Side chain assignment with CN-NOESY in XEASY

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One can go to the simultaneous 15N,13C-resolved [1H, 1H]-NOESY directly rather than the HCCH-spectra to complete the side-chain assignment after backbone and HB resonance assignment were obtained. Separately recorded 15N, 13C(aliphatic), and 13C(aromatic) NOESYs can also be combined and used as a simultaneous NOESY, however, extra effort may be needed. Although the risk is low, it is suggested to confirm the assignment from NOESY by using HCCH due to the risk of misinterpretion of inter-residue NOE as intra-residue NOE.

  • Pros
    • Fast. Since it is necessary to double check the chemical shift in NOESY spectra before NOE peak picking and interpretion in NOESY, the side chain assignment can be done while check the chemical shift in NOESY and will not incease much extra time.
    • Redundant information. In addtion to the correlations observed in HcCH-COSY/TOCSY type spectra, NOE of side-chain resonances to intra-and sequential-amide resonances provides addtional information for peak pattern recognization, which also reduce the risk.
  • Cons
    • Redundant information. There is risk of misinterpretation of inter-residue NOE as intra-residue.
    • May need experience.

One can start side-chain assignment as following after correct backbone assignment and HA/HB assignments were obtained.

  1. Process the simultaneous NOESY as a single 13C NOESY spectra. In this case, the 15N chemical shift is calibrated as 13C shift. The real chemical shift can be easily calculate back based on the acquisition parameters. It is recommended to analyze the simultaneous NOESY as a real single NOESY.
  2. In UBNMR, run makeNoePeaks to generate a starting simultaneous NOESY peakList including intra- and sequential NOEs considering previous assigned backbone and HA/HB chemical shifts and also averaged chemical shifts from BMRB for all assignable unassigned side-chain resonances. The 15N chemical shift has already been converted to pseudo-13C chemical shift which will exactly fit the spectra.
  3. In XEASY, use ns to load two copies of the Simultaneous 15N,13C-resolved (1H,1H) NOESY and display the two copies in orthogonal views (X-axis :w1(13C/15N), Y-axis: w2(1H); Z-axis: w3(1HC/HN) & X-axis: w3(1HC/HN), Y-axis: w2(1H); Z-axis: w1(13C/15N) use ls, lc, and lp, respectively, to load noe.seq, bbsc.prot and simnoesyI1.peaks; use sp, se and gs to display [w2(1H),w3(1H)]-strips and [w2(1H),w1(13C)]-strips residue by residue.
  4. In XEASY, start from one view (X-axis: w1(13C/15N), Y-axis: w2(1H); Z-axis: (1HC/HN)) of the NOESY, check [w2(1H),w3(1HN)] (both intra- and sequential)-[w2(1H),w3(1HA)]-[w2(1H),w3(1HB)]-strips to confirm assignments for backbone and HB resonance. Adjust peak position and resort strips if necessary. Do same check in the other view (X-axis: w1(13C/15N), Y-axis:w2(1H); Z-axis: w3(1HC/HN)).

    Example Figure 1 for Ile 12 side-chain assignment by using NOESY This is how it looks after this step. The strips are displayed in the order of HN, N, HA, CA, HB, CB, QG2, CG2, HG12, CG1, HG13, CG1, QD1, CD1, HN (i+1) and N (i+1), where the name represent the x-dimesion of the strip, i+1 stands for the next residue. Since the assignment of HN, N, HA, CA, HB, CB HN(i+1) and N(i+1) are correct, the peak labels are on top of the real NOE peaks for these atoms and these strips are displayed correctly. However, the remaining strips are from average chemical shift from BMRB and the peak labels are off from peaks, for which we need to find out the correct position.
    <img src="%ATTACHURLPATH%/sc1a.jpg" alt="sc1a.jpg" width='983' height='731' /> 
  1. In XEASY, guess the 1H chemical shift for other side-chain groups based on peak pattern from strips [w2(1H),w3(1HN)]-[w2(1H),w3(1HA)]-[w2(1H),w3(1HB)] and BMRB chemical shift for these unassigned groups, start from the group with unique 1H or 13C shift. Adjust the peak position for these unassigned groups, then resort and redisplay the strips.

    Example Figure 2 for Ile 12 side-chain assignment by using NOESY This is how it looks after adjusting peak position of QD1, QG2, HG12 and HG13 peak labels to observed peaks that are possibly the real side-chains resonances. This is based on the peak patterns observed in strips of assigned chemical shifts, which need knowledge of peak pattern for different residues.
    <img src="%ATTACHURLPATH%/ile2.jpg" alt="ile2.jpg" width='983' height='731' /> 
 Example figure 3 for Ile 12 side-chain assignment by using NOESY  This is how is looks after strips resort and redisplay, the temporary assigned QD1, QG2, HG12 and HG13 need to be confirmed from the 13C chemical shift. 

   <img src="%ATTACHURLPATH%/ile3.jpg" alt="ile3.jpg" width='983' height='731' /> 
  1. IN XEASY, check strips for unassigned groups from the other view (X-axis: w1(13C/15N), Y-axis:w2(1H); Z-axis: w3(1HC/HN)), check if a corresponding possible 13C shift could be found for the temporary assigned 1H shift.
    1. Adjust the 13C shift peak position if there is one within the expected 13C shift range and gives an expected peak pattern, resort and redisplay the strips for assignment confirmation. Go to the next unassigned group for this residue.

      Example figure 4 for Ile 12 side-chain assignment by using NOESY This is how it looks after adjustment of peak position in 13C dimension. Fortunately, corresponding possible 13C shift are observed for all temporary assigned side-chain resonances QD1, QG2, HG12 and HG13. Adjust 13C chemical shift by adjust peak position in CG2, CG1 and CD1 strips.
    <img src="%ATTACHURLPATH%/ile4.jpg" alt="ile4.jpg" width='983' height='731' /> 
 Example figure 5 for Ile 12 side-chain assignment by using NOESY This is how it looks after strip resort and redisplay. Now all strips of residue Ile look resonanable and the side-chain assignment of Ile12 is done in simultaneous NOESY and will be confirmed in HCCH. 

   <img src="%ATTACHURLPATH%/ile5.jpg" alt="ile5.jpg" width='983' height='731' /> 
    1. Re-adjust the 1H shift peak position and repeat the previous step search for the corresponding 13C shifts, repeat these steps to get the most reasonable assignment. Or go to the next residue and come back to the side-chain assignment of this residue later.
  1. Continue on the next residue and repeat these steps until finishing all residues.
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