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Biomolecular NMR - Experiments

  • Hydrogen exchange effect on T1 and T2 measurements are reduced by steady-state pulse (sspul); Kim et. al. (2013) JBNMR 55, 249. SK versions use sspul in Agilent and Bruker sequences.
  • For T2 measurement of large proteins, the relaxation time per a CPMG cycle is made half.
  • A new NOE build-up pulse scheme is included; Ferrage et. al. (2010) JMR 207, 294.
  • Pseudo-3D acquisition in Bruker; the relaxation parameter can be arrayed via vd lists or vc lists and it is encoded as an additional dimension (F1QF).
  • Interleaved 2D acquisition in Bruker; in N15 NOE measurement, F1I is used for options of 1H saturation and no saturation.
  • Bruker topspin 3 introduced changes of sequences; TC (temperature compensation), shaped 1H pulses, and reducing the CPMG cycle.

 

NMR ExperimentBruker Sequence
15N-T1 HSQC hsqcf3T1sk
Acquire pseudo-3D; F3(1H) - F2(15N) - F1(vd list)
Use sspul and TC
vd list = 0.01, 0.02, .... (s)
Modified from hsqct1etf3gpsitc3d
15N-T1 TROSY trosyf3T1sk
Acquire pseudo-3D; F3(1H) - F2(15N) - F1(vd list)
Use sspul and TC
vd list = 0.01, 0.02, .... (s)
Modified from trt1etf3gpsi3d.2
15N-T2 HSQC hsqcf3T2sk
Acquire pseudo-3D; F3(1H) - F2(15N) - F1(vc list)
vc list = 1, 2, 3.... for loops of CPMG
Use sspul and TC
Relaxation time = 8ms*vc
Modified from hsqct2etf3gpsitc3d.2 that uses relaxation time = vc*16*(0.45ms+0.5p30) = vc*7.92ms
15N-T2 TROSY trosyf3T2sk
Acquire pseudo-3D; F3(1H) - F2(15N) - F1(vc list)
Use sspul and TC
vc list = 1, 2, 3.... for loops of CPMG
Relaxation time = 8ms*vc
Modified from trt2etf3gpsitc3d.2 that uses relaxation time = vc*16*(0.45ms+0.5p30) = vc*7.92ms
15N-NOE HSQC hsqcf3NOEsk
Acquire interleaved-2D; F2(1H) - F1(15N) - F1I(l0)
F1I needs 2 times td1
Use d1=2 and d20=4 for NOE
Use the 1H saturation scheme by Ferrage(2010) JMR, 207,294
xau calibsk for PLDB6
15N-NOE TROSY trosyf3NOEsk
Acquire interleaved-2D; F2(1H) - F1(15N) - F1I(l0)
F1I needs 2 times td1
Use d1=2 and d20=4 for NOE
Use the 1H saturation scheme by Ferrage(2010) JMR, 207,294
xau calibsk for PLDB6
15N-T2HE HSQC
(T2 Hahn echo to check Rex)
hsqcf3T2HEsk
Acquire pseudo-3D; F3(1H) - F2(15N) - F1(vd list)
Use sspul and TC
vd list = 0.01, 0.02, .... (s)
Relaxation time= 7.68ms*vd/0.01
xau calibsk for PLDB19
15N-T2HE TROSY
(T2 Hahn echo to check Rex)
trosyf3T2HEsk
Acquire pseudo-3D; F3(1H) - F2(15N) - F1(vd list)
Use sspul and TC
vd list = 0.01, 0.02, .... (s)
Relaxation time= 7.68ms*vd/0.01
xau calibsk for PLDB19

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