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Structure determination by NMR
NMR principles
Data acquisition
Spectra process
xwinnmr、nmrpipe、nmrview、Topspin
Assignment
Data Analysis
Structure determination
InsightII、Xplor、CNS
Structural analysis
Procheck、Molmol、Pymol
~~NMR Experiments studies~~
Sample prepare
• High concentrated protein
– 10mg-30mg
• Proton labeling
– H1
– H1-N15
– H1-N15-C13
• Limitation
– Protein molecular size <25 Kda
Modern Fourier transform NMR
spectrometer
Coil and superconductor
LN2 and LHe2 tank
Spectra process and Assignment
 Chemical shifts in proteins
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.
Well-dispersed 1D Spectrum
Hα,H2O
HN, aromatic
CH,CH2,CH3
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.
Two-dimensional Fourier Transform NMR
• COSY (correlation spectroscopy)
– The original 2D experiment. Used to identify nuclei that share a
scalar (J) coupling. The presence of off-diagonal peaks (crosspeaks) 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.
Two-dimensional Fourier Transform NMR
2D COSY: thru bond
Spin system identification
2D COSY of isoleucine
Spin system
identification
2D NOESY: thru space
Secondary structure elements have characteristic
NOE patterns
Sequential assignment
Spectra process and Assignment
 2D NMR spectroscopy
Spectra process and Assignment
 2D NMR spectroscopy
 2D TOCSY
Spectra process and Assignment
 2D NMR spectroscopy
 2D NOESY and TOCSY
Spectra process and Assignment
 Assignment
Spectra process and Assignment
 Assignment – TOCSY : identify spin system
HN91 HN92 HN93
0
g
b
b
b
4
0
a
a
a
Ha91
Ha93
Ha92
10
6
10
0
10
7
Spectra process and Assignment
 Assignment - sequential assignment
Spectra process and Assignment
 Assignment – NOESY : sequential assignment
Spectra process and Assignment
 Assignment - sequential assignment
TOCSY : Amide to Aliphatic Region
N’-ACGSC RKKCK GSGKC INGRC KCY-C’
NOESY and TOCSY : Amide to a Region
H
O
H
H
N
C
C
N
C
N
C
H
H
O
KWRRWVRWI
Chemical Shift Table for 20 Common Amino Acids
Chemical Shift Table for 20 Common Amino Acids
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 (50100 a.a.)
Isotope-labeling of proteins (II)
15N, 13C labeling
• Grow proteins on minimal media (M9) with
15NH Cl as the sole nitrogen source and 13C4
glucose as the sole carbon source.
• $1000-$10000 for mM sample.
• Structure elucidation of larger proteins (100250 a.a.)
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.)
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.
2D 1H-13C HSQC
H1-N15 label HSQC
Assigned HSQC
Triple Resonance Experiment Use for
Sequence Assignment
• HNCA & HN(CO)CA
• HNCO & HN(CA)CO
• NHCBCA & CBCA(CO)NH
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
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
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.
HCCH-TOCSY for side-chain proton
assignments
Strip plot extracted from a
3D HCCH-TOCSY
spectrum obtained with
uniformly 13C-labeled
HP0495 in D2O
Structure determination by NMR
NMR principles
Data acquisition
Spectra process
xwinnmr、nmrpipe、nmrview、Topspin
Assignment
Data Analysis
Structure determination
InsightII、Xplor、CNS
Structural analysis
Procheck、Molmol、Pymol
Data Analysis and Structure determination
 Data Analysis
 NOESY – distance restrain
 CSI – chemical shift index
 Structure determination
 principles
Data Analysis
 NOESY
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
Data Analysis
 NOESY
H : Slowly exchanging
(kNH < 0.02 min-1 ) amide
protons
the observed crosspeaks
Hydrogen bonds
NOE restrain : 20-30/per residues
Data Analysis
 CSI
Data Analysis
 CSI
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.
Data Analysis
 Calculation
Data Analysis
 Calculation
• Start: The temperature is set to 1000-3000 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.
Data Analysis
 Calculation
Protein NMR Structure Determination
Protein in solution
~0.5 ml, 2 mM concentration
NMR spectroscopy
1D, 2D, 3D, …
Sequence-specific
Resonance assignment
Extraction of Structural
information
Sample preparation:
cloning,
Distances
between
protein expression
Secondary
protons
(NOE),
purification,
structureangles(J
of
Dihedral
characterization,
protein
coupling),
H-bond
isotopic
labeling.
(Amide-proton
exchange rate ),
Structure calculation
RDC restraints
Structure refinement
Final 3D
structures
The Completeness of Assignment is an Determinant for
NOESY Assignment
residue
N
C
Ca
Cb
other
Q1
124.279 (8.379)
175.880
60.337 (4.111)
27.906 (2.834, 2.302)
Cg, 31.404 (2.644, 2.644)
D2
114.136 (7.959)
174.728
52.070 (4.746)
42.126 (3.154, 3.154)
W3
124.678 (9.602)
178.494
58.251 (5.635)
32.468 (3.526, 3.317)
C1, 128.925 (7.384); C3, 124.926 (8.290); C2, 123.330 (7.286); C2, 114.589
(7.308); C3, 120.036 (6.811); N1, 129.962 (10.193)
E4
120.757 (8.707)
178.990
59.771 (3.782)
27.665 (2.021, 2.021)
Cg, 34.591 (2.422, 2.200)
T5
118.196 (8.910)
175.760
65.742 (3.942)
67.089 (3.739)
Cg2, 22.548 (1.248)
F6
122.999 (8.796)
177.890
61.930 (4.228)
39.135 (3.615, 3.171)
C1, 132.878 (7.165); C2, 132.878 (7.165); C1, 129.972 (7.024); C2, 129.972
(7.024); C, 128.127 (6.834)
Q7
117.315 (8.118)
178.649
59.520 (3.647)
30.229 (1.193, 1.193)
Cg, 34.492 (1.851, 1.851); N2, 107.564 (6.195, 4.472)
K8
118.131 (7.449)
178.680
58.946 (4.036)
32.895 (1.807, 1.770)
Cg, 25.188 (1.487, 1.487); C, 29.128 (1.710, 1.710); C, 42.023 (2.944, 2.944)
K9
115.307 (8.261)
176.634
57.489 (4.167)
34.724 (1.626, 1.626)
Cg, 26.475 (1.090, 1.090); C, 29.549 (1.398, 1.398); C, 41.720 (2.882, 2.882)
H10
106.834 (7.803)
173.998
55.370 (4.846)
30.286 (2.767, 2.000)
C2, 122.074 (6.786); C1, 137.835 (8.755)
L11
120.994 (8.311)
176.088
55.178 (5.406)
41.738 (2.156, 2.156)
Cg, 26.267 (1.788); C1, 24.285 (1.103); C2, 24.285 (1.103)
T12
114.206 (8.237)
171.678
58.813 (5.003)
69.998 (3.775)
Cg2, 19.537 (1.220)
D13
125.154 (8.271)
175.311
52.472 (4.874)
39.360 (3.060, 2.766)
T14
114.229 (8.106)
172.387
58.915 (4.802)
69.593 (4.067)
Cg2, 19.268 (0.947)
K15
124.290 (8.239)
176.948
57.949 (3.623)
32.306 (1.440, 1.440)
Cg, 24.692 (0.736, 0.405); C, 29.281 (1.423, 1.423); C, 41.742 (2.741, 2.741)
K16
120.841 (7.904)
174.770
53.666 (4.277)
30.617 (1.680, 1.680)
Cg, 24.350 (1.234, 1.234); C, 28.913 (1.545, 1.545); C, 41.894 (2.953, 2.953)
V17
121.789 (6.052)
175.994
62.623 (3.325)
32.235 (1.441)
Cg1, 21.082 (0.405); Cg2, 19.392 (0.105)
K18
128.767 (8.665)
176.794
53.827 (4.488)
29.301 (1.886, 1.886)
Cg, 19.141 (1.548, 1.548); C, 24.296 (1.748, 1.748); C, 42.046 (3.062, 3.062)
C19
122.110 (8.066)
174.536
58.997 (3.692)
40.173 (3.027, 2.339)
D20
118.616 (8.880)
177.547
57.451 (4.453)
38.424 (3.056, 2.905)
Structural Statistics of
the Best 20 Structures
Ramachandran Plot
3D Structure Determination of RNase 3 from
Rana catesbeiana
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.
References
•
Derome, A. “Modem NMR Techniques for Chemistry Research” Pergamon, 1987.
•
Clore, G.M. and Gronenbron, A.M. (1994) Protein Science, 3,372-390 “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.
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科儀新知1994年六月份。
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Yee, A. et al. (2002) PANS, 99, 1825-1830 “An NMR approach to structure proteomics”.
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Clore, G.M. and Gronenbron, A.M. (1998) TIBTECH, 16, 22-34 “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, 5891-5898.
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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, 357-406.
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Staunton, D., Owen, J. and Campbell, I. D. (2003) NMR and Structural Genomics. Acc. Chem. Res.
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