Design, principles and building blocks of Design, principles and building blocks of heteronuclear heteronuclear NMR pulse sequences NMR pulse sequences Michael Sattler Michael Sattler EMBL Heidelberg EMBL Heidelberg GSF GSF- National Research Center for Environment and Health National Research Center for Environment and Health Technical University Technical University München nchen Prog. NMR Spectrosc. (1999) 34 , 93-158. http://www.embl.de/nmr/sattler EMBO Practical Course: EMBO Practical Course: Structure determination of biological macromolecules by solution Structure determination of biological macromolecules by solution NMR NMR Biozentrum Biozentrum Basel, July 6 Basel, July 6- 13 2007 13 2007 Contents Contents • Heteronuclear Heteronuclear NMR: motivation NMR: motivation • Basic pulse sequence elements and 2D correlations Basic pulse sequence elements and 2D correlations • RF pulses: calibration, selective pulses RF pulses: calibration, selective pulses • Sensitivity enhancement, gradients, coherence selection Sensitivity enhancement, gradients, coherence selection • Water Water- flip back flip back • Spin Spin- state selection, TROSY state selection, TROSY • Triple resonance experiments Triple resonance experiments • Isotope editing and filtering Isotope editing and filtering
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Design, principles and building blocks of Design, principles and building blocks of
δδ((1313C): evolution during tC): evolution during t11
J(J(11H,H,1313C): active during C): active during ΔΔ
J(H,H): not activeJ(H,H): not active
J(C,C): active !J(C,C): active !
Relaxation during tRelaxation during t11: T1(: T1(11H), T2(H), T2(1313C)C)
Basic building blocks: Basic building blocks: heteronuclearheteronuclear correlationcorrelation
Relaxation during tRelaxation during t11
T2MQ (IxSy) > T2(S)
Methyl TROSY
T1 I-spin (1H)
T2 S-spin (13C)
T2 S-spin (13C)
Bax et al JMR (1990) 86, 304-318
synchronous decoupling!
Transfer Transfer amplitudesamplitudes forfor antiphaseantiphase//inin--phasephaseconversionconversion in CH, CHin CH, CH22 and CHand CH33 spinspin systemssystems
•• AdiabaticAdiabatic””fastfast”” passage: keep magnetization and passage: keep magnetization and rfrf field field colinearcolinear: |: |ddζζ/dt/dt| | «« ωωeffeff
•• NonNon--linear pulse phase modulation linear pulse phase modulation frequency sweep: frequency sweep: ddΔωΔω/dt/dt
•• broadbroad--band inversion with low powerband inversion with low power
•• problem: problem: adiabaticityadiabaticity requires long requires long ττPP
Instead of precessionaround z-axis with:φfree = ΔΩ∗ τp
ω = 2ω1 1effθ
ω1
ΔΩ = √3ω1
ΔΩ = 3√ ω1
Mx180°-pulse:τπ =π/ω1
0 −ΔΩ
φBSP(0) = −ΔΩ * τπ = −√3 ω1 * π/ω1 = −√3π ≈ 48.2°
1313CC’’ 1313CCαα
rotating coordinate system at :−ΔΩ
x
y
ΔΩ τπx
y
x
y
ΔΩ τπx
y
ΔΩ τπ
180°-pulse at −ΔΩ
360° on-resonance=̂
free evolution
((1313CCαα))
Bloch Siegert shift: inversion simulationMz -> Mz
[%]
100
50
0
-50
[kHz] 4 0 -4 -8
Q3 (2 msec, 1650.4Hz, freq: 2kHz, -3kHz)
W. Bermel, Bruker
Bloch Siegert shift: inversion simulationMz -> Mz
[%]
100
50
0
-50
[kHz] 4 0 -4 -8
Q3 (2 msec, 3300.8Hz, freq: 2kHz, -3kHz)
W. Bermel, Bruker
Bloch Siegert shift: inversion simulationphase
[°]
200
100
0
-100
[time] T/2 T
Q3 (2 msec, 1650.4Hz, freq: -3kHz)
without pulse, with Q3 pulse, difference
My (freq: 2kHz)
W. Bermel, Bruker
BSP compensationBSP compensation
ΔΩ/2π [kHz]
BSP [°]
120
-80
-60
-40
-20
0
20
40
60
80
100
10 12 14 16 18 20 22 24 26 28 30
Bloch-Siegert phasefor a band-selective(± 5kHz) G3 pulse
Determine phase empirically(0th and 1st order)
Use amplitude-modulatedinversion pulse (0th order)
Use additional pulse(all orders are corrected)
φ+BSP(0)
δCα
C'
G3, 250 s, 18 kHzphase modulation
μ
TT t12
t12
Intrinsic correction(all orders are corrected)
φδ'
G3, 700 s, 18 kHzamplitude modulation
μ
TT t12
t12
BSP
φTT t1
2t12 δCα
C'
TT t12
t12
G3 shape withadded BSP correction
φBSP = ω12/(2ΔΩ)*τP
Calculate φBSP for each subpulse
of a shaped pulse and add it to
the phase to compensate for the
BSP.
JMR (1992) 100, 604; JMR (2000) 146, 369.
Experimental determination of Bloch Experimental determination of Bloch SiegertSiegert PhasePhase
•• Zero order phase correction Zero order phase correction δδ00 applied to the phase of a flanking 90applied to the phase of a flanking 90oo pulsepulse
•• First order phase correction by addition of a delay First order phase correction by addition of a delay δδ11
Bloch Siegert shift: inversion simulationMz -> Mz
[%]
100
50
-50
[kHz] 4 0 -4 -8
Q3 (2 msec, 3300.8Hz, freq: 2kHz, -3kHz)
with BS compens.
without
W. Bermel, Bruker
BSP
φTT t1
2t12
ΔΩ/2π [kHz]
BSP [°]
120
-80
-60
-40
-20
0
20
40
60
80
100
10 12 14 16 18 20 22 24 26 28 30
Bloch-Siegert phasefor a band-selective(± 5kHz) G3 pulse
BSP compensation (intrinsic or add. pulse)BSP compensation (intrinsic or add. pulse)
GradientsGradients in in heteronuclearheteronuclear NMR NMR experimentsexperiments
x x
Gz
I
y
x
y
xa
b
y
xa
b
a
b
y
x
Spin echo with gradients
Δ2
Δ2
x x y
Gz
S
I
I: Iz
Iz
-Iy
-Iy 2I Sx z -2I Sz y2I Sz z2I Sz z
Iy Iy
I-S:
y
x
Spoil/purge gradients
Coherencerejection
Coherenceselection
Water Water suppressionsuppression methodsmethods
Concentration [Concentration [11H] in HH] in H22O O ≈≈ 110 M, concentration 110 M, concentration biomoleculebiomolecule ≈≈ 1010--33 MM
PROBLEMS: PROBLEMS: dynamic range (receiver); radiation dampingdynamic range (receiver); radiation damping
•• PresaturationPresaturation
depends on Bdepends on B00 homogeneity (shimming)homogeneity (shimming)
signals with near solvent frequency are suppressed as well (e.g.signals with near solvent frequency are suppressed as well (e.g. HHαα in proteins)in proteins)
reduces S/N of exchangeable protons due to saturation transferreduces S/N of exchangeable protons due to saturation transfer
difficult to combine with triple resonance/multidifficult to combine with triple resonance/multi--pulse sequencespulse sequences
•• SpinSpin--lock, gradient spoil pulses, WATERGATElock, gradient spoil pulses, WATERGATE☺ can be combined with watercan be combined with water--flipflip--backback
suppression of signals near watersuppression of signals near water
•• HeteronuclearHeteronuclear gradient echoesgradient echoes☺ excellent waterexcellent water--suppression with sensitivity enhancement, combine with watersuppression with sensitivity enhancement, combine with water--flipflip--backback
•• PostPost--acquisitionacquisition
☺ apply lowapply low--pass filters to eliminate signals at 0 pass filters to eliminate signals at 0 ±± ωω, i.e. water on, i.e. water on--resonanceresonance
suppresses signal near water as wellsuppresses signal near water as well
•• can be recorded in Hcan be recorded in H22O due to excellent water suppression by O due to excellent water suppression by heteronuclearheteronuclear gradient echogradient echo
•• 11H H ↔↔ X transfer can be optimized simultaneously for X transfer can be optimized simultaneously for 1313C and C and 1515NNbut: some relaxation loss for but: some relaxation loss for 1313C due to longer delay.C due to longer delay.
•• poor waterpoor water--suppression, since E/AE cannot be implemented without sensitivitsuppression, since E/AE cannot be implemented without sensitivity loss.y loss.
•• building block for simultaneous 3D/4D NOESY experiments.building block for simultaneous 3D/4D NOESY experiments.
MultiMulti--dimensionaldimensional NMR experimentsNMR experiments• To resolve signal overlap with increasing molecular weight• 1/√2 loss of S/N per indirect dimension, but increased resolution
Can be recorded in HCan be recorded in H22O due to excellent water suppression by O due to excellent water suppression by heteronuclearheteronuclear gradient echogradient echo
HBHDHE (aromatic side chain assignments)HBHDHE (aromatic side chain assignments)
JACS (1995) 115, 11054
Cβ
H
γδ ε
δ ε
H H
H H
H
H
Cα
•• Delay Delay ξξ for for 1,1,--1 1801 180o o 1313C pulse to decouple C pulse to decouple 11J(CJ(Cαα,C,Cββ) )
•• 1313C pulses are selective with excitation nulls at C pulses are selective with excitation nulls at 1313CCββ and and 1313CCarar, respectively, respectively
Relative sensitivity of triple resonance experimentsRelative sensitivity of triple resonance experiments
•• HMQC rather than HSQC for HMQC rather than HSQC for 1313C C edited NOESY to minimize offedited NOESY to minimize off--resonance effects for resonance effects for 1313C C rfrf pulsespulses
•• Alternatively use adiabatic Alternatively use adiabatic inversion pulses, i.e. at higher fieldsinversion pulses, i.e. at higher fields
Isotope edited/filtered experiments: single filterIsotope edited/filtered experiments: single filter
1H-12C and 1H-13C can be separated into subspectra
add ψ = +/- x → 1H-12C filteredsubtract ψ = +/- x → 1H-13C edited
To use as a filtered/edited pulse sequence:To use as a filtered/edited pulse sequence:ΔΔ’’ ++ ΔΔ”” = 1/(J) = 1/(J) (single J(single J--filter)filter)ΔΔ’’’’−− ΔΔ’’ = 0= 0
((11HH--1212C and C and 11HH--1313C can be separated into C can be separated into subspectrasubspectra))