1D Excitation Sculpting zgesgp
  1. Copy an existing 1D zgesgp experiment (pulse program: zgesgp). Or in an new experiment "rpar P3919GP all" and change the pulse program to zgesgp.

  2. Type eda, in the acquisition parameter window check and adjust td, ns, ds, sw.

  3. Type ased, in the pulse program parameter window, enter calibrated p1, pL1 values, and O1 determined from 1D zgpr experiment.

  4. In gs mode adjust p12 to achieve minimum FID area integral.

  5. Start the experiment by rga and zg;

  6. Process the FID by typing “dfp”, and phase the spectrum when necessary.

1D 31P

  1. Type "rpar P31CPD" in a new data set, or use "edc" command to copy from an exisiting 1D 31P experiment. Another option is "rpar P31" depending on decoupling choice.

  2. Use "getprosol" command to load the default phosphorous power parameters.

  3. In the acquisition parameter window check and adjust td, ns, ds, sw, d1.

  4. Estimate receiver gain using command rga, then start the experiment using zg command;

  5. Process the FID by typing “efp”, and phase the spectrum when necessary.

 

1D Watergate with 15N Decoupling Option zgwg.df
  1. Copy an existing 1D experiment (pulse program: zgwg.df) by typing “edc”.

  2. Type eda, in the acquisition parameter window check and adjust td, ns, ds, sw, O1P and O2P.

  3. Type ased, in the pulse program parameter window, enter calibrated p1 and pL1 values; set pcpd2 220 us and corresponding pL12 12 dB if applying decoupling. Otherwise pL12 should be 120 dB.

  4. Start the experiment by rga and zg;

  5. Process the FID by typing “dfp”, and phase the spectrum when necessary.

1H-15N HSQC (Tutorial link)hsqc
  1. Go to an existing HSQC experiment (pulse program: hsqcetfpf3gp), type “edc” to copy its parameters and generate a new experiment number. Or in a new experiment number "rpar HSQCETFPF3GP".

  2. Type “eda”, set parameters td, ns, ds, sw, O1P and O3P. For 13C and 15N labeled sample, ZGOPTNS should be "-DLABEL_CN".For only 15N labeled sample, leave ZGOPTNS blank.

  3. Type “ased” to set power length and power level for both 1H and 15N, including p1, pL1, p21, pL21, pL12, and pcpd2.

  4. Estimate receiver gain using rga command, and Start the experiment using command zg;

  5. Process the FID by typing “xfb”. Phase the spectrum when necessary.

1H-13C HSQC hsqcetgp
  1. Go to an existing experiment (pulse program: hsqcetgp), type “edc” to copy its parameters and generate a new experiment number. Or in an new experiment number "rpar HSQCETGP".

  2. Type “eda”, set parameters td, ns, sw, O1P and O2P, CNST2. Note that CNST2 can be different for different C-H bond.

  3. Type “ased” to adjust power length and power level for both 1H and 13C, including p1, pL1, p3, pL2, pL12, and pcpd2.

  4. Start the experiment by rga and zg; Process the FID by typing “xfb”. Phase the spectrum when necessary.

  5. Tutorial link

1H-15N HET SOFAST (SOFAST-HMQC)sofast
  1. Go to an existing SOFAST experiment (pulse program: sofast), type “edc” to copy its parameters and generate a new experiment number. Or "rpar SFHMQCF3GPPH" in an new experiment entry.

  2. Type “eda” to check and adjust parameters such as td, ns, sw, O1P and etc.

  3. Type “ased” to adjust power length and power level for both 1H and 15N, including, pL1, p3, pL2, pL12, and pcpd2.

  4. To calibrate shape pulse sp15 and sp20, type “stdisp” to open shapetool window, and calculate PC9 (3000 us at 120°) and Reburp1000 (2000us at 180°) pulses. sp value = shapetool value + p1 in dB.

    Shape Pulse Calculation Steps: choose the shape pule in shapte tool window, then go to Analysis/Integrate Shape, put in correct shape pulse length, angle, and 90 degree pulse length, hit "Enter" key. The calculated shapetool value will be shown as "Change of power level"

    Note that for Bruker pulse programs, you may use "getprosol 1H pulselength powerlevel" command, to let TopSpin calculate the power related parameter for you.

  5. In the new Bruker SOFAST pulse programs (starting TopSpin 3.5), there are CNST54, and CNST55, which may be adjust to achieve better receiver gain RG.
  6. Start the experiment by rga and zg;

  7. Process the FID by typing “xfb”. Phase the spectrum when necessary.

     

2D TOCSY dipsi2esgpph
  1. Go to an existing 2D-Tocsy experiment (pulse program: dipsi2esfbgpph), type “edc” to copy its parameters and generate a new experiment number. Or in a new experiment, "rpar DIPSI2ESFBGPPH".

    Pulse program dipsi2esfbgpph is based on dipsi2esgpph and have f2 and f3 channels included.

  2. Type "edasp" to check /adjust spectrometer routing.

  3. Type “eda”, set parameters td, ns, ds, sw, O1P, O2P and O3P if necessary.

  4. In acquisition window, if sample is unlabeled, leave ZGOPTNS blank. If sample is N15 labeled, set ZGOPTNS "-DLABEL_N". If sample is N15 and C13 labeled, set ZGOPTNS "-DLABEL_CN".

  5. Type “ased”, adjust power length and power level for 1H. If sample is labeled, you will need to check 15N and/or 13C power level and power length according.

  6. Type "pulse" to calculate pL10 based on targeting p6 value. Use p6 35 us.

  7. Set d9 (Tocsy mixing time) 90 ms for a protein sample; set pL32 70dB for water presaturation

  8. In gs mode, adjust O1P and p12 to achieve minimum FID area integral for water suppression.

  9. Start the experiment by rga and zg.

  10. Process the FID by typing “xfb”. Phase the spectrum when necessary.

 

2D NOESY noesyesgpph
  1. Go to an existing 2D-Noesy experiment (pulse program: noesyesfbgpph), type “edc” to copy its parameters and generate a new experiment number. Or in a new experiment, "rpar noesyesfbgpph".

    *Pulse program noesyesfbgpph is based on noesyesgpph and have f2 and f3 channels included.

  2. Type "edasp" to check and adjust spectrometer routing.

  3. Type “eda”, set parameters td, ns, ds, sw, O1P, O2P and O3P if necessary.

  4. In acquisition window, if sample is unlabeled, leave ZGOPTNS blank. If sample is N15 labeled, set ZGOPTNS "-DLABEL_N". If sample is N15 and C13 labeled, set ZGOPTNS "-DLABEL_CN".

  5. Type “ased”, adjust power length and power level for 1H. If sample is labeled, you will need to check 15N and/or 13C power level and power length according.

  6. Set d8 (NOESY mixing time) around 90 ms for biomolecules, or 300-500 ms for small molecules; set pL32 70dB for water presaturation.
    Users may consider using 2D-ROSEY for medium size molecules, MW 1000-3000 Da, mixing time 200ms.

  7. In gs mode, adjust O1P and p12 to achieve minimum FID area integral for water suppression.

  8. Start the experiment by rga and zg.

  9. Process the FID by typing “xfb”. Phase the spectrum when necessary.

Your may copy a data set from "expts" folder, or use rpar command to read paramenter set in the bruker library.

Make sure the constants (cnst21, cnst22, cnst23) are consistent through out all your 3D Triple resonance experiments, also set O2P the same as the constant stated in the pulseprogram. If one constant is set different from one experiment to another, the resulting spectra may show same signal with different chemical shifts in carbon dimemsion.

It is highly recommended to run a 1H-13C 2D of the 3D experiment first, just to make sure all the paramenters are set correctly. Signals in HNCA, HNCOCA, HNCACB, and CBCACONH should overlap well. Signals from HNCO and HNCACO should overlap well.

Saturation Transfer Difference (STD) - NMR experiment procedure
The best application of STD-NMR is ligand/drug screening, to identify ligand(s) that interacts with the host from a compound pool (multiple compounds) . In this system, the host is usually larger molecules, such as proteins and nucleic acids.

Samples: (using a DNA sample and one ligand as an example)

  1. DNA sample alone. For reference spectrum only, so low concentration is OK.
  2. Ligand sample alone.For reference spectrum only, so low concentration is OK.
  3. DNA+Ligand. Suggested molar ratio =<1:20, such as 0.1mM:2mM.
    * All samples should have the same buffer composition.

NMR Procedure:

  1. Run standard proton 1Ds on all three samples. The spectrum of ligand alone should closely match the spectrum of DNA+ligand, since in the latter sample ligand is in large excess.
    • Have sufficient number of scans (ns) to see all signals, especially for DNA signals in sample c.
    • The reference spectra will give you an idea the distribution of ligand and DNA signals. Since ligands are usually small, their signals are fewer. Therefore, it is usually not difficult to select a DNA signal for irradiation (on resonance frequency), which should be far from ligand signal positions.
  2. Set up STD experiment for sample 3 (DNA+Ligand).
    1. Create a new data set, then “rpar STDDIFFESGP.3”. This is a STD experiment with water suppression by excitation sculpting pulse.
    2. Use command “getprosol 1H XX XXX” to set up all the pulse parameters.
    3. Check O1p, which may not be at 4.7 if organic solvent is in the buffer system.
    4. Saturation time D20 =2s by default. May need adjustment if necessary.
    5. SPW9 40-60dB. My experience is that 40dB worked better than 60dB.
    6. Number of scans ns should be high, >=64
    7. Create an irradiation frequency list and save in AcquPars/FQLIST/FQ2LIST, to define the irradiation frequency (on resonance frequency). The frequency should be on a DNA proton signal which is NOT close to any ligand signals.
      The FQ2LIST file should have three numbers, magnet frequency, on resonance frequency and off resonance frequency.
    8. Run the STD experiment.
    9. Process the data using AU program “stdsplit”.
  3. NMR result:
    1. The first 1D should look like a standard 1D, and the second one would show ligand signals if the STD experiment worked.
    2. If no ligand signals appeared after trying different irradiation frequencies, either the ligand may not interact with the host, or the affinity may be very strong. The strong affinity can be identified by comparing 1D spectra of the DNA alone with the DNA in the presence of the ligand.