Molecule : Isotope Labelling

Setup Molecule Isotope Labelling Patterns

This popup is used to specify the isotopic incorporation of molecules in different samples. This is typically done in the earlier stages of a project, before the labelling information is required for assignment etc.

Although the user can employ the standard isotope labelling schemes when performing operations in Analysis, the setup of molecular isotope patterns allows a greater level of control and allows the isotopic labelling to be automatically set according to context. The standard labelling schemes, which are also used as references for molecule specific patterns, may be created and edited via the Reference Isotope Schemes popup.

A given molecule can be represented by several different samples, each with distinct isotope patterns. Each sample is associated with the NMR experiments (and hence spectra) that were performed on that sample. When assigning a peak, if the peak’s experiment has been linked to a sample with labelling information then the isotopic proportions hat should be considered are automatically known.

Labelled Molecules

The first tab allows the user to setup the samples (labelled versions) of each molecule that are available. Each molecule version will appear as a row in the top table. The user should set the “Experiments” of each sample to specify which NMR experiments used that labelled version on the molecule.

Each sample of a molecule may refer to only a single labelled version or a mixture of different labelled forms (of the same molecule), e.g. both fully labelled and unlabelled. These different forms are referred to as “Patterns” and can be used in any proportion, to create a sample with a mixed composition.

The Sample Composition table allows the user to add patterns (labelled versions of the molecule) to the currently selected mixture. Often the user will add a single pattern based on a standard labelling scheme; this is the simplest setup. However, any number of patterns may be added and their proportions in a mixed sample may be adjusted by changing the “weight” of each. It is possible for a given pattern to be re-used in a number of different samples. For example a natural abundance version of the molecule may appear on its own in a sample, but as a mixture together with an enriched pattern in another sample.

Labelling Patterns

The second tab allows the user to specify any deviations of a labelling pattern (version of a molecule) from a standard labelling scheme. An initial pattern will contain residues that are associated with an initial scheme; the residues isotopomers are set to be the same as in this scheme. However, the user may adjust any of the residues in the pattern to use a different scheme. Here the term “isotopomer” is used to refer to a single isotope pattern of an individual residue, and there may be several such patterns present for a given type of residue, so that a polymer molecule will generally be a random mixture of the different individual forms.

A residue within a pattern may deviate from a standard scheme by adjusting the proportions of the different isotopomers; this is set via the “Weighting” value after selecting the required isotopomer in the pulldown menu. Adjusting these weightings preserves the internal correlations between atoms within isotopomers; only the relative abundance of separate isotopomer forms is affected.

The individual atomic incorporations for a residue may be adjusted if the “Isotopomer” pulldown is set to “Override Scheme”, but this looses any atom pair information that was specified via an isotopomer; only average incorporations are set. For example there may be no single isotopomer with both CA and CB labelled at the same time, even though the average shows incorporation for both sites.

Examples

These examples illustrate how to setup various kinds of isotope labelling “sample”, starting from a project with molecule specifications but without existing labelling information. After creation each sample should be associated with the required NMR experiments (which were run on that sample), by double clicking in the “Experiments” column of the Labelled Samples table and checking the required options.

Simple Single Scheme Labelling

As an example of specifying a molecule with a single pattern from the standard “1,3 13C Glycerol” scheme, the user would first select the required molecule (to the right of “For molecule”) and press [New Sample]. In the lower right pulldown menu the user then selects “13Glycerol” and then presses [New Pattern From Scheme]. The molecule in this sample will have the labelling specified by one pattern made according to the selected scheme.

Mixture of Labelled and Unlabelled

As an example of a mixture, say that a sample of a molecule was composed of a 1:3 ratio of fully labelled and unlabelled molecules (two different patterns in a mixture). The user would make one sample for the molecule using [New Sample], then setup the Sample Composition to have two patterns, one based on a uniform labelling scheme (e.g. “uni_15N13C2H”) and one for the “NatAbun” scheme; select each scheme in the lower right pulldown menu and for each make [New Pattern From Scheme]. Labelling patterns “A” and “B” will be created. For the pattern that corresponds to the “NatAbun” basis scheme set the weight to 3 (the weight for the other pattern will remain at 1.0). This labelled sample specification is nor ready to used because the individual residues do not need any further adjustment.

Labelling a Specific Residue

As an example of a molecule that is unlabelled except at one specific sequence position, where the residue has 15N, the user would first make a New Sample for the required molecule and add a New Pattern from the “NatAbun” scheme; this will make Pattern “A”.

The user should then go to the second “Labelling Patterns” tab (at the top) and ensure that the correct molecule and Pattern “A” are selected in the upper pulldown menus. The sequence of the molecule will be displayed in the upper table. To set the labelling for the only labelled residue, the user scrolls down until the residue is visible, then selects the row for the residue in the table (highlighting the row). Next, in the pulldown menu to the bottom right of the residue table, the user selects the scheme “uni_15N” and then presses the adjacent [Set Residue Composition From Scheme:]. The residue will now be listed in the table as using the “uni_15N”, and not “NatAbun” like all the others.

Main Panel

button Clone: Clone popup window

button Help: Show popup help document

button Close: Close popup

Labelled Molecules

Setup isotope labelling at the molecule level; the versions of a present molecule in different samples

Labelled Samples

Table 1
Molecule The name of the molecule (sequence template) that the isotope labelled sample refers to
Sample The serial number of the sample (mixture of labelling patterns) in the CCPN project
Sample Name A user editable name for the isotope labelled sample (Editable)
Experiments The NMR experiments that are asscociated with the isotope labelling sample; can be set at any time by the user (Editable)
Labelling Patterns One or more labelling patterns (versions of a molecyle), in diifferent proportions, constiture the sample
Chains The molecular system chains (assignable entities in a complex) that are associated with the labelled molecule
Sequence Part of the residue sequence of the labelled molecule
Mol Mass The estimated average molecular mass of the molecule (albeit a mixture of different patterns)

button Delete Sample: Delete the selected isotope labelling molecule specification

button New Sample: Make a new, intially bank, isotope labelled sample, to which molecule labelling patterns may be added

pulldown For molecule: Selects which molecular sequence template to use when making a new specifcation of an isotope labelled sample

Sample Composition

Table 2
Labelling Pattern A code letter for a specific labelling pattern version of the molecule sample
Basis Scheme The standard isotope labelling scheme on which the labelling pattern is primarily based (for mosed residue)
Weight The relative weighting of this labelling patterns compated to other patterns in the sample sample; for a mixture of molecule versions (Editable)
% Composition The relative composition of this pattern compated to others in the sample
Mol Mass The estimated molecular masss of the version of the isotopic molecule represented by the pattern

button Remove Pattern: Remove the selected molecule labelling pattern from the sample/mixture; the pattern is deleted if it is no longer used in any sample

button Edit Pattern: Edit the selected molecule labelling pattern (isotopic version), in terms of residue and atom incorporations

button Add Existing Pattern: Add a molecule labelling pattern to the current sample specification, using a pre-constructed pattern

pulldown *None*: Selects which extsing molecule labelling pattern to add to the selecte sample

button New Pattern From Scheme: make a complete new isotope labelling pattern for the molecule using the selected isitope labelling scheme

pulldown *None*: Selects which standard isotope labelling scheme to use as a template to make a new molecule specific labelling pattern

Labelling Patterns

Setup the specific residue and atom labelling patterns in a given molecule version

pulldown Molecule: Selects which molecule to view the residue labelling patterns for

pulldown Pattern: Selects which residue labelling to view from those available for the selected molecule

button New Pattern From Scheme: make a new labelling pattern (isotope version of the molecule) using the selected standard labelling scheme

pulldown *None*: Selects which standardised isotope labelling scheme to use as a template for creating a to make a new molecule specific labelling pattern

Residue Pattern

Table 3
Seq Id The sequence ID number of the labelled residue in the molecule; a separate numbering system to the numbering in chains
Residue Code The code of the residue identifying which type it is
Schemes The names of the standard isotope labelling schemes on which the residues labelling is based within this specific pattern
Isotopomers Tne numbers of the isotopomer versions of this residue that specify labelling; from the standard scheme

button Set Natural Abundance: Set the selected residue labelling to have natural abundance isotopic incorporation labels.

button Set Residue Composition From Scheme: Set the selected residue to have labelling based upon isotopomers from the selected standard scheme

pulldown *None*: Selects which standardised isotope labelling scheme to base the residues isotopomer labelling upon

Residue Composition

pulldown Chem Element: Selects which chemical element to display atomic isotope incorporation levels for

check Show Exchangeable: Sets whether to display water exchangeable atoms in the atom isotope table

pulldown Isotopomer: Selects which isotopomer version, from those set for the residue to show atom istopes for; can be set to override the schem istopomers

float 1.0: Specifies what relative weighing the selected residue isotopomer has compared to others for the same residue

Table 4
Atom The name of the atom within its residue, for which to set isotopic abundances, within the selected labelling pattern
Weighting ¹²C The amount of ¹²C isotope incorporation; can be a ratio, percentage or fraction (the value used is relative to the sum of all weights) (Editable)
Weighting ¹³C The amount of ¹³C isotope incorporation; can be a ratio, percentage or fraction (the value used is relative to the sum of all weights) (Editable)
% ¹²C The percentage ¹²C isotope incorporation for this atom, calculated using stated weights
% ¹³C The percentage ¹³C isotope incorporation for this atom, calculated using stated weights

button Reset Residue To Orig Scheme: Resets the selected residue to use atom isotope incorporation levels from the isotopomers of the set scheme; removes any atom level edits

button Propagate Abundances: Spread the istotope incorporation levels from the last selected atom (in the table) to all selected atomes; overrides isotopomer levels

button Set Natural Abundance: Set the selected atoms to have natural abundance isotope incorporation levels