Make an automatic sequential assignment

Instructions

To perform an automated sequential assignment in Analysis, use the Automated Seq. Assignment popup. This popup is accessed through M: Assignment: Automated Seq. Assignment. The {Instructions} tab gives an overview of what requirements need to be met before one can start an automatic sequential assignment:

Setup

Go to the {Settings} tab to set basic input parameters (as shown in the example figure below) for the automated sequential assignment:

• • Select the Peak Lists to include in the automatic assignment procedure in the ‘Peak Lists’ panel.

  • To include or exclude a peak list from the procedure, simply toggle the |Use?| parameter.

  • Set the peak matching tolerances in ‘Peak-Peak Match Tolerances’

Finding resonances from peaks

The next step is to go to the {Spin Systems} tab and let Analysis automatically find resonances from the peak lists and the experiment types, and to assign them to the most likely atom types.

To do this, go to the {Spin Systems} tab, which at first presents us with a table listing all the spin systems in the project. To find resonances and assign them to the most likely atom types, click [Find Resonances From Peaks]. After confirmation, Analysis sorts the peak lists and starts making its assignments. After a few seconds, a list of Intra and Inter residue resonances is presented:

What is important here is that Analysis comments under |Comments| where it has found potential problems in the procedure in how to find and assign resonances. Start to inspect a problem by selecting the related spin system, and click [Show Peaks] or [Go To Root Location].

Automation

The next step is to go to the {Automation} tab and set parameters for the automation using the program Nexus:

• • Set the “Gaussian 13C variance” and the “Gaussian 1H variance”. These variances are similar to tolerances used in matching peaks, albeit that they are a bit more sophisticated by using Gaussian probabilities instead of hard tolerances.

  • Click [Run Nexus] to let the program do its automatic sequential assignment predictions.

After some time the assignment results and their reliability is plotted versus the sequence in the {Score Graph} tab.

Inspect and confirm predictions, and commit assignments

To inspect and confirm predictions, go to the {Predictions} tab. In this tab, the residues are listed, with their |Prediction Score|, |Best Matching Spin System| and |Confirmed Spin System| etc..

To inspect predictions, you can display strips of the sequentially assigned spin systems by selecting residues of interest, setting the category of matching in the lower left pulldown menu named “Any Available” and click [Strip Selected Using:]. The window for display is chosen in “Window:” in the {Settings} tab.  

To commit assignments, select the assignments that you think are correct and click [Confirm Selected]. To add the confirmed assignments to the project, click [Commit Assignments].