Restraints and Violations

Introduction

In CcpNmr Analysis (Analysis), many kinds of NMR data can be used to generate restraints and the restraints themselves are of different types, such as distance or angles etc. Analysis has a management system which displays tables for any kind or restraints in the CCPN data model. Restraints are typically used as input data for structure calculation programs.

Creation of Restraints

Structural restraints can be created with various tools available in the CCPN software. For example distance restraints may be made via the Make Distance Restraints functionality (using through space experiments like NOESY) and the Make H Bond Restraints functionality. Dihedral angle restraints can be made from chemical shift information using the DANGLE functionality and from scalar couplings using the 3J H-Ha Coupling functionality.

Import and Export of Restraints

The FormatConverter can be used for import and export of restraints in a textual file format specified by the user. This is a convenient management system for Analysis where the user can, for example, export restraints for use in different structure calculation programs and then import structural information back to Analysis, to help with violation analysis and NOE peak assignment.

Restraint Sets

Commonly, several sets of structures are generated during repeated cycles of structure calculation. Each set of structures derives from a specific set of restraints, and new structures are used to make an improved restraint set for the next round. Clearly, the user needs to store the restraints that are used together each time, which makes it easier to keep track of which input data were used to generate which structures. To achieve this in Analysis, each set of restraints is stored as a separate entity, the Restraint Set. Restraint Sets are composed of Restraint Lists which in their turn are composed of Restraints (see Figure 1).

A Restraint Set often represents all 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. The idea is that the user creates the Restraint Sets and modifies them only until it is time to use them for structure calculation. In essence, a Restraint Set takes a “frozen” 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. It is a good practice to always make a new Restraint Set if assignments have changed.

Restraint Lists

A Restraint List groups together any number of Restraints of the same type (i.e. angle and distance restraints cannot be mixed in a list). Restraint Lists can 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 splitting a Restraint List:

• • To separate 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 contains Restraints that represent multiple, ambiguous assignments.

  • To split a Restraint List into violated and not violated Restraints during violation analyses in aim to determine which restraints are incompatible (e.g. mistaken assignment, minor conformation or artifact) with a structural model.

Restraints

Restraints are listed in Restraints Lists. Resonances may indicate a single unambiguous possible connection between two different pairs of resonances (usually 1H), or they may be ambiguous and have additional, alternative possibilities for the restrained resonances.  In Analysis, the first resonance possibility is represented by a green row (in the restraint tables) and alternatives are represented as grey rows. Note that such ambiguous restraints can represent logical uncertainty (before an NOE is resolved) or real physical ambiguity where a peak is caused by two or more overlapping pairs of resonances. A Restraint can be deleted from a Restraint List.

Restraints are linked to peaks through Resonances, which makes it possible to navigate between the Restraints and the spectrum peaks, that they are derived from. It is also possible to both refresh a Restraint according to the latest peak assignment or to refresh the peaks according to the Restraints (see Figure 1) (e.g. after an ARIA or CYANA run that removes restraint ambiguity). It should be noted that the link between restraints and peaks can generally only be maintained if the restraints were created by CCPN or CCPN-complicant software.

Figure 1. A Restraint Set is a container for Restraint Lists which is a container for Restraints. Restraints and Peak Assignments are both influenced by Resonances.

Violations

A violation records an inconsistency between a Restraint and a structure. Essentially this means that a structural value is outside the bounds of the Restraint. Any Restraints that were violated in a structural analysis will be coloured; red, orange or yellow depending on severity. Restraint violation analyses may be performed by the structure calculation software itself (e.g. ARIA) or inside Analysis, given a suitable structure. Further details about the violations can be found, such as the amount of violation and the proportion of structures (in an ensemble) affected. Different functionalities can be used to analyse Restraints and Violations dependent on the Restraint type, e.g. one functionality for distance restraints allows the user to switch between the “NOE sum” method and the “Minimum” method. The “Minimum” method 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.

Violation Lists

A Violation List is a grouping of violations within a Restraint Set that relate to a given structure. The violations listed may relate to restraints that belong to several different Restraint Lists. Often there will only be a single Violation List for a given set of restraints. However, further lists can be present; if more than one structure was analysed or if the violations are calculated using a different method.