Solution Structure Determination by NMR and Distance Geometry Calculations
1. Pure Protein (0.3 mL, 0.5 - 1 mM; ~ 10 mg)2. Sequence-specific resonance assignments3. Sequence-specifice distance constraints
disulfide bondsspin-spin coupling constantsinternuclear distance constraints from NOEsresidual dipolar couplingshydrogen bonds standard bond lengths; bond angles
4. Distance Geometry Calculations5. Constrained Energy Minimization; Refinement
Basic Principles
Nuclear Spin and Magnetization
Magnetization and Coherence
Fourier Transform NMRErnst & AndersonRev. Scientific Instrumentation 37: 93 (1966)
Nuclear Relaxation
Spin-Spin Coupling
Chemical Shift with Spin-Spin Coupling
1D NMR Spectrum of a Protein
2D Correlated SpectroscopY
AV-COSY
Nuclear Overhauser Effect (NOE)
Def: The change in the intensity of resonance i upon irradiation of resonance j due to “cross relaxation” between spins i and j through space
2D NOESY Spectrum of a Protein
Some Useful 2D NMR Experiments
1H-13C Heteronuclear Correlation Spectrum
Solution Structure Determination by NMR and Distance Geometry Calculations
1. Pure Protein (0.3 mL, 0.5 - 1 mM; ~ 10 mg)2. Sequence-specific resonance assignments3. Sequence-specifice distance constraints
disulfide bondsspin-spin coupling constantsinternuclear distance constraints from NOEsresidual dipolar couplingshydrogen bonds standard bond lengths; bond angles
4. Distance Geometry Calculations5. Constrained Energy Minimization; Refinement
WR41
Protein NMR Structures
C-TmZip
ER14
MMP-1
IL13
FGF-2
WR90
WR64
LC8
ER115
N-TmZip
JR19
ZR18
OP3
WR33
ZR31 ER75Z-domain
IR24
ER75: Ecol YgdK (nmr)ER30: Ecol SufE (xray)IR24: Hinf IscU (nmr)SR17 Bsub IscU (nmr)
N N
C C
YgdKG. Liu et al. (Szyperski)
Gram neg IscU
IscU, SufE,YgdK Domain Families are Structurally
Similar
YgdK cf SufEA. Kuzin et al. (Hunt)
Ramelot et al, J. Mol. Biol. 2004Goldsmith et al., J. Mol. Biol. 2004Liu et al. submitted
T. Ramelot et al. (Kennedy)
Gram pos IscU
G. Kornhaber et al. (Montelione)
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