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