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.
- Import a CARA template, import a sequence file, add your spectra to the spectra subsection. Use MonoScope to quickly check their orientation.
- 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.
- Use StripScope to link your strips together and map them onto the sequence. The StripScope page describes this.
- You can use additional spectra as they become available to resolve difficult cases.
Example:
- 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.
- 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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