Assignment : Pick & Assign From Roots

Pick and Assign Peaks Based on Locations in Root Spectra

This popup window is designed to help in the early stages of peak picking and resonance assignment. The main idea is that certain “root” spectra, typically an HNCO or 15N HSQC, with initialised peak lists are used as a basis for locating and assigning peaks in other spectra that overlap in the relevant dimensions. For example you may use the amide peak positions in a 15N HSQC spectrum to pick peaks in restricted regions of a 3D HNCACB spectrum and copy amide resonance assignments to the amide dimensions of the 3D spectrum where they overlap with the HSQC peaks.

Often the root spectrum, that is the source of peak position and assignment information, will only be assigned in an anonymous way; the peaks will have spin system and resonance numbers (e.g. “{7}[4][5]”) and not link to any particular residues or atoms. For this tool it doesn’t matter whether the root peaks carry full assignments or not, all that is important is that there are some peaks to specify locations and some form of assignment to copy. The easiest way to setup initial assignments on a root spectrum, after picking some peaks, is to use the Initialise Root Resonances option.

In normal operation the user chooses a peak list to act as the the source of the root assignments and positions, and maybe a spectrum window to view those peaks in. Next the assignable spectra that are to be the target for peak picking and/or assignment are chosen. Firstly, the user adds and removes spectrum windows to the “Target Windows” list, the spectra to be operated on are then selected from those that are visible in these windows. By selecting different kinds of window different kinds of spectra may be operated on, so for example the user could both work with HCN spectra and HHN spectra with the same HSQC root. This tool is largely visual and it is important to be able to see the locations that are bing considered (e.g. amide), hence the user is only presented with assignable spectra that can potentially be seen. The “Assignable Spectra” section is filled with the spectra for the target windows that may be assigned, and the user double-clicks in the “Active?” to set whether the individual spectra should be operated on or not (for peak picking and assignment).

One windows and spectra are setup is is advisable to consider the “Tolerances” tab that controls how wide a search region is used to pick and assign peaks relative to the positions of the reference, root peaks. The user can also setup exclusions to avoid picking peaks near the water signal (e.g. in 15N HSQC-NOESY) or homonuclear diagonal.

The last “Link Peaks” tab is the one that remains active while the user is actually running the the peak picking and assignment functions. Here, the main table lists all of the peaks in the root peak list that are used for position and assignment references. Clicking on a row in this table, assuming the relevant navigations are checked above, will cause the location of any root and target spectrum windows to move in order to show the root (e.g. amide) location for the selected row. Even if peaks are not peaked or assigned this tool may be used for efficient coordination in window. Typically clicking on an HSQC peak will present an amide position in 3D target windows, locating the X and Z axes, so that the user can see the column of peaks that may be picked/assigned.

For a single selected root location, the user may transfer assignments or pick peaks and transfer assignments, by clicking the appropriate buttons in the “Pick & Assign Functions” section. This is one-by-one way of working is the safest because the user is presented will the spectra for each location and will be able to view the result of the peak picking and assignment. The equivalent “Process-all Functions” will work though all root locations in the table picking and assigning peaks en masse, according to the set tolerances, in a quick but less controlled manner.

The “Assign Non-root Resonances” options are present so that you can give a starting assignment to the assigned spectra in the dimension that does not match the root location. For example, you could add 13C resonances to an HNcoCA spectrum or 1H resonances to the indirect dimension 1H dimension of a 15N HSQC-TOCSY. These “non-root” resonance numbers will all be new and unique, thus this operation should only be used for types of experiment where the is one peak for each non-root resonance. For example HNcoCA has one peak per CA resonance by HNCA usually has two, so the function is only recommended for the former.

Caveats & Tips

This tool can be operated in two slightly different ways, according to the preference of the user and the quality of the spectra. The “Process-all” functions can be used to start with, given fairly strict tolerances, but the user should then go through each root position checking for and tidying up mistakes (picking noise & artefacts for example). Alternatively the root locations could be picked and assigned one-by-one so the user can spot problems as they occur.

Although the same tolerances, set via the “Tolerances” tab, are used for both peak picking and for resonance assignment some peaks that can be picked may not be assigned with the same settings. Whereas the peak picking is done relative to the root location the assignment, in common with the rest if Analysis, is relative to the current chemical shift average and thus may differ from the root location. Widening the tolarances a little, or increasing the chemical shift weighting of the root spectrum can allow assignments to be made if they were previously out of bounds.

Main Panel

button Clone: Clone popup window

button Help: Show popup help document

button Close: Close popup

Windows & Spectra

Selection of source or “root” for assignments, the target spectra and which spectrum windows to navigate within

pulldown Root Peak List: Selects which peak list is considered as the “root”; the positions and assignments that will be used to pick/assign related spectra

pulldown Root Window: Selects which spectrum window is used to navigate to the location of root peak selected in the “Link Peaks” table

Target Windows

button Remove Target Window: Remove the selected target window from consideration, so that it is no longer used for navigation and assignment

button Add Target Window: Add the window selected in the adjacent pulldown list as a source of assignable spectra and a target for navigation

pulldown *None*: Selects a spectrum window, from those not already selected, that may be used for assignment/navigation

Table 1
# The serial number of the spectrum window
Name The name of the spectrum window used for navigation and providing assignable spectra
Selected Mapping When there are multiple options, states which axes of the window (X, Y, Z...) correspond to the directly bound “root” dimensions (Editable)

Assignable Spectra

Table 2
Spectrum The “experiment:spectrum” name for the spectrum which may be used for peak picking & assignment; must be present in the above windows
Active? Whether the spectrum is considered active for the processes of peak picking and/or assignment; inactive ones will not be affected (Editable)
Assign Non-root dim? Whether spectrum dimensions that do not map to the root (typically non-amide) may be assigned; common for HNCO, HNH-TOCSY etc. (Editable)
Experiment Type The full CCPN experiment type of the spectrum; should be something with an obvious relation to the root peak list
Dimensions The kinds of isotope (or otherwise) present on the dimensions of the spectrum

Tolerances

Settings that relate to assignment and peak picking tolerances when going from “root” positions to related spectra

float Root Dim 1: Sets the upper limit to the dimension 1 assignment tolerance, within which peaks may be picked and assignments made, relative to the root location

float Root Dim 2: Sets the upper limit to the dimension 2 assignment tolerance, within which peaks may be picked and assignments made, relative to the root location

float 4.95: Sets the lower bound to the 1H ppm exclusion zone, usually representing a water signal, within which no peaks will be picked or assigned

float 4.95: Sets the upper bound to the 1H ppm exclusion zone, usually representing a water signal, within which no peaks will be picked or assigned

int 1: When using multi-dimensional cross marks to indicate peak positions, sets how many marks persist from subsequent peak navigations

float 0.25: Sets the width of the exclusion zone around the 1H-1H homonuclear diagonal, within which no peaks will be picked or assigned