This popup allows the user to run an automated sequential protein backbone assignment with the program Nexus. After inspection of the results, the user can discard or commit automated assignments to the project.

In order to run an automated backbone assignment the following requirements need to be met:

• • The project must contain one or more of the following types of NMR spectra that are typically used for assigning sequential connectivities in proteins:

    • HNCA

    • HNCO

    • HNCACB

    • HNCB

    • HNC

    • HN(ca)CB

    • HN(co)CA

    • HN(co)CACB

    • HN(coca)CB

    • CBCAcoNH

    • CAcoNH

    • CcoNH

  • Peaks must have been picked in the backbone spectra and be assigned as follows (note that genuinely missing peaks are generally tolerated):

    • The amide nitrogen dimension of all peaks must be (anonymously) assigned to N.

    • The assigned amide nitrogen resonances are contained in spin systems

    • Multiple peaks that are assigned to the same nitrogen are assumed to correspond to the same residue

To run the assignment first a number of options need to be set in the {Options} tab, such as the spectra to be used and the tolerances for matching peak positions. Subsequently, resonances are searched, categorised into inter and intra-residual peaks, and curated by the user by manual inspection of the resonance categorisation in the {Spin Systems} tab. Following the setup of the resonances, the automated sequential backbone assignment routine is run ({Automation} tab), and results are displayed in the {Score graph} and {Predictions} tabs. The {Predictions} tab is used to inspect the result and to discard or confirm an automated assignment.

Options

The {Options} tab allows the user to specify the settings used for running the automated backbone assignment. These include general settings, peak-peak matching tolerances and selection of spectra used in the routine.

The Assignment: Automated Backbone Sequence popup (Options).

General

This panel contains the general options that are used in the automated sequential backbone assignment procedure.

Chain

Specifies the chain used for the automated assignment.

Shift List

Specifies the chemical shift list used for the automated assignment.

Window

Specifies the navigation window used for the automated assignment.

Peak-Peak Match Tolerances

This panel contains the options to set the peak to peak matching tolerances.

13C (ppm)

Specifies the peak to peak matching tolerance in the 13C dimension in ppm.

1H (ppm)

Specifies the peak to peak matching tolerance in the 1H dimension in ppm.

Peak Lists

This panel includes the peak lists table used for the automated sequential protein backbone assignment.

Peak Lists table

This table lists all the peak lists that can be used for the automated sequential protein backbone assignment. 

Spectrum

The name of the spectrum associated with the peak list.

Experiment Type

The experiment type of the spectrum associated with the peak list (see also Experiments: Experiment Prototypes).

Use?

If set to 'Yes', the peak list is used in the automated sequential protein backbone assignment.

Num Peaks

The number of peaks in the peak list.

Spin Systems

The {Spin Systems} tab is used for automatically finding resonances for spin systems and assigning them to atom types based on the peak lists set in the {Options} tab. Finding the resonances and their atom type assignments is done using the [Find Resonances From Peaks] button, and the resulting assigned resonances are listed in the Spin Systems table from which the automated atom type assignments can be inspected and modified.

The Assignment: Automated Backbone Sequence popup (Spin Systems).

Spin Systems table

This table lists the spin systems in the project with their and their assigned resonances. A distinction is made between intra and inter residue resonances.

#

The spin system serial number.

Residue

The residue assignment of the spin system.

Intra residue resonances

The atom types and chemical shifts of the resonances assigned as intra residue resonances.

Inter residue resonances

The atom types and chemical shifts of the resonances assigned as inter residue resonances.

Comments

Comments generated by Analysis during the resonance finding procedure (see [Find Resonances From Peaks]).

Find Resonances From Peaks

This command is used to start the search for intra and inter residue resonances and their atom type assignments based on the selected peak lists. The procedure only adds missing resonances, and leaves existing assignments unchanged. The Spin Systems table is updated with the new assignments, and any problematic assignments are highlighted in red and commented in the |Comments| column.

Show Peaks

Opens the Peaks: Selected Peaks popup for inspection of the peaks of the selected spin system.

Go To Root Location

Navigates to the root location (H, N) of the selected spin system in the spectrum window specified in the {Options}. Note that the spectrum window has to be open for navigation.

Automation

The {Automation} tab is used for setting additional parameters and running of the actual automated sequential protein backbone assignment using the Nexus program.

The Assignment: Automated Backbone Sequence popup (Automation).

CcpNmr Nexus

This panel contains the parameters and action button for the Nexus program.

Keep existing assignments?

If selected, existing assignments are kept during the automated assignment procedure.

Run Nexus

Starts a Nexus run for automated sequential protein backbone assignment using the spectra and options specified by the user. Intermediate results for each iteration are displayed grahically in a transient Nexus result popup.

Gaussian <dimension> variance (ppm)

Specifies the Gaussian variance to be used in Nexus for <dimension>. This parameter is used to generate matching scores between peaks of potential sequential neighbours, where one peak position is compared to the height of a Gaussian centred at the other peak position (peak intensities are not considered). The variance should be set to be roughly the same as the observed line width of the peaks.

In the routine, peaks with a slight degree of overlap will be considered, even if the match is poor.

If the Gaussian variance is set too large, high-confidence regions of assignment in the sequence will generally not be negatively affected, however there is an increasing chance of the poorer matching regions giving a false positive result or at least obscuring the right answer: Peaks that certainly do not originate from the same resonances give rise to some overlap.

If the Gaussian variance is set too small only the best matching regions will give any result, and hence some sequential assignments might be missing. Also, the number of alternate possibilities suggested in the problematic regions will diminish, providing fewer indications where these problematic regions are. With good quality data, lowering the Gaussian variance may improve the convergence of the procedure.

Number of iterations

Specifies the number of iterations used in the Nexus run.

Score Graph

The {Score graph} tab provides the results of the Nexus run in the form of a graph (see also Columns Graph for the graph display and print options). The user is provided a direct overview of the quality of the assignments by colour coding the sequential assignments according to their reliability as Good, Mediocre and Bad.

The Assignment: Automated Backbone Sequence popup (Score Graph).

Predictions

The {Prediction} tab lists all the individual predicted sequential assignments from the Nexus run, and allows the user to inspect, edit, confirm and commit the assignments to the project.

The Assignment: Automated Backbone Sequence popup (Predictions).

Predictions table

This table contains all the sequential assignment predictions made by Nexus.

Residue

The name of the residue in the selected protein sequence.

Assigned Spin System

The spin system that is assigned to the residue. The assignment is made after committing a confirmed assignment to the project using [Commit Assignments].

Confirmed Spin System

The spin system of which the assignment to the given residue is confirmed using the [Confirm Selected] button.

Prediction Score

The calculated score for the prediction of the assignment. the higher the score, the more reliable the prediction is.

Best Matching Spin System

The spin system that is the best match assignment to the given residue.

1st Alternative Spin System

The spin system that is the first best alternative for assignment to the given residue.

2nd Alternative Spin System

The spin system that is the second best alternative for assignment to the given residue.

Score Threshold

The threshold above which assignment predictions are confirmed when using the [Confirm Above Threshold] button.

Confirm Above Threshold

Confirms all the assignment predictions with scores above the threshold specified under Score Threshold.

Confirm Selected

Confirms all the selected assignment predictions.

Clear Confirmation

Deletes all the assignment confirmations of the selected residue.

Strip Selected

Strips the selected spin systems in the navigation window selected in the {Options} tab.

Commit Assignments

Commits all the selected confirmed assignments to the project. Note that non-confirmed assignments are not committed.

Instructions

The {Instructions} tab contains the basic instructions for running the automated sequential protein backbone assignment.

The Assignment: Automated Backbone Sequence popup (Instructions).

Basic Instructions

Before you start it is assumed that:

a) You have backbone spectra from the allowed types: HNCA, HNCO, HNCACB,

   HNCB, HNC, HN(ca)CB, HN(co)CA, HN(co)CACB, HN(coca)CB, CBCAcoNH,

   CAcoNH, CcoNH.

b) You have picked peaks in your backbone spectra (although in general genuinely

   missing  peaks will be tolerated).

c) The picked peaks have all been assigned to (anonymous) backbone nitrogen

   resonances, in their amide nitrogen dimensions.

d) Multiple peaks assigned to the same amide nitrogen relate to the same residue.

e) The assigned amide nitrogen resonances are in spin systems.

Steps b)-e) may be easily achieved by running Assignment::Initialse Roots on an

HSQC then Assignment::Link Peak Lists to transfer amides and spin systems to

backbone experiments.

To run the assignment:

1) Check General options: Make sure you are working with the correct molecule

   chain, chemical shift list and window (to display sequentially assigned

   strips in).

2) Set the ppm tolerances. Within these tolerances peaks may be attributed to

   the same resonance, although if two match the closest will be used.

3) Toggle the "Use" column values to specify which spectra will be analysed.

4) In the backbone Spin Systems tab click [Find Resonance From Peaks]

5) Correct any problems in the spin system table - indicated by red rows.

   Typical issues include:

   * Having too many peaks e.g. 3 peaks for one amide in HNCA

   * Peaks don't match within tolerances: e.g. an HNcoCA peak doesn't match any

     of the HNCA peaks

6) In the Automation tab adjust the parameters as required and click [Run Nexus].

   MARS functionality will be incorporated in due course.

7) When the assignment algorithm is finished you will be presented with a graph of

   assignment reliability.

8) In the Predictions tab you must confirm all assignments before you commit them.

   Either set a threshold and commit en masse by clicking on 

   [Confirm Above Threshold], or confirm your manual selections via the button

   [Confirm Selected]. Use the [Strip Selected] option with a few sequential,

   confirmed assignments to check the tentative assignments in the spectra.

9) Click [Commit Assignments] to do the real assignment of your peaks and spin

   systems to residues of your molecular chain.