Homonuclear Assignment
We will assume a standard set of homonuclear 2D spectra: 2D-[H,H]-COSY (or 2D-[H,H]-TOCSY with a short mixing time), 2D-[H,H]-TOCSY with a long mixing time and 2D-[H,H]-NOESY. This guide will follow the standard methodology for homonuclear assignments as detailed in Kurt Wüthrich, NMR of Proteins and Nucleic Acids, 1986, Wiley ISBN -471-82893-9. See here for 15N-labelled proteins.
All work will be done using the HomoScope tool.
- Open the COSY with HomoScope and zoom into the fingerprint region (HN=x-axis, HA=y-axis)
- Pick HN/HA peaks in the fingerprint region.
- Extend systems into the sidechain using either of two methods.
a. Use the Horizontal or Vertical ruler to mark the HA shift and search for correlations to it. Then select the H/HA peak and use extend horizontally (eh) or "extend vertically" (ev) to extend the assignment of the spin-system to the HB protons.
b. Use the diagonal to search for signals correlating to the HA.Click on the diagonal peak and then navigate up with "PageUp?" or right with "Ctrl-PageUp?" to search for an aligning correlation peaks corresponding to the HA/HB signals. Type "ev" or "eh" to extend assignment vertically or horinzontally.
- Check whether your assignments give consistent results in the TOCSY. Switch to the TOCSY with right-click "Select Spectrum" or use ShortCuts "ns" and "ps".
- If you are able to decide on the type of spin system based on the assigned peaks, use Assignment->Set System Type... to define it.
- Link the Systems together using the NOESY spectrum
Display the NOESY spectrum. Zoom in the HN/HA-region. All intraresidual peaks are already labelled. Pick one of the unpicked HN/HA peaks. You can use the Picking->Propose Peak... function to get a list of suggestions for assignments. If you decide that two residues are really sequential neighbours, use Assignment->Link Systems... to link them. You need to enter the Spin System ID for both.
- Once a fragment is sufficiently long, map it onto the sequence using Assignment->Show Alignment...
This mapping function takes the Spin System Types you set into account, thus strongly reducing the number of theoretically possible matches.
- Assign Prolines with HA/HA or HA/HD NOEs.
15N-labelled protein
Usually, you follow the same methodology as for homonuclear assignement, but you use 15N-HSQC, 3D 15N-resolved TOCSY and 3D 15N-resolved NOESY spectra. You can use PolyScope, which is HomoScope's 3D-extension and follow the procedures as described above. Alternatively, follow this procedure:
- Use SynchroScope to pick systems (HN-N) in the 15N-HSQC and label spins HN, N, HA, HB, HB2, HB3 in the 3D 15N-resolved TOCSY
- Switch to the 3D 15N-resolved NOESY and pick remaining spins and label as sequential: HN-1, HA-1, HB-1 etc.
- Use StripScope to link the strips together. Sequence mapping is not as reliable with protons, because the different amino acid types are less distinct in their 1H chemical shifts. CA/CB provide a much sharper discrimination, so be very careful when using this. Use PolyScope's Spin System Type feature to support/confirm sequential assignments.
- Use SystemScope to extend your system and finish the assignments.
PolyScope is better suited for the initial mapping work, but in later phases of the assignment, the above combination (and especially SystemScope) can be quite powerful.
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