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
Peak List: For spectra with (mostly) one peak per residue, selects which peak list to initialise; by adding anonymous resonance and spin system numbers
0.02: The upper limit in the difference between 15N ppm positions for two peaks to be considered as a potential amide
Follow Amides: Sets whether to follow the H-N locations of potential amide peaks when clicking in rows of the table
Window: Selects the spectrum window in which to show positions of potential amide peaks
Mark Amides: Whether to put a multi-dimensional cross mark though the H-N positions of the selected peak pair
Clone: Clone popup window
Help: Show popup help document
Close: Close popup
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 |
Confirm Selected: Confirm the selected peak pairs as being amide side chain peaks; a common nitrogen by different hydrogens
Unconfirm Selected: remove any conformation for the selected peak pairs being amide side chain peaks
Update Table: Manually force an update the table of potential pairs of amide side chain peaks