Collecting a 15N Edited HSQC

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Collecting a Superb 15N edited HSQC (NHSQC)

  1. Concentrate your protein as much as possible. It should be at least 50uM for 2D work and around 1mM for 3D work.
  2. Spike in 10% D2O.
  3. Place in a Shigemi tube (if low volume) or in any other NMR tube. Cap and seal.
  4. Insert your sample into the spectrometer. Create a new dataset.
  5. Open experimental parameter set ‘NHSQC’. If you can’t find it, open hsqcetf3gpsi. Also, double check that this is the pulse program.
  6. Edit the number of points in the direct and indirect dimension. Should be 2k and >= 128.
  7. The number of dummy scan should be at least 16.
  8. The number of scans should be adjusted depending on the concentration. For 1mM protein, 2 scans is ok. For 500uM protein, 8 scans will be necessary.
  9. Edit your sweep width in the indirect dimension. A larger sweep width will prevent folding of the spectrum but will decrease digital resolution.
  10. Edit your 15N center position. It should be between 115-120 ppm.
  11. Edit the ZGOPTNS. Add –DLABEL_CN if the protein is double labeled. This will tell the pulse program to decouple 13C.
  12. Check experiment time with ‘expt’.
  13. Lock ‘lock h2o+d2o’.
  14. Tune ‘atma’.
  15. Shim ‘topshim’ or ‘topshim 3d’.
  16. Find p1 ‘pulsecal’.
  17. Calculate receiver gain ‘rga’.
  18. Zero go ‘zg’.
  19. Process the specrum with ‘xfb’.
  20. Edit phasing manually with ‘ph’.
  21. Rule of thumb - If you can collect an amazing NHSQC in 10 min you are ready for 3D work.
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