Resonance Assignment/CARA/Backbone assignment GFT

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HA And HB Assignment in (4,3)D HABCAB(CO)NHN with CARA

  1. Run GFT_CreateHABProjSpins Lua script to create GFT spins like <nop>CApHA-1 and <nop>CBpHB-1. Known CA and CB chemical shifts and average HA and HB chemical shifts from assigned residue types will be used. IMPORTANT! This script will overwrite existing CA+/-HA-1 and CB+/-CB-1 spins!
  2. Use <nop>PolyScope or <nop>SynchroScope to move these spins to their appropriate positions. Create these spins if they are not present in a spin system. You can also use <nop>StripScope.
  3. Run GFT_HABCAB2HAHBCACB Lua script to create HA-1 (or HA2-1 and HA3-1) and HB-1 (or HB2-1 and HB3-1) spins. It should also create CA-1 an CB-1 spins if they were missing and report inconsistencies. IMPORTANT! This script will overwrite existing HA-1 and HB-1 spins!
  4. Run CopyProjectedSpinsToOriginSystem2 or CopyProjectedSpinsToOriginSystem Lua script to copy HA-1 and HB-1 to HA and HB spins of successor systems. In CARA jargon "projected spins" refers to spins with non-zero offset.


HA And HB Assignment in 3D HBHA(CBCACO)NH with CARA

  1. Make sure the HBHA(CBCACO)NH is loaded into repository.
  2. If necessary, adjust the calibration in the H-N plane to match that of 15N-resolved NOESY. Also, adjust the calibration in the Hα/Hβ dimension to match 15N- and 13C-resolved NOESY spectra.
  3. Proceed with picking HA-1 (or HA2-1 and HA3-1) and HB-1 (or HB2-1 and HB3-1) spins in assigned fragments. Use HA-1 and HB-1 for spins with degenerate shifts.
    • Make sure that the spins are matching the preceding residue type. For example, if the previous residue is Ile, you should pick HB-1, as HB2-1 would be incorrect.
    • The standard BioPack pulse sequence ghbha_co_nh.c employs 1H multiplicity editing. Thus, cross-peaks of CH2 groups will have the opposite sign that of CH and CH3 (Ala HB). The advantage of this is the additional information on amino acid typing. The drawbacks are possible mutual signal cancellations, within Ser and Thr spin systems, or between spin systems, which overlap in the H-N plane.
  1. Create empty spin systems for each unassigned residue preceding an assigned fragment. This is, typically, the case with prolines. Link each empty system to adjacent assigned fragments.
  2. Run CopyProjectedSpinsToOriginSystem Lua script to copy "projected spins" (with offset -1) to preceding spin systems. You have to run it 6 times, for HA-1, HA2-1, HA-3, HB-1, HB2-1 and HB3-1.
    • Empty spin systems will thus become populated - this is the reason for creating them in the previous step.
    • Existing HA, HA2, HA3, HB, HB2 and HB3 spins (if any) will be updated with new chemical shifts. You may want to preserve this information by saving the repository before running CopyProjectedSpinsToOriginSystem.


Additional backbone assignment and verification

  1. Verify the backbone assignment by tracing HA(i) <-> HN(i+1) and HB(i) <-> HN(i+1) connectivities in the 15N-resolved NOESY spectrum.
  2. Pick HA-1 (or HA2-1 and HA3-1) and HB-1 (or HB2-1 and HB3-1) spins in unassigned systems, if your backbone assignment is incomplete.
  3. Try to complete the backbone assignment by matching 15N-resolved NOESY strips. If reliable assignment cannot be established, postpone completion until the side-chain assignment is complete.


%COMMENT%

-- Main.AlexEletski - 06 Jul 2007

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