the “Restraints” tab, if a violation list is selected there. The data is merely presented in a different way. Selecting a specific row in the table allows the user to [Show Restraint], to view the restraint that was violated in the “Restraints” tab. Other functions allow the user to jump to the peak, that gave rise to the restraint, which may be the underlying source of the problem.

Caveats & Tips

Having large restraint sets in a CCPN project can make loading the CCPN data into Analysis slow, and takes up more memory. Thus it is advisable to delete any restraint sets that are no longer in use,

If the restraint table is slow to update, consider switching off any structure selection. Extracting values from large structure ensembles can take a noticeable amount of time.

The [Calculate Violations] function will not work until a Structure is selected.

Although the CCPN violation analysis tries to do a good job it can never be exactly the same as analyses performed during a structure calculation. Most of the reason for this is due to the stereo-specific resolution of prochiral atoms. For example, Analysis can only determine which of HBa or HBb atoms actually goes with the HB2 or HB3 sites by taking a poll (based on minimising violation) after the structure calculation is complete, and this prochiral resolution may differ from what actually occurred in the calculation (minimising energy).

Main Panel

pulldown Restraint Set: Which set of restraints is currently active in the popup; a grouping for restraint and violation lists

button Clone: Clone popup window

button Help: Show popup help document

button Close: Close popup

Restraint Sets

Documentation missing

Table 1
# Serial number of restraint set
Restraint Lists Number of restraint lists, of all types, in the set
Violation Lists Number of violation lists in the set
Fixed Resonances Number of resonances (fixed in assignment) used to describe restraints in the set
Fixed Atom Sets Number of atom groups (fix in assignment) used in the set’s restraints
Fixed Resonance Sets Number of fixed resonance to atom set assignments
Chain States Number of alternate chain conformations explicitly covered

button Import: Disabled option, import restraints from file. Use FormatConverter

button Delete: Delete the selected restraint set with all its restraints and violations

Restraint Lists

Documentation missing

Table 2
# The restraint list serial number, within its containing restraint set
Type The restraint list type; distance, dihedral, h-bond etc.
Name The human name to identify the restraint list (Editable)
Restraints The number of individual restraints in the list
Experiments The NMR experiments used to derive the restraints
Details User editable textual comment for the list (Editable)
Unit The units of the restrained quantity

button Show Restraints: Show a table of individual restraints for the selected restraint list

button Export ARIA list: Export the selected restraint list (if distance type) as a CNS .tbl file for ARIA v1

button Create Equiv. Peaks: Make peaks equivalent to restraints. Requires that the restraints were initially made in CCPN

button Delete Selected Lists: Delete the selected restraint lists

button Merge Lists: Merge the selected restraint lists into a single list (if the same type)

button Split Ambig/Unambig: Split the last selected restraint list into two new lists; one which has ambiguous (excluding prochiral ambiguity) atom restraints and one which does not

button Split Violated: Split the last selected restraint list into two new lists; one with violated restraints, one with non-violated

Restraints

Documentation missing

pulldown Restraint List: Select the specific list to display restraints for; number:type:name

pulldown Violation List: Select which violation list, if any, to display restraint violations from

pulldown Structure: Select which loaded structure to use for restraint value comparison

pulldown Value Method: Select how values are generated from a structure: “NOE sum” is used where ambiguous distance restraint options all contribute NOE peak intensity

Table 3
# Serial number of restraint & any ambiguous item (restraint:item)
Resonances The name of the resonances restrained (fixed within the restraint set)
Value The target or best value for the restraint, e.g. optimum distance
Upper Limit The lower bound value for the restraint
Lower Limit The upper bound value for the restraint
Error An error value for the restraint, often just the with between bounds
Peaks The number of CCPN peaks used to derive the restraint information
Mean Viol The average value of a violation over a structure ensemble
Viol Fraction The fraction of a structure ensemble’s conformations (models) violated by the restraint
Struc Value The value of the data being restrained in the selected structure ensemble
Orig. Data The data value used to derive the restraint e.g. relative peak intensity
Weight The relative strength weighing of the restraint (not used by all structure calculation protocols)
Details User-editable textual comment for the restraint (Editable)
Items The number of ambiguous or alternative possibilities on the restraint

button Delete Items: Delete the selected restraint items; assignment possibility not necessarily whole restraint

button Delete Restraints: Delete all (whole) restraints for selected items, even where only one item of an ambiguous restraint is selected

button Show Restraints for Selected Peaks: If present in the table, highlight the rows of the restraints derived from peaks selected in spectrum windows

button Update Assignment From Peak: Update the items (assignment possibilities) for a restraint from any linked CCPN peak

button Show Peaks: Show a table of peaks from which the selected restraints were derived

button Show Selected On Structure: Show the connections of the selected restraints on a graphical structure display

button Assign Peak: Assign the peak from which a restraint is derived

button Show Violations: Show a table of individual violation records for selected list

button Calculate Violations: Calculate restraint violation records for restraints using selected structure

button Export ARIA List: Export the restraints as a CNS .tbl file for ARIA v1

button Delete List: Delete the entire restraint list currently on display

button Setup Resonances: For imported restraints add any possible CCPN resonance links

button Merge Duplicates: Merge any restraints that represent the same atoms pairings together, e.g. from reciprocating return NOE peaks

button Update Peak Assignments: Update the assignments of any linked CCPN peaks according to the current restraint items

Violations Lists

Documentation missing

Table 4
# The serial number of the violation list
Violations The number of violation records in the list
Structures The number of structural models used in violation calculation
Details User-editable textual comment for violation list

button Show Violations: Show a table of individual violation entries for the selected list

button Delete Lists: Delete the selected violation lists

Structures Analysed

Table 5
# Number of structure ensemble used in violation analysis
Models The number of the conformational models used in the violation analysis
Chains The molecular chains present in the structure
Generation Any structure generation group to which the structure belongs

Violations

Documentation missing

pulldown Violation List: Select which violation list to show violation items for

Table 6
Restraints List The restraint list that the violations were calculated for
Restraint The restraint that was analysed in terms of value & bounds
Amount The amount violated; difference from the nearest restraint bound
% Violated The % of conformational models violated in structure
Type The type of the restraint analysed; distance, dihedral etc.
Peaks The number of peaks used to derive the constraint
Calc Value The structure-derived value use to calculate the violation
Value Error The error in the violation amount

button Assign Peak: Documentation missing

button Show Peaks: Documentation missing

button Show Restraint: Documentation missing

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