Violation analysis and structure validation

Restraints & Violations

Load project CcpnCourse3c. In the Structure: Restraints & Violations popup (M: Structure: Restraints & Violations), choose Restraint Set "2" at the top; these are the results from the ARIA run (set 1 was the input) and choose the {Restraint Lists} tab. You will see that ARIA has generated one or more restraint list for each peak list and restraint list that was used as input data. Choose one of the rows labelled "REJECT", which represents the peaks/restraints that ARIA rejected, and select the {Restraints} tab. From the "Violation List:" pulldown menu at the top of the table select "1" and you will hopefully see that many of the rows become red, indicating that these restraints caused violations:

If you change the Restraint List pulldown menu to a list that is not labelled as "REJECT" you will see that there are far fewer violations:

Choose a restraint row for one of the violated (red/orange) restraints and click [Show Peaks]. This will open a table containing the peak (or peaks) that gave rise to the restraint. Note that  a restraint may be linked to more than one peak, for example where there are symmetry related peaks that correspond to the same close resonance pair. In this Selected Peaks table click on the peak row and then the [Find Peak] button at the top. You will be whisked to the point in the spectra where the peak resides. From here you may choose to look at the peak assignment by using the 'a' key with the mouse over the peak. Note you can directly assign a peak via a restraint by using the [Assign Peak] button from below the Restraints table.

Making and Checking NOESY Assignments with Structures

An alternative, more detailed, method of looking at NOE assignments is via the M: Assignment: NOE Contributions option. This is designed to make the whole process of comparing NOE peaks to a structural model efficient. Select this and in {Peak List & Display Settings} choose the "NOE Peak list" for the NOESY experiment you are currently looking at, then double click the "Use?" column in the lower table for the relevant window, so that it becomes "Yes". Also set 'Mark Peaks' to true. Now move to the {Peak Assignments} tab, select the peak in the spectrum window (left click & drag) and in the Assignment: NOE Contribution popup, click [Selected Peak]. This will show you on the structure and in terms of the close chemical shifts (and distances given the selected structure) what the likely NOE assignments are.

By clicking on the rows in the NOE peaks table you will see that several things happen automatically: The view of the selected windows (in this case the selected Window 4) zooms and marks the selected peak; the graphical structure view highlights the possible atom connections that the peak could represent; and the lower table of the Assignment: NOE Contributions popup shows the structurally possible, shift-matched resonance pairs ordered in terms of shift and geometric distance. In the lower table clicking on an assignment row and [Assign Selected] sets that assignment for that peak. Also, selecting [Predict Peaks] will use the entered structure and the known chemical shift values to predict the positions of peaks, near to the selected real peak, which correspond to close resonance pairs. These artificial peaks are labelled with the structural distance and are contained in an entirely separate list to the real peaks (so nothing is contaminated and they can be removed easily).

Note that if we change peak assignments then we have the choice of making (or letting ARIA make) a new set of restraints. Alternatively we can curate the existing restraints using the [Update Assignment From Peak] button (see M: Structure: Restraints & Violations, {Restraints}), which will alter the distance restraint to reflect this new assignment.

The final part of these tutorial exercises is to look at how we can use the CING software to analyse and validate or NMR and structure data.

Open an existing project

If the project "CcpnCourse3c" is not already open start Extend-NMR on the command line by typing:

-> extendNmr

When the Extend-NMR menu bar has appeared select M: Project: Open Project.  Navigate to find and select the CcpnCourse3c project, then click [Open]. This loads the data that resulted from an ARIA calculation.

Setting Up a CING Run

In the Extend-NMR GUI select the {CING} tab and click the green [New Run] button in the upper right corner. This run specification will contain all of the data that CING will analyse. When a new run is made the {Structures} table will fill with the various models from an ensemble:

Accepting this, we move on to the {Shifts & Measurements} tab. Here click [Add Measurement List] to add the shift list to the CING analysis. Move to the {Peak Lists} tab, and add the N-NOESY:182:2 peak list (this is the one that ARIA made) by selecting it from the lower right pulldown menu and selecting [Add Peak List]. Finally in the {Restraint Lists} tab add restraint list "Distance-2:1". This restraint list is the one that came back from ARIA and consequently lives in the second restraint set. Note that in general you may select as much data as you like, but we are just selecting a smaller subset for demonstration purposes.

Select the {Run Settings} tab below the CING logo. To submit the analysis press the [Submit Project] button. Note that this may take some time, so you may wish to let the demonstrator make a real submission and view some previously calculated results, thus sparing the iCING server. Although, feel free to use this server generally. Click [Check Run Status] to view the status of your submitted project.

To view some precalculated validation data look at the following URL in a web browser:

http://nmr.cmbi.ru.nl/CASD-NMR-CING/data/GR/CGR26ACheshire/CGR26ACheshire.cittp

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