Assignment : Initialise Root Resonances

Add Starting Resonances and Spin Systems to Root Spectra

The purpose of this popup window is to setup certain kinds of spectra at the very start of the assignment process. This initialisation involves adding new, anonymous resonances and spin system (residue) assignments to picked, but unassigned peaks. The kinds of spectra that have their peak lists processed in this way are normally those that, on the whole, have one peak for each residue in the molecular system. This typically involves 15N HSQC and HNCO spectra where you get one peak for each NH group. In these instances two resonance are added for each peak; one for the 15N dimension and one for the 1H dimension, both resonances for the peak are then added to the same spin system (a resonance grouping that stands-in for a residue).

The initial resonances and spin system groups that have been added to an initialised “root” spectrum then serve as the basis for assigning related spectra, often with higher dimensionality. The general principle is that the positions of the peaks, and hence the chemical shifts of their assigned resonances, serve as guides to pick, locate and assign peaks in other spectra that share the same resonances in some of their dimensions. For example peaks in a 15N HSQC spectrum can be used to link equivalent peaks in a 3D HNCA spectrum or 3D 15N HSQC-NOSEY spectrum of the same sample because peaks in the 3D spectra derive from the same amide groups. Once related spectra have been linked via assignments to a common set of resonances and sequential assignment has been performed the resonances and spin systems will no longer be anonymous; the resonances will be assigned to specific atoms, and the spin systems will be assigned to residues.

One complication of the initialisation process is that not all peaks in the root spectra relate to unique residues or spin systems. The most common such examples are peaks that derive from NH2 (amide) side chain groups of Glutamine and Asparagine residues. In an 15N HSQC spectrum an NH2 group will give rise to two peaks one for each hydrogen, but these peaks share the same nitrogen and thus appear at the same 15N frequency. When a root spectrum is initialised such peaks must be taken care of in the appropriate way; the two peaks of an NH2 group share the same nitrogen resonance and the same spin system number.

The “Amide Side Chain Peaks” table in the popup window lists all of the pairs of peaks that have similar 15N resonance positions, and which lie in the chemical shift regions for NH2 groups. The purpose of the table is for the user to confirm or deny that the pairs of peak really are NH2 groups, and not coincidental matches. The user can locate a potential NH2 peak pair by selecting an appropriate spectrum window and, assuming “Follow Amides” is set, clicking on the row for the peak pair. If the peaks look like a genuine NH2 pair (similar intensity, deuterium sub-peak etc) then the peaks are may be confirmed by double clicking in the “Confirmed?” column.

With any NH2 groups confirmed, the peak list is initialised via [Initialise Peak List!] at the top; all peaks will have new resonances and spin systems added, and peaks from NH2 groups will be linked appropriately.

Caveats & Tips

This initialisation tool is currently limited to the following types of experiment: 15N HSQC, HNCO, HNcoCA, 13C HSQC.

Only potential NH2 peak pairs with 15N positions that are within the stated tolerance are shown. This tolerance can be reduced to make the amide search more specific, although this may be at the expense of detecting NH2 pairs that are distorted due to peak overlap.

Main Panel

pulldown Peak List: For spectra with (mostly) one peak per residue, selects which peak list to initialise; by adding anonymous resonance and spin system numbers

float 0.02: The upper limit in the difference between 15N ppm positions for two peaks to be considered as a potential amide

check Follow Amides: Sets whether to follow the H-N locations of potential amide peaks when clicking in rows of the table

pulldown Window: Selects the spectrum window in which to show positions of potential amide peaks

check Mark Amides: Whether to put a multi-dimensional cross mark though the H-N positions of the selected peak pair

button Clone: Clone popup window

button Help: Show popup help document

button Close: Close popup

button Initialise Peak List!: For the stated peak list, considering confirmed amide side chain peaks, add spin system and resonance numbers to all peaks

Amide Side Chain Peaks

Table 1
# Number of the peak pair for the table
Confirmed? Whether the peak pair is confirmed as being from an amide side chain; a common nitrogen by different hydrogens (Editable)
15N Δppm The difference in 15N ppm position between the two peaks of the pair
Peak 1 The assignment annotation of the first peak in the pair
Peak 2 The assignment annotation of the second peak in the pair
Shift N The average 15N position of the peaks in the pair
Shift H1 The 1H chemical shift of the first peak in the pair
Shift H2 The 1H chemical shift of the second peak in the pair

button Confirm Selected: Confirm the selected peak pairs as being amide side chain peaks; a common nitrogen by different hydrogens

button Unconfirm Selected: remove any conformation for the selected peak pairs being amide side chain peaks

button Update Table: Manually force an update the table of potential pairs of amide side chain peaks

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