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Heteronuclear Backbone assignment

We will consider the case of a 13C,15N-labelled protein with the following recorded spectra: 15N-HSQC, HNCA, HNCACB, CBCA(CO)NH. Of course, you can use different or additional spectra, any spectra connecting backbone residues can be used.

  1. Import a CARA template, import a sequence file, add your spectra to the spectra subsection. Use MonoScope to quickly check their orientation.
  2. Use SynchroScope to calibrate your 3D's onto the 2D and among each other. Then follow the spin picking and assignment procedure as described on the SynchroScope page. Note: You can also use PolyScope to do this. PolyScope does not require Unique Key Identifiers.
  3. Use StripScope to link your strips together and map them onto the sequence. The StripScope page describes this.
  4. You can use additional spectra as they become available to resolve difficult cases.


    • Add the 15N-resolved-[1H,1H]-NOESY and 15N-resolved-[1H,1H]- TOCSY to the repository.
    • Pick HN/HN and HN/HA NOEs in SynchroScope and assign them as intraresidual (HA) or sequential (HA-1) by comparing to the TOCSY.
    • Use StripScope to link the strips together using the sequential NOEs.
  5. Prolines cannot be assigned with this methodology because they have no N-HN anchors.

    Strategies to assign Prolines.

    • Use SystemScope to enter the Proline Spin System via sequential NOEs (HA/HA or HA/HD) from the succeeding spin-system or via a correlation spectrum (HACAN, ...).
    • Use HomoScope to pick CD/HD* peaks which occur in a unique region of the [1H,13C]-HSQC and then use SystemScope to expand the Spin System.
    • Use (H)C(CO)NH and H(CCO)NH to assign the proline sidechain {CA-1,CB-1,CG-1,CD-1} and {HA-1, HB2-1 ...} from the system suceeding the PRO: System(PRO+1). Then use the script CreateOriginSystemsFromProjectedSpins.lua to transfer the spins CA-1 -> CA, CB-1 -> CB ...from System(PRO+1) into a new system which is automatically linked as predecessor to the System(PRO+1).
    • Use the CBCA(CO)NH to get CA-1 and CB-1 chemical shifts of the System(PRO+1). Run the script CreateOriginSystemsFromProjectedSpins.lua to create the PRO spin system. Open a HC(C)H-TOCSY with SystemScope(rotated) and select the new PRO system. Find the attached protons and assign the rest of the sidechain.

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