Resonance Assignment/CARA/Backbone assignment GFT

Picking Spins in (4,3)D GFT HNNCABCA and CABCA(CO)NHN Spectra

Open (4,3)D GFT HNNCABCA and CABCA(CO)NHN in SynchroScope as you did with 3D HNCO. You can either open four separate SynchroScope windows, or open only one and switch between (4,3)D subspectra there.

Pick CABCA spins just like you did with C-1 spins in HNCO. It is recommended to start with (4,3)D CABCA(CO)NHN to pick CAmCA-1 CApCA-1, CAmCB-1 and CApCB-1 spins first. Then pick CAmCA CApCA, CAmCB and CApCB spins in (4,3)D GFT HNNCABCA.

Picking new spins is not as slow as in XEASY. However, it would make sense to add a script, which would create initial CABCA spins to speed up the process.

See the this page for details and examples on how to use SynchroScope: http://www.cara.ethz.ch/Wiki/SynchroScope

Calculating CA and CB Chemical Shifts

Once the GFT spins have been picked as completely as possible, run the GFT_CABCA2CACB Lua script.

IMPORTANT! Back up the repository before running the script. Missing CA and CB spins will be created, and the chemical shifts of the existing CA and CB spins will be updated.

You will need to select the project (most likely it will be the only one) and a spectrum. The spectrum is needed to use the correct carrier offset (~ 43 ppm) of the projected dimension, saved as an attribute. See also the page on loading GFT spectra.

• GFT_CABCA2CACB pop-up:
  

    <img src="%ATTACHURLPATH%/gftcacbcalc.png" alt="gftcacbcalc.png" width='217' height='184' />

The script will produce a log in the terminal window, reporting large deviations (default threshold: 0.5 ppm), missing spins and possible glycines.

At present it seems difficult to reconcile this approach with UBNMR due to:

• Nomenclature differences - H vs HN
• Two sets of SRDs SRD-I and SRD-II in XEASY/UBNMR approach
• Inconvenience - using UBNMR would require reading/writing to disk; Lua scripts operate on the data in memory.

To verify connectivities in 15N-resolved NOESY it may be useful to calculate HA-1 and HB-1 spins first.

%COMMENT%

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

1. Run GFT_CreateHABProjSpins Lua script to create GFT spins like CApHA-1 and 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 PolyScope or 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 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