Calibrating Lew Kay Pulse Sequences

First load in the calibration template files from kay/vnmrsys/data/A__templates. These should contain good starting values and may have the arrays already set up for you. Type in “au” to get them started and make changes from there.

Finding tof

Locate the optimal tof using BioPack water pulse sequence.
Or use Kay’s sequence t2pul_lek_500 as follows:
  • load file cal_tof_t2pul_lek_sel4.fid
  • This is essentially the s2pul Varian sequence and includes the option of water presat.
  • Array tof to find minimum in water signal with fsat='y'

1H pw90

Load file cal_H1pw_hsqc_ydw.fid for starting parameters.
  • Use hsqc_ydw_cal to calibrate 1H pulse without water presaturation using 15N HSQC. (Recommended for multidimensional experiments)
  • Set nt=8.
  • set sph to shape desired (e.g. 'hard' or any shape file)
  • tpwrx = power of 1H pulse to be calibrated (e.g. 58 on 800; 53 on 500)
  • tofNH is the position of carrier for this one pulse, set to one of the HN resonances using movetof and then reset tof back
  • Array pwh to find pw90 at nullset pwx2x=0 (N15 pulse)
  • Beware: dpwr2 is the power for continuous wave 15N decoupling and should be around 42. dhpwr2 is for 15N pulses (see below).
  • On the 500 the proton pw90 was found to be 7.75 with a tpwrx of 53. On the 800, a protein sample pwx was found to be 8.7 at tpwrx=58 compared to 7.8 using t2_pul_lek sequence. This is consistent with BioPack results.

1H flip-back pulse calibration

Load file cal_pwsl_t2pul_lek_sel3.fid
  • t2pul_lek_sel3_500
  • Used to calibrate a water flipback pulse after a 90°. See notes from Jianjun. tpwrsl set to 10, array pw_sl to find minimal water (~1000-1500)

15N pw90 calibration

Load file cal_N15pw_hsqc_ydw.fid
  • Use hsqc_ydw_cal to calibrate 15N pulse on protein samples (Recommended procedure )
  • A rectangular ('hard') or shaped 15N pulse can be calibrated. Set spn = 'hard' or name of shape file.
  • Set set pwh=0 (i.e. set proton calibration pulse to 0)
  • Set power with dnx; find pw by arraying pwx2x; a 90¡ pulse leads to a null.dnx = -16, and pwx2x = 0 gives a regular 15N HSQC.
  • On the 500: the 90° pwx2x of 36.5 was found with a dnx power of 57.
  • On the 800: the 90° pwx2x was 36.5 usec at dnx of 60.

15N decoupling power calibration

  • For 15N decoupling on the 500, the pwx2x was 207 usec at a dnx of 42, giving a dmf2 of 4830 and a field strength of 1207.7 (±24 ppm). Use dmf2adf to set dmf2. At a dnx power of 44, the pw90 was 160 usec, giving a dmf2 of 6250 Hz, and a field strength of 1562 Hz.
  • For 15N decoupling on the 800, pwx2x was 247.5 at dnx of 42 giving a field strength of 1012 Hz (±12.5 ppm). Use dmf2adj to set dmf2. At a dnx or 44, pwx2x was 192.5 giving a field stength of 1299 Hz (± 16 ppm).

13C pw90 calibration

Load file cal_C13_hnco_ydw.fid
  • Use hnco_ydw_cal for calibration of any 13C pulse (hard or shaped) on a protein sample (RECOMMENDED)
  • A null in the hnco_ydw_cal 1D spectrum occurs when pwco90x = 90°
  • spcx specifies the pulse shape
  • d_cx specifies the power for the pulse being calibrated.
  • On the 500, a d_cx power gave a pwco90x of 12.75 usec.
  • On the 800: a d_cx power of 61 gave a pwco90x of 14.4 usec.

Alternative 1H pw90 (using H2O signal)

t2pul_lek_500 (or 800 version)
  • This is essentially the s2pul Varian sequence and includes the option of water presat.
  • Array pw to find null at 360°. Divide pw360 by 4 to obtain pw90.
  • Find a power that gives a pw90 around 8 usec.
  • On the 800: tpwr=56 gives a pw90 of 7.9; tof = -298.
  • On the 500: tpwr=53 gives a pw90 of ~7.6; tof=-172.

Alternative 15N pulse calibration methods

  • Use hetcal_echo_lek2_500 on benzimide (small molecule standard)
  • This is essentially the same as the Varian pwxcal sequence.
  • Turn off presat with fsat = 'n'
  • Finding pwN90 with N15 standard:
  • Set ss=2, nt=4, dhpwr2=57 (or whatever), dm2 = 'nnnn' and array pwx2 to find 90° null and calculate 15N pw90 at the N15 power setting of dhpwr2.

Alternative 13C pulse calibration

  • Use Varian pwxcal or hetcal_echo_lek2_500 for calibration of standard small molecule sample (methanol auto test sample) as described above for 15N.
  • Finding pwC90 using methanol autotest sample
  • hetcal_echorm_500 pulse sequence
  • Set dof and proton pw90 and power
  • Set dhpwr=54, dm='nnnn', dmm='cccp'
  • Measured splitting is 142 Hz, so set jxh=142 (otherwise phase is distorted)
  • Set dhpwr=61 and array pwx1 to 90° null for C13.

15N tof calibration methods

Find N15 dof2 by calculation based on ratio of 1H and 15N gyromagnetic ratios (Recommended procedure). This is accomplished by typing dof2cal(ppm desired), where "ppm desired" is the carrier position (in ppm) that you desire. But first you must correctly reference the 1H dimension.
Finding N15 dof with N15 standard:
With benzimide standard (indirect detection #2 sample)
Set tof = 2414.4 (on the 500)
Set pwx2 = 0 so that you see only decoupling
Set dm2 = 'nnny', dmm2 = 'cccc' for continuous wave 15N decoupling, dpwr2=35
Array dof2 (e.g. Ð3000, -500, -250, 0, 250, 500) and locate the frequency setting which provides coalescence of the 1H doublet caused by N15 coupling. Fine tune with increments of 25 Hz at dpwr2=33. Should be about dof2=350 on 500.

13C dof calibration

Find C13 carrier frequency (i.e. dof) by calculation based on ratio of 1H and 13C gyromagnetic ratios (Recommended). Use macro dofcal(with ppm of desired offset).
Find dof using t2pul_lek_500 (autotest sample)
dpwr=35, dm='nnny', dmm='cccc'
array dof=5000,-5900,-5875,.. to locate frequency which collapses the splitting
Ranjith found dof=-5775 which corresponds to 49.5 ppm.