Making Distance Restraints
Introduction
The next part of the exercise is to look at how we can use a CCPN project to get peaks lists and restraints (both dihedral and distance) that can be used in an ARIA structure calculation, and how we can pass intermediate structural information back into Analysis to help with violation analysis and NOE peak assignment.
Open an existing project
For this last section we will be using programs that are part of the Extend-NMR software collection. The Extend-NMR project has its own graphical interface, but many of its components are also available from within Analysis. If CcpNmr Analysis is not already open, start it up on the command line by typing:
-> analysis
from the main menu select M: Project: Open Project. Navigate to find and select the CcpnStructCourseB project, then click [Open]. This project is at the same point that we left the last project if everything went according to plan.
You might get a warning that various files have moved location. You might also get a dialog with a list of spectra paths (because those also have moved location). If the paths are all in grey then just click the [All Done!] button at the bottom. If any path is in red then Analysis cannot find the corresponding spectrum data file, so either you need to tell Analysis where it is (by double clicking the path cell and navigating to the correct location) or accept that that particular spectrum will not have its contours displayed. When the project data is loaded select M: Window: window2 and a blue NOESY spectrum will hopefully appear.
To make a list of distance restraints from the assigned peaks in an NOE peak list first go to M: Structure: Make Distance Restraints. At the top of the popup, change the peak list to C-NOESY:173:1 and set the Restraint Set pulldown to "1" . We can leave all of the other parameters alone for demonstration purposes. The 'Restraint Distance Params' section would allow us to specify how the NOE peak intensities relate to the distance bounds of any generated distance restraints. The default method is to calculate a target distance as peak volume raised to the power of -1/6 multiplied by some scaling factor, such that the reference intensity (in this case defaults to the peak list's average volume) exactly corresponds to the reference distance (in this case 3.2 Angstroms). The upper and lower bounds of the distance restraint are calculated as fractional changes from the calculated target distance (default is 20% above and below) while observing absolute minimum and maximum values for the bounds (1.72 & 8.00 Angstroms respectively by default). The {Residue Ranges} and {Chem Shift Ranges} tabs would allow you to make only restraints for specific assigned regions of your molecule or for specific shift ranges.
Making Restraints From Assigned Peaks
Press [Make Assigned Restraints] to calculate restraints for assigned peaks from the selected peak list. After a short pause you will see the Structure: Restraints and Violations popup appear. This shows that you have one Restraint set (a way of grouping related restraints and violations) containing a H-bond restraint list, which was already loaded via the FormatConverter, and a distance restraints list of 600+ new restraints. Click on the row of a restraint list in the central table and then click on the {Restraints} tab. Note that you can also get to this popup via the M: Structure: Restraints and Violations command.
In the {Restraints} tab you will see the restraints listed. Each restraint has one green-coloured row. Note some restraints also have following grey rows. These grey rows are alternative distance pairs for restraints that are ambiguous, i.e. a possible connection between two different pairs of 1H resonances. 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. This project has already been through one ARIA run, which is why we have so many peaks with ambiguous assignments.
Making Restraints From Unassigned Peaks
There is a second common way to generate distance restraints, which is to match the chemical shifts of resonances to NOE peak positions, thus generating potentially highly ambiguous distance restraints. Such restraints would typically be used as input for an iterative structure generation program like ARIA, where they would eventually be filtered to select only the correct contributing resonance pairs. Firstly, we could leave the matching of chemical shifts to the ARIA program by handing the program peak lists rather than restraint lists, which is what we we will demonstrate for the N-NOESY data. However, it is also possible to make such restraints in CCPN. Accordingly, the {Shift Match Tolerances} and {Network Anchoring} tabs in the M: Structure: Make Distance Restraints popup allow you to generate such distance restraints for peaks which do not have assignments. Generate distance restraints based on shift matching by firstly set the peak list to "C-NOESY:173:1" click [Make Shift Match Restraints] This command uses the current settings, but {Chem Shift Ranges} and {Shift Match Tolerances} are only relevant for this command.
In the case of the shift-matching method potentially ambiguous distance restraints are generated by simply matching peak positions to close chemical shifts. In the case of network anchoring method, chemical shifts are also matched to peaks, but the ambiguous possibilities are refined by selecting only NOE assignments from amongst the possibilities that are supported by other, assigned NOEs or covalent structure. Say, for example, that a peak could arise from a number of resonance pairs. Two resonances A & B are more likely to be a correct assignment for the peak if we know that they are close to (or bound to) the same intermediary resonance, C and therefore must be close to each other.
Merging and Splitting Restraint Lists
To prepare these newly generated restraint lists for the ARIA calculation we will merge and split them in order to generate restraints that are separated into "Unambiguous" (Unambig) and "Ambiguous" (Ambig) categories. In ARIA we do this so that the "Ambiguous" restraints and peaks follow a different protocol; they enter the calculation after the unambiguous, more certain, restraints have formed the initial structure.
In the M:Structure:Restraints and Violations, {Restraint Lists} tab, merge the two lists that derive from the C-NOESY experiment by clicking on the two relevant rows (probably numbers 2 & 3) while holding down the 'Ctrl' and click [Merge Lists] at the bottom followed by [OK]. You will see that the restraints have been combined and there is now only one list from the C-NOESY. Then for the remaining, enlarged restraint list, select its row and click [Split Ambig/Unambig]. These are now ready for input to ARI:
Note: It is also possible to split both the lists into "Ambig" and "Unambig" at the first stage (two Ambig and two Unambig lists as a result) and then merge the four lists into two.
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