2. Structure determination by NMR
NMR principles
Data acquisition
Spectra process
xwinnmr、nmrpipe、nmrview、Topspin
Assignment
sparky
Data Analysis
Structure determination
InsightII、Xplor、CNS
Structural analysis
Procheck、Molmol、Pymol

3. ~~NMR Experiments studies~~

4. Sample prepare High concentrated protein Proton labeling Limitation
10mg-30mg
Proton labeling H1
H1-N15
H1-N15-C13
Limitation
Protein molecular size <25 Kda

5. Modern Fourier transform NMR spectrometer
Coil and superconductor
LN2 and LHe2 tank

6. Spectra process and Assignment
Chemical shifts in proteins

7. Spectra process and Assignment
Chemical shifts in proteins
the a-proton is always around 4 ppm；
the aromatic protons are around 7 ppm ；
the backbone amides at 8 ppm.

8. Well-dispersed 1D Spectrum
HN, aromatic
Hα,H2O
CH,CH2,CH3

9. Why do we go beyond one dimension?
To resolve the crowded signals in 1D spectrum by spreading them into other dimensions.
To elucidate the “through-bond” and “through-space” relationships between the spins in the molecules.

10. General scheme for two-dimensional NMR spectroscopy.

11. Dependence of the NMR signal on the evolution period, t1.

12. The functions of the different periods of a two-dimensional NMR experiment are summarized as follows:
Preparation: The desired nonequilibrium state of the spin system is prepared from the initial (equilibrium) state of the spin system. The preparation period in its simplest form consists of a single pulse that generates transverse magnetization, but more complex sequences of pulses can be used to prepare other coherences, such as multiple quantum coherences, and to perform solvent suppression.
Evolution: The off-diagonal components of the density operator prepared in step (1) evolve under the Hamiltonian, He . During the course of the experiment, the incrementable time t1 normally begins at an initial value and increases in discrete steps to a maximum value, t1max. The Hamiltonian, He , may be the free-precession Hamiltonian or may include applied rf fields. The frequencies with which the detected coherence evolves during t1 results in signals appearing at those frequencies in the F1 dimension of the final two-dimensional spectrum. This process is known as F1 frequency labeling of the coherence.
Mixing: During the mixing period, coherence is transferred from one spin to another. The mixing period is the key to establishing the type of correlation between the two dimensions and consequently dictates the information content of the spectrum. Depending on the type of experiment, the mixing period consists of one or more pulses and delays.
Acquisition: The FID is recorded in the conventional fashion. As discussed in Section 4.3, if more than one coherence transfer pathway is feasible, phase cycling or field gradient pulses are used to determine which coherence transfer processes contribute to the final spectrum.

13. Schematic generation of a three-dimensional NMR experiment from the combination of two two-dimensional NMR experiments.

14. The development of a three-dimensional data set from a two-dimensional data set.

16. Two-dimensional Fourier Transform NMR
14 mer

17. COSY (correlation spectroscopy)
The original 2D experiment. Used to identify nuclei that share a scalar (J) coupling. The presence of off-diagonal peaks (cross-peaks) in the spectrum directly correlates the coupled partners.
NOESY (Nuclear Overhauser Effect Spectroscopy)
A 2D method used to map NOE correlations between protons within a molecule. The spectra have a layout similar to COSY but cross peaks now indicate NOEs between the correlated protons.

18. Two-dimensional Fourier Transform NMR
14 mer

19. 2D COSY: thru bond
Stop here 12/9/08

20. Spin system identification 2D COSY of isoleucine

21. Spin system identification

22. The five regions of the COSY spectrum containing the fingerprint cross-peaks.

23. TOCSY Total Correlation Spectroscopy
HOHAHA(homonuclear Hartmann–Hahn) spectroscopy

24. Pulse sequence and coherence level diagram for the TOCSY experiment.

25. Sections of H2O TOCSY spectra acquired with mixing times of 48 (left), 83 (center), and 102 ms (right).

26. Cross-Relaxation NMR Experiments
NOESY (Nuclear Overhauser Effect Spectroscopy)

27. Pulse sequence and coherence level diagram for the NOESY experiment.

28. 2D NOESY: thru space

30. Secondary structure elements have characteristic NOE patterns

31. Sequential assignment Side chain assignment

32. Sequential assignment

33. Spectra process and Assignment
2D NMR spectroscopy

34. Spectra process and Assignment
2D NMR spectroscopy
2D TOCSY

35. Spectra process and Assignment
2D NMR spectroscopy
2D NOESY and TOCSY

36. Spectra process and Assignment

37. Spectra process and Assignment
Assignment – TOCSY : identify spin system
HN91
HN92
HN93 g b b b 4 a
Ha91 a
Ha93 a
Ha92 10 6 10 10 7

38. Spectra process and Assignment
Assignment - sequential assignment

39. Spectra process and Assignment
Assignment – NOESY : sequential assignment

40. Spectra process and Assignment
Assignment - sequential assignment

41. TOCSY : Amide to Aliphatic Region
N’-ACGSC RKKCK GSGKC INGRC KCY-C’

42. NOESY and TOCSY : Amide to a RegionH O

43. KWRRWVRWI

44. Chemical Shift Table for 20 Common Amino Acids

45. Chemical Shift Table for 20 Common Amino Acids

46. heteronuclear multiple-quantum coherence (HMQC)
Heteronuclear single-quantum coherence (HSQC)
TROSY

47. Isotope-labeling of proteins (I) 15N labeling
Grow proteins on minimal media (M9) with 15NH4Cl as the sole nitrogen source.
$100-$1000 for mM sample.
Structure elucidation of medium-sized proteins ( a.a.)

48. Isotope-labeling of proteins (II) 15N, 13C labeling
Grow proteins on minimal media (M9) with 15NH4Cl as the sole nitrogen source and 13C-glucose as the sole carbon source.
$1000-$10000 for mM sample.
Structure elucidation of larger proteins ( a.a.)

49. Isotope-labeling of proteins (III) 15N, 13C, 2H labeling
Grow proteins on minimal media (M9) with 15N2H4Cl as the sole nitrogen source and 13C,2H-glucose as the sole carbon source in deuterated water.
Re-exchange deuterium on amide nitrogen to protons.
Strain must be adapted to grow on D2O.
> $10000 for mM sample.
Structure elucidation of larger proteins (> 200 a.a.)

50. Isotope-labeling of proteins (IV) Site-specific labeling
Add labeled amino acids to non-labeled media.
Assuming that the amino acid is not metabolized, all residues corresponding to that amino acid will be labeled in the protein.
Technique is interesting when structural or dynamic information is only required for specific residues. Thereby, the complete assignment of the protein may be circumvented.

51. H1-N15 label HSQC

52. Assigned HSQC

53. 2D 1H-13C HSQC

54. 2D 1H-13C HSQC

55. 2D 1H-13C HSQC

56. Triple Resonance Experiment Use for Sequence Assignment
HNCA & HN(CO)CA
HNCO & HN(CA)CO
NHCBCA & CBCA(CO)NH

57. HNCO and HN(CA)CO
Regions generated from Tyr56 to Glu63 are shown here.
Red contours ：former residues
Black contours ：intra-residues.
Black and Red contours ：intra- and inter-residue cross peaks are overlapping

58. HNCA and HN(CO)CA
Regions generated from Tyr56 to Glu63 are shown here.
Red contours ：former residues
Black contours ：intra-residues.
Black and Red contours ：intra- and inter-residue cross peaks are overlapping

59. HNCACB + CBCA(CO)NH
Regions generated from Tyr56 to Glu63 are shown here.
The black lines show the scalar connectivities by Cα atoms and the blue lines show the scalar connectivities by Cβ atoms.

60. HCCH-TOCSY for side-chain proton assignments
Strip plot extracted from a 3D HCCH-TOCSY spectrum obtained with uniformly 13C-labeled HP0495 in D2O

61. Sparky - NMR Assignment and Integration Software

62. Structure determination by NMR
NMR principles
Data acquisition
Spectra process
xwinnmr、nmrpipe、nmrview、Topspin
Assignment
sparky
Data Analysis
Structure determination
InsightII、Xplor、CNS
Structural analysis
Procheck、Molmol、Pymol

63. Data Analysis and Structure determination
NOESY – distance restrain
CSI – chemical shift index
Structure determination
principles

64. Data Analysis
NOESY

65. Data Analysis NOESY medium range NOEs : i to i+2, i+3, i+4
long range NOEs
: i to i+5…….
filled circles < 6.0 Hz, open circles > 7.0 Hz
filled diamonds
kNH < 0.02 min-1

66. Data Analysis
NOESY
H : Slowly exchanging (kNH < 0.02 min-1 ) amide protons
the observed crosspeaks
Hydrogen bonds
NOE restrain : 20-30/per residues

67. Data Analysis
CSI, chemical shift index

68. Data Analysis
CSI

69. Structure determination
Calculation
There is no method for a "direct" or ab initio calculation of a structure from NMR data. We have to include assumptions to make up the lack of experimental data. We therefore have to provide e.g. bond distances and angles for amino acids.
NMR structure calculation cannot result in the structure. Instead structure calculation is repeated many times, producing a large number of structural models. All the models that satisfy the experimental constraints are assumed as being representative of the protein.

70. Data Analysis
Calculation

71. Data Analysis Calculation
Start: The temperature is set to Kelvin which is very hot. At this extreme temperature different conformations of the polypeptide convert into each other very fast. In a completely random manner a large number of conformations are sampled.
We let the protein hop and shake around under these unnatural conditions to allow it to sample as many conformations as possible. The NOE distances are always switched on to force the protein to preferentially choose conformations that agree with the NOESY distances.
After a while the temperature is slowly reduced over quite some time to room temperature. While the system cools down we slowly reintroduce a correct description of the protein.
In the end, we simulate the protein as correct as it is possible on a computer.
The structure at the very end of the protocol is saved.

72. Data Analysis
Calculation

73. Protein NMR Structure Determination
Protein in solution
~0.5 ml, 2 mM concentration
Sample preparation:
cloning,
protein expression
purification,
characterization,
isotopic labeling.
Distances between
protons (NOE),
Dihedral angles(J
coupling), H-bond
(Amide-proton
exchange rate ),
RDC restraints
NMR spectroscopy
1D, 2D, 3D, …
Secondary
structure of
protein
Sequence-specific
Resonance assignment
Extraction of Structural
information
Structure calculation
Final 3D
structures
Structure refinement

74. The Completeness of Assignment is an Determinant for NOESY Assignment
residue N C C C
other Q1
(8.379)
(4.111)
(2.834, 2.302)
C, (2.644, 2.644) D2
(7.959)
(4.746)
(3.154, 3.154) W3
(9.602)
(5.635)
(3.526, 3.317)
C1, (7.384); C3, (8.290); C2, (7.286); C2, (7.308); C3, (6.811); N1, (10.193) E4
(8.707)
(3.782)
(2.021, 2.021)
C, (2.422, 2.200) T5
(8.910)
(3.942)
(3.739)
C2, (1.248) F6
(8.796)
(4.228)
(3.615, 3.171)
C1, (7.165); C2, (7.165); C1, (7.024); C2, (7.024); C, (6.834) Q7
(8.118)
(3.647)
(1.193, 1.193)
C, (1.851, 1.851); N2, (6.195, 4.472) K8
(7.449)
(4.036)
(1.807, 1.770)
C, (1.487, 1.487); C, (1.710, 1.710); C, (2.944, 2.944) K9
(8.261)
(4.167)
(1.626, 1.626)
C, (1.090, 1.090); C, (1.398, 1.398); C, (2.882, 2.882)
H10
(7.803)
(4.846)
(2.767, 2.000)
C2, (6.786); C1, (8.755)
L11
(8.311)
(5.406)
(2.156, 2.156)
C, (1.788); C1, (1.103); C2, (1.103)
T12
(8.237)
(5.003)
(3.775)
C2, (1.220)
D13
(8.271)
(4.874)
(3.060, 2.766)
T14
(8.106)
(4.802)
(4.067)
C2, (0.947)
K15
(8.239)
(3.623)
(1.440, 1.440)
C, (0.736, 0.405); C, (1.423, 1.423); C, (2.741, 2.741)
K16
(7.904)
(4.277)
(1.680, 1.680)
C, (1.234, 1.234); C, (1.545, 1.545); C, (2.953, 2.953)
V17
(6.052)
(3.325)
(1.441)
C1, (0.405); C2, (0.105)
K18
(8.665)
(4.488)
(1.886, 1.886)
C, (1.548, 1.548); C, (1.748, 1.748); C, (3.062, 3.062)
C19
(8.066)
(3.692)
(3.027, 2.339)
D20
(8.880)
(4.453)
(3.056, 2.905)

75. Structural Statistics of the Best 20 Structures

76. Ramachandran Plot

77. 3D Structure Determination of RNase 3 from Rana catesbeiana

78. References http://www.cis.rit.edu/htbooks/nmr/inside.htm
“Spin Dynamics: Basics of Nuclear Magnetic Resonance” by Malcolm H. Levitt
“Protein NMR Spectroscopy: Principles and Practice” by Cavanagh, John, and Fairbrother, Wayne J, and Palmer, Arthur G, III, 2006.
“High-Resolution NMR Techniques in Organic Chemistry” by J.-E. Ba¨ckvall, J.E. Baldwin and R.M. Williams, 2009.
Wuthrich, K. “NMR pf protein and Nucleic Acids” Wiley-intersciences, 1986.

79. References
Derome, A. “Modem NMR Techniques for Chemistry Research” Pergamon, 1987.
Clore, G.M. and Gronenbron, A.M. (1994) Protein Science, 3, “Structures of Large Proteins, Protein-Ligand and protein –DNA Complexes by Multidimensional Heteronuclear NMR”.
Croasmun, W.R. and Carlson, R.M. “Two-Dimensional NMR Spectroscopy-application for Chemists and Biochemists” VCH, 1994.
CraiK, D.J. “NMR in Drug Design” CRC Series in Analytical Biotechnology, 1996.
Reid, D.G. “Protein NMR Techniques” Methods in Molecular Biology, 1997.
科儀新知1994年六月份。
Yee, A. et al. (2002) PANS, 99, “An NMR approach to structure proteomics”.
Clore, G.M. and Gronenbron, A.M. (1998) TIBTECH, 16, “Determining the Structures of Large Proteins, Protein Complexes by NMR”.
Clore, G. M. and Gronenborn A. M. (1998) New Methods of Structure Refinement for Macromolecular Structure Determination by NMR. Proc. Natl. Acad. Sci. USA. 95,
Gardner, K. H. and Kay, L. E. (1998) The Use of 2H, 13C, 15N Multidimensional NMR to Study the Structure and Dynamics of Proteins. Annu. Rev. Biophys. Biomol. Struct. 27,
Staunton, D., Owen, J. and Campbell, I. D. (2003) NMR and Structural Genomics. Acc. Chem. Res. 36,