Transcript Slide 1

NMR Assignments
What is the NMR Assignment Issue?
• Each observable NMR resonance needs to be assigned or associated with the atom in the protein
structure.
 NMR spectra of proteins are complex, where the complexity increases with the size or
number of residues of the protein
13C & 15N isotope enrichment to simplify the NMR spectra need to assign these NMR
 Use
resonances
1
13C and 15N NMR resonances to assign
 a typical protein will have hundreds of H,
Model
ATOM
ATOM
ATOM
ATOM
ATOM
ATOM
ATOM
ATOM
ATOM
ATOM
ATOM
ATOM
ATOM
ATOM
ATOM
ATOM
ATOM
ATOM
ATOM
ATOM
1H
NMR Spectra
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
1
N
CA
C
O
CB
CG
OD1
OD2
1H
2H
3H
HA
1HB
2HB
N
CA
C
O
CB
OG
ASP
ASP
ASP
ASP
ASP
ASP
ASP
ASP
ASP
ASP
ASP
ASP
ASP
ASP
SER
SER
SER
SER
SER
SER
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
20.897
20.874
20.456
21.196
22.271
22.154
22.132
22.088
21.578
19.948
21.182
20.170
22.757
22.854
19.279
18.826
17.435
16.466
18.770
18.165
9.212
8.930
7.476
6.691
9.155
9.514
8.602
10.695
8.579
9.056
10.199
9.590
9.961
8.252
7.108
5.701
5.682
6.118
4.994
5.857
Protein PDB File
4.618
3.154
2.921
2.361
2.570
1.088
0.278
0.789
5.083
5.016
4.777
2.670
3.100
2.674
3.349
3.152
2.511
3.095
4.507
5.461
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
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0.00
NMR Assignments
... Ala 70
Ser 71
O
... H2N
CH
C
Leu 72 ...
O
H
N
CH
CH3
O
H
N
C
CH2
OH
CH
Ser 71
15
55.5 ppm O N 114.8 ppm
HN 7.08 ppm
Ha 3.76 ppm
CH
H
N
C
13CO
CH
13Cb
64.8 ppm
Hb 3.73 ppm
15N
125.6 ppm
HN 8.20 ppm
H
N
C
13CO
171.9 ppm
13Cb
17.5 ppm CH3
Hb 1.45 ppm
...
CH3
Leu 72 ...
O
13Ca
... H2N
OH
CH3
... Ala 70
119.3 ppm
HN 7.76 ppm
C
CH2
Again, as illustrated here, the goal is to explicitly
assign each H, C, & N in the protein’s primary
sequence with its corresponding NMR resonance
15N
CH
CH
CH2
OH
OH
13Cb
CH
CH3
42.9 ppm
Hb 1.52 ppm
13Cd
CH3
...
170.9 ppm
CH2
13Ca
59.9 ppm
Ha 4.35 ppm
58.6 ppm
Ha 4.09 ppm
C
13CO
178.1 ppm
13Ca
O
13Cg
27.9 ppm
Hg 1.65 ppm
25.4 ppm; 25.7 ppm
Hd 0.82 ppm; 0.98 ppm
NMR Assignments
Predicting NMR Chemical Shifts
• A ever-growing number of computer programs are being developed to predict chemical shifts
from structure or sequence.
 SHIFTS, SHIFTX2, SPARTA+, Camshift, PPM, 4DSPOT, shAIC, etc.
 Empirical models based on high quality structures with NMR assignments, and molecular
dynamics
J. Biomol. NMR 2010 48(1):13.
J. Biomol. NMR 2012 54(3):257
NMR Assignments
How Are NMR Assignments Made For a Protein?
• Requires the collection and analysis of multidimensional NMR data
2D, 3D, 4D NMR spectra
• This in turns requires software to assist in the processing and analysis of the data
 ongoing effort to develop software to automate NMR assignments
 not “100%” efficient but significantly aids in the manual assignment

Assignment Table
Resi due
D1
E2
D3
E4
R5
W6
T7
N8
N9
F10
R11
E12
Y13
N14
L15
.
.
.
N
120.1
128.9
116.1
113.3
123.8
127.8
109.9
115.2
116.7
110.1
121.2
119.0
122.1
121.5
127.7
(8.08)
(9.93)
(8.90)
(7.45)
(7.97)
(9.27)
(9.32)
(8.42)
(7.88)
(7.57)
(8.03)
(8.56)
(8.28)
(8.45)
(9.02)
CO
179.1
176.6
176.5
175.6
173.1
177.1
174.8
174.5
173.3
177.2
175.0
177.9
173.2
175.7
176.9
Ca
53.8
56.0
56.2
52.9
54.3
55.4
59.6
50.9
52.1
58.2
55.8
52.3
61.9
54.7
58.0
(4.37)
(4.55)
(4.73)
(4.71)
(4.43)
(5.50)
(4.85)
(5.06)
(4.94)
(4.46)
(4.06)
(3.79)
(3.80)
(4.89)
(4.48)
Cb
39.9
30.9
38.9
27.2
28.9
30.8
71.3
38.7
38.0
38.5
29.7
27.4
41.2
39.2
41.2
(3.00,0.58)
(2.11,1.78)
(2.70,2.34)
(1.23,0.63)
(1.84,1.47)
(3.11)
(4.34)
(3.13,2.70)
(3.33,3.01)
(2.63,2.67)
(1.83,1.67)
(1.16,-0.05)
(2.99,2.52)
(2.26)
(1.96,1.64)
Ot her s
Cg 36.2(2.75,2.41)
Cg 34.8(2.57,1.79)
Cg 26.2(1.59,1.16);Cd 42.6(3.09,3.02)
Cg 20.2(0.73)
Cg 27.4(1.45,1.35);Cd 43.1(3.13)
Cg 36.6(2.19,1.97)
Cg 27.2(1.20);Cd 27.8(0.87);Cd 27.8(0.78)
NMR Assignments
NMR Data Processing Software
• Needs to specifically handle format of multidimensional NMR data
2D, 3D, 4D NMR spectra
• NMRPipe, Felix, ACD and others
 all have similar functions and capability
 all handle common instrument data formats (Bruker, Varian)
 choice is primarily based on personal preference

NMRpipe:
- UNIX/LINUX
- simple script to
process NMR data
- mimics flow of
processing steps
- uses UNIX pipe
functionality to pass
data between one
function to the next
NMR Assignments
NMR Data Processing Software
• Main steps in the processing process include:
window function (SP), zero fill (ZF), Fourier transform
(FT), phase (PS), transpose (TP)
• Other steps include
 removing solvent (SOL), linear prediction (LP) and data
extraction (EXT)
• These steps are simply repeated for each dimension of the
NMR data
Standard Processing Script for 3D NMR Data

X
Processing
steps for
X,Y,Z
dimensions of
3D spectra
Y
Z
NMR Assignments
NMR Data Processing Software
• Because of the exponential increase in time to collect nD NMR spectra, the number of data points
collected for the indirect FIDs are kept to a minimum
 1D NMR ~few mins.  2D ~few hours  3D ~ few days
 1D NMR 8-32K pts  2D 2K x 512 pts  3D 2K x 128 x 80 pts
• Two major impacts:
 FIDs in indirect dimension are typically truncated  artifacts in the spectra
 FIDs in indirect dimension have very low resolution
• These issues are addressed in processing the data
 ZF, SP, LP
FT
NMR Assignments
NMR Data Processing Software
• A main goal in applying a window function for a nD NMR spectra is to remove the truncation by
forcing the FID to zero.
Truncated FID with spectra “wiggles”
Apodized FID removes
truncation and wiggles
NMR Assignments
NMR Data Processing Software
• Some common window functions with the corresponding NMRPipe command
NMR Assignments
NMR Data Processing Software
• Want to maximize digital resolution, number of data points in each dimension

time constraints are a practical limitation for nD NMR data
NMR Assignments
NMR Data Processing Software
• Improve digital resolution by adding zero data points at end of FID
essential for nD NMR data
 no significant gain after one ZF, just interpolation between points

8K data
8K FID
No zero-filling
8K zero-fill
16K FID
8K zero-filling
NMR Assignments
NMR Data Processing Software
• Linear Prediction
extrapolate FID data in time domain
 enhances resolution
 works best for data without significant relaxation
 assumes sinusoid shape
 a set of coefficients is found such that linear combination of a group of points predicts the
next point in the series.
 number of coefficients determine the number of NMR signals (damped sinusoids) that
can be predicted
 LP is usually limited to extending data to about twice its original size
 forward linear prediction - points immediately after each group are predicted
 backward linear prediction - points immediately before each group are predicted
 forward-backward linear prediction - combines results from separate forward- and
backward-linear prediction calculations.

LP
NMR Assignments
NMR Data Processing Software
• Linear Prediction
model (set of coefficient) can be applied to predict a new synthetic point
 uses a group of existing points from the original data
 new point along with group from the original data is used to predict yet another point
 process can be continued indefinitely
 becomes unstable when group contains all synthetic points
 Mirror Image LP
 LP order (number of coefficients) must be as large as the number of signals to extract,
but smaller than half the original data size.
 For constant time data, (no decay) can temporarily add the data's mirror image
complex conjugate for the LP calculation and then discard it.
– time increment must be the same between each point
– either 0,0 or 90,-180 phase correction

LP
Progress in Nuclear Magnetic Resonance Spectroscopy (1988), 20(6),515-626
NMR Assignments
NMR Data Processing Software
• Effects of Combining Linear Prediction with Zero Filling

significant improvement in resolution for nD NMR data collected with minimal data points
NMR Assignments
NMR Data Processing Software
• uniform data sampling
avoids under-sampling frequencies
 FT algorithms expect uniform spacing of digital data

The Nyquist theorem
Need to sample twice as fast (DW)as the fastest frequency
Traditional NMR acquires EVERY
data point with a uniform time-step
between points.
Reason why nD NMR experiments
take so long, why FIDs in indirect
dimensions are truncated and the
spectra have low resolution and
sensitivity
NMR Assignments
NMR Data Processing Software
• Non-uniform data sampling
significant improvement in resolution and sensitivity for nD NMR data
 Don’t need uniform sampling, just need alternative to FFT to process the data.
 The sampling non-uniform scheme is the primary decision and impact on the spectra

exponential in t1 and linear
in t2
Exponential in both
t1 and t2
randomly sampled from an
exponential distribution in
t1 and t2
Random in t1 and t2.
Graham A. Webb (ed.), Modern Magnetic Resonance, 1305–1311.
NMR Assignments
NMR Data Processing Software
• Non-uniform data sampling
VERY IMPORTANT POINT, tn is no longer defined by DW and number of points
 tn is now user defined since DW is no longer relevant.
 Avoid FID truncation, maximize resolution

voltage
time
Traditional NMR
FID is truncated because number
of points and DW determine how
much of the FID can be collected
NUS NMR
FID is under-sampled, but the
entire FID is sampled.
NMR Assignments
NMR Data Processing Software
• Non-uniform data sampling
Both noise (N) and signal to noise (SNR) are proportional to the total evolution time
 Optimal setting is 1.3T2 of the evolving coherence
 Maximize sensitivity

Magn. Reson. Chem. 2011, 49, 483–491
NMR Assignments
NMR Data Processing Software
• Non-uniform data sampling
What is the optimal sampling density?
 Increase enhancement by increase exponential bias, eventually regenerate truncated FID
 Highly resolved spectra is pT2

TSMP – time constant for the exponential
weighting of the sampling.
 - enhancement
lw – line width
Magn. Reson. Chem. 2011, 49, 483–491
NMR Assignments
NMR Data Processing Software
• Non-uniform data sampling
A 1.5 to 2.0 bias to early data points and a 4x reduction yields a 2x enhancement
 Or a 3T2 with a 3x reduction yields a 1.7 enhancement

Truncated FID
Sampling Density/LW = TSMP/T2
Magn. Reson. Chem. 2011, 49, 483–491
NMR Assignments
NMR Data Processing Software
• Non-uniform data sampling
Different sampling schemes have different performances at different sampling densities
 Sinusoidal Poisson Gap is currently the best – random sampling, while minimizing gap size
particularly at the beginning and end of the FID
 Some drastic sampling densities at 1% or less.

Top Curr Chem. 2012 ; 316: 125–148
NMR Assignments
NMR Data Processing Software
• Non-uniform data sampling
Dramatic gain in resolution for 48 kDa
protein with only 3% sampling of the
Nyquist matrix
Same experimental time for US and NUS

J Biomol NMR. 2009 November; 45(3): 283–294.
NMR Data Processing Software
• Non-uniform data sampling
How is the time-domain data processed?
 Use the partial data to reconstruct the full Nyquist grid then process as normal (nmrPipe)

maximum entropy reconstruction is a common approach

forward maximum entropy (FM), fast maximum likelihood reconstruction (FMLR)

multi-dimensional decomposition (MDD); and compressed sensing (CS)

MddNMR: http://www.enmr.eu/webportal/mdd.html

Newton: http://newton.nmrfam.wisc.edu/newton/static_web/index.html

RNMRTK: http://rnmrtk.uchc.edu/rnmrtk/RNMRTK.html

mpiPipe: Available by contacting the Wagner Group

NMR Assignments
NMR Data Processing Software
• Solvent Removal (SOL)
protein NMR spectra are typical collected in water
 the large solvent signal can interfere with the interpretation of the NMR data
 Carrier frequency is usually centered on the water signal
 the signal associated with the water resonance can be filtered or subtracted from the
time domain of the FID

SOL
NMR Assignments
NMR Data Processing Software
• Solvent Removal (SOL)
with Solvent Subtraction
without Solvent Subtraction
NMR Assignments
NMR Data Processing Software
• Phase Correction (PS)
Because of the challenges of phasing nD NMR data and the baseline artifacts that first-order
phase corrections are known to cause, typically phase corrections are set to 0,0 or 90-180 by
proper delays in the pulse sequence
 A number of methods of data collection are used to obtain phase correction in the indirect
dimensions
 Fourier transformed data contains a real part that is an absorption lorentzian and an
imaginary part which is a dispersion lorentzian


we want to maintain the real absorption mode line-shape

done by applying a phase factor (exp(iQ)) to set F to zero

this is what we are doing when we phase the spectra
NMR Assignments
NMR Data Processing Software
• Phase Correction (PS)

Phase of the peak is determined by the relative phase of the pulse and the receiver
to obtain correct phasing in the indirect dimension, we need to collect both sine and cosine
modulated data
 alternate both the phase of the pulse relative to the receiver and the storage of this data
between real (sine) and imaginary (cosine)

NMR Assignments
NMR Data Processing Software
• Phase Correction (PS)
Phase of the peak is determined by the relative phase of the pulse and the receiver
 Also determines the order in which the data is stored.
 Some Common Phase Cycle Schemes:
o
0
 STATES – phase cycles the 90 -pulses prior to t1 incrimination by 90
o
o
 TPPI – phase cycles both the receiver and the 90 -pulses prior to t1 by 90 for each t1
increment
o
o
 States-TPPI – phase cycles both the receiver and the 90 -pulses prior to t1 by 180 for
each t1 increment
 Echo-antiecho – uses gradients to reduce the number of phase cycling steps and
combines N (echo) and P(antiecho) coherence selection

NMR Assignments
NMR Data Processing Software
• Phase Correction (PS)
Experiment
Increment
Pulse Phase
Receiver Phase
TPPI
(4k + 1)
t1(0) + (4k)D
x
x
(4k + 2)
t1(0) + (4k + 1)D
y
x
(4k + 3)
t1(0) + (4k + 2)D
-x
x
(4k + 4)
t1(0) + (4k + 3)D
-y
x
STATES
(4k + 1)
t1(0) + (4k)2D
x
x
(4k + 2)
t1(0) + (4k)2D
y
x
(4k + 3)
t1(0) + (4k + 1)2D
x
x
(4k + 4)
t1(0) + (4k + 1)2D
y
x
States-TPPI
(4k + 1)
t1(0) + (4k)2D
x
x
(4k + 2)
t1(0) + (4k)2D
y
x
(4k + 3)
t1(0) + (4k + 1)2D
-x
-x
(4k + 4)
t1(0) + (4k + 1)2D
-y
-x
NMR Assignments
NMR Data Processing Software
• Phase Correction (PS)
The phase introduced by a gradient of duration τG to coherence of order p which involves k
spins with gyromagnetic ratios gk is given by:
φ(r) = r Gz τG Sk( pkγk)
Complex Fourier transformation and combination of the two signals yields a purely
absorptive spectrum with frequency sign discrimination.
NMR Assignments
NMR Data Processing Software
• Data Conversion (bruk2pipe)
Prior to processing the NMR data by NMRPipe is a requirement to convert the file format
 This process requires defining some important experimental parameters
 number of points, sweep width, phase cycling, etc

bruk2pipe -in 1/ser -bad 0.0 -noaswap -DMX -decim 16 -dspfvs 12
-xN
2048 -yN
40
-zN
-xT
1024 -yT
20
-zT
-xMODE
Complex -yMODE Echo-AntiEcho
-zMODE
-xSW
8928.571 -ySW
2189.142
-zSW
-xOBS
600.182 -yOBS
60.823
-zOBS
-xCAR
4.773 -yCAR
117.086
-zCAR
-xLAB
1H -yLAB
15N
-zLAB
-ndim
3 -aq2D
States
-out 1/FID/HNCO%03d.fid -verb -ov
\
128 \
64 \
STATES-TPPI\
3333.333 \
150.942
\
179.715 \
CO \
\
Phase cycling determines how the data is stored and retrieved
States - odd data points are written to the real data array, even data points to the imaginary data
array.
source 1 2 3 4 = real 1 3 + imaginary 2 4
TPPI - data are copied to the real data array.
source 1 2 3 4 = real 1 2 3 4
Echo-antiecho - 4 data points are mixed and written to the real and imaginary data arrays.
source 1 2 3 4 = real 1+3 4-2 + imaginary 2+4 1-3
States-TPPI - Same as States, but every second real and imaginary data point is multiplied by -1.
source 1 2 3 4 = real 1 -3 + imaginary 2 -4
NMR Assignments
NMR Data Processing Software
• NMR data analysis/visualization
NMRDraw, NMRViewJ, PIPP, etc
 Again, most programs have similar functionality, choice is based on personal preference
 display the data (zoom, traces, step through multiple spectra, etc)
 Peak-picking – identify the X,Y or X,Y,Z or X,Y,Z,A chemical shift coordinate
positions for each peak in the nD NMR spectra

Peak Picking List
Peak#
1
2
3
4
5
6
7
8
9
10
11
12
.
.
.
15
N (ppm)
127.747
127.803
114.644
121.299
119.425
126.940
121.296
122.376
133.054
127.974
122.890
117.582
1
H (ppm)
9.537
9.405
9.312
9.287
9.225
9.181
9.107
9.090
8.983
8.934
8.944
8.928
NMR Assignments
NMR Data Processing Software
• NMR data analysis/visualization
Peak Picking
 Critical for obtaining accurate NMR assignments
 Especially for software for automated assignments
 Only provide primary sequence and peak-pick tables
 Two General Approaches to Peak Picking
 Manual
– time consuming
– can evaluate crowded regions more
effectively
 Automated
– pick peaks above noise threshold
OR
– pick peaks above threshold with
characteristic peak shape
– only about 70-80% efficient
J. OF MAG. RES. 135, 288–297 (1998)
– crowded overlap regions and noise
regions (solvent, T2 ridges) cause problems
– noise peaks and missing real peaks cause
problems in automated assignment software

NMR Assignments
NMR Data Processing Software
• NMR data analysis/visualization

What is the Statistical likelihood that a signal is a peak?
100 simulated spectra containing
a single peak with random noise.
A successful identification
occurred if the known peak has
the highest intensity that is at
least 1.414 times greater than the
next intense peak.
A signal intensity of 1
corresponds to a SNR of 80.
J Biomol NMR (2013) 55:167–178.
NMR Assignments
NMR Data Processing Software
• Automated NMR assignments
AutoAssign, CONTRAST, GARANT, PASTA, etc
 uses peak lists, primary protein sequence, details of NMR experiments
 tries to mimic “skilled user”, uses databases of previous assignments, etc
 Automated analysis of NOESY data is a sub-set of the NMR assignment issue with
programs designed to specifically address this need
 AutoStructure, CANDID, ARIA, ROSSETTA, etc

From, peak-lists and protein
sequence, software attempts to
make the assignment.
Not 100% success rate, still
need user intervention to
complete/correct assignments.
Most problems arise from
quality of peak-list: noise,
missing peaks, etc.
Need to Know How
Assignments are Made!
NMR Assignments
NMR Assignment Protocol
• 2D NMR Experiments
Kurt Wüthrich Nobel prize in 2002 for developing NMR to determine 3D structures of
proteins.
 Wüthrich “NMR of Proteins and Nucleic Acids” 1986, John Wiley & Sons
 Applicable for proteins of <100 amino acids
 Primarily dependent on three 2D experiments: NOESY, COSY, TOCSY
• Sequence-Specific Resonance Assignments in Proteins (Backbone Assignemnts)

H3C
Takes advantage of short
sequential distances between
CaiH, CbiH and NHi+1
CH3
CbiH O
Ni
Cia
dbN
daN
Ci
Ni+1
dbN
H
daN
H
dNN
dNN
H
dNN
NMR Assignments
2D NMR Experiments
• 2D COSY
Correlation Spectroscopy
1
3
 Correlates H resonances that are scalar coupled ( J)
i
i
 Identifies which NH resonances are bonded to CaH resonances
 separated by three-bonds
 chemical shift evolution based on J occurs during t 1
 requires the sample be in H2O (90/10 H2O/D2O) to observe NH
 all three-bond couplings observed, not just NH-Ca
 spectra is symmetric
 strength of cross peak depends on strength of coupling constants
 all predicted peaks are not necessarily observed
–weak couplings
– obscured by solvent, noise
– overlap or degenerate peaks

NMR Assignments
2D NMR Experiments
• 2D COSY

Typical Small Protein COSY
NMR Assignments
2D NMR Experiments
• 2D NOESY
Nuclear Overhauser Spectroscopy
1
 Correlates H resonances that close in space (≤5Å)
 also contains COSY peaks
 NOE intensity builds up during mixing time (t m), ususally 100-150 ms
i+1 resonances with CaHi resonances
 Correlates NH

NMR Assignments
2D NMR Experiments
• 2D NOESY

Typical Protein NOESY (Lysozyme)
Both NHi-Cai and
NHi+1-Cai are present
NMR Assignments
2D NMR Experiments
• Making the Sequential Assignments

Connecting COSY (NHi-Cai) peaks with NOESY (NHi+1-Cai)
i
i
 COSY experiment allows you to identify the NH -Ca cross peaks in the NOESY
experiment
 N-terminal amino acid only has one cross peak associated with its NH chemical shift
The Backbone Walk
NOESY cross peak
COSY cross peak
NHi+1-Cai
NHi-Cai
A24
NHi-Cai NHi+1-Cai
T27
Y28
NHi-Cai
NHi+1-Cai
F25
D26
NHi-Cai
NHi-Cai
D26 A24
NHi+1-Cai
F25 T27
NH Chemical Shifts (ppm)
Y28
Biochemistry 1989, 28, 1048-1054
NMR Assignments
2D NMR Experiments
• Verifying the Sequential Assignments and Side-Chain Assignments

The accuracy of the backbone assignments from connecting COSY (NHi-Cai) peaks with
NOESY (NHi+1-Cai) can be verified by proper assignment of the side-chain with the
backbone assignments.
 know the primary sequence of the protein
 therefore, know what amino acid is residue (i) and what amino-acid should be (i+1)
 amino acid type indicates the number and type or chemical shifts that should be
observed for the residue
As example:
Gly – no side chain
Ala – single methyl (1.39 ppm)
Val – two g methlys (0.97 & 0.94 ppm)
one Hb (2.13 ppm)
NMR Assignments
2D NMR Experiments
• Connectivity Patterns
• COSY TOCSY patterns
for the 20 amino acids
• Side-chain assignments
involves “matching”
the expected patterns
and typical chemical
shift ranges
• Some connectivity
patterns are not unique
and can only eliminate
some possible
assignments
In real data, overlapping or missing cross-peaks are common.
Connectivity pattern may not exactly match predicted.
NMR Assignments
2D NMR Experiments
• Connectivity Patterns
Leu - expected
Ca
Cb Cg Cd
Leu - actual
Ca
Cb Cb/Cg
Cd
Structure induces chemical shift changes which perturbs the pattern and induces overlap.
But, the data has to be consistent with the amino-acid spin system or the assignment is
probably incorrect
NMR Assignments
2D NMR Experiments
• Connectivity Patterns
NMR assignments should be consistent
with expected trends
 significant differences should be
explained by the structure
 (ring current, h-bonds, etc)

NMR Assignments
2D NMR Experiments
• 2D TOCSY

TOtal Correlation SpectroscopY
 cross peaks are generated between all members of a coupled spin network
– NMR resonances for the complete side-chain spin systems is obtained
 coherence transfer period occurs during a multi-pulse spin-lock period
 length of spin-lock determines how “far” the spin coupling network will be probed
 1/(10 JHH) should be used for each transfer step
 not all correlations are observed
COSY
TOCSY
Spin-Lock Pulse (~14ms)
NMR Assignments
2D NMR Experiments
• 2D TOCSY
• What happens during the spin-lock time cannot be described in terms of vector models or product
operators, because it relies on strong coupling
• Under strong coupling, chemical shift differences between different spins become negligible
 Two states ab and ba become identical in energy
 Instead of transition of single spins, the coherences now involves transitions of combinations
of spins
 Under this condition, a coherence of one spin is actually in resonance with a coherence of its
coupling partner(s) (all with the same frequency), and will oscillate back and forth between
all coupled spins
NMR Assignments
2D NMR Experiments
• 2D TOCSY
Typical Small Protein TOCSY
 Side-chain spin systems are
correlated with NH resonance

Boxed regions indicate side-chain
spin systems for His and Ile,
respectively
Bull. Korean Chem. Soc. 2001, Vol. 22, No. 5 507
NMR Assignments
3D NMR Experiments
• Takes advantage of 13C and 15N labeling
• Extends assignments to proteins in the 20-25 kDa range
• Extends Connectivity by Scalar Coupling (J) into 3D dimensions
1
13
1
15
 Primarily uses one-bond heteronuclear coupling ( H- C, H- N)
1
3
 J generally stronger than J
2D 1H-15N HSQC is the root experiment of most of the standard triple-resonance (1H,
13C, 15N) NMR experiments
• 3D NMR simplifies data and removes overlap by spreading information into third dimension
• Requires multiple experiments (≥ 6) to “walk through” the backbone assignments similar to the
2D COSY & NOESY experiments
• Requires a similar number of additional experiments to obtain the side-chain assignments

NMR Assignments
3D NMR Experiments
• 2D 1H-15N HSQC experiment
• correlates backbone amide 15N through one-bond coupling to amide 1H
• in principal, each amino acid in the protein sequence will exhibit one peak in the 1H-15N
HSQC spectra
 also contains side-chain NH2s (ASN,GLN) and NeH (Trp)
 position in HSQC depends on local structure and sequence
 no peaks for proline (no NH)
Side-chain NH2
NMR Assignments
3D NMR Experiments
• Consider a 3D experiment as a collection of 2D experiments
z-dimension is the 15N chemical shift
• 1H-15N HSQC spectra is modulated to include correlation through
coupling to a another backbone atom

Cbi-1 O
Cbi
O
Ci
Ni-1
Cai-1 Ci-1
Ni
Cai
H
H
H
H
• All the 3D triple resonance experiments are then related by the common
1H,15N chemical shifts of the HSQC spectra
• The backbone assignments are then obtained by piecing together all the
“jigsaw” puzzles pieces from the various NMR experiments to reassemble
the backbone
NMR Assignments
3D NMR Experiments
• Amide Strip
3D cube
2D plane
amide strip
Strips can then be arranged in backbone sequential order to visual confirm assignments
NMR Assignments
3D NMR Experiments
• 3D HNCO Experiment
common nomenclature  letters indicate the coupled backbone atoms
i
i-1 (carbonyl carbon, CO or C’)
 correlates NH to C
 no peaks for proline (no NH)
• Like the 2D 1H-15N HSQC spectra, each amino acid should display a single peak in
the 3D HNCO experiment
1
15
 identifies potential overlap in 2D H- N HSQC spectra, especially for larger
MW proteins
 most sensitive 3D triple resonsnce experiment
 may observe side-chain correlations

Cbi-1 O
1J
i-1
N
i-1
Ca
i-1
C
NC’
Ni
1J
H
H
H
Cbi
O
Cai
Ci
NH
H
NMR Assignments
3D NMR Experiments
• 3D HNCO Experiment
NMR Assignments
3D NMR Experiments
• 3D HNCO Experiment
One expanded plane or slice from a 3D
HNCO experiment, where the 15N
chemical shift is 118.21 ppm
A total of 128 planes, with a digital
resolution of 0.28 ppm per plane for the
entire experiment.
slice through
3D cube
NMR Assignments
3D NMR Experiments
• 3D HN(CA)CO Experiment
correlates NHi to COi
i
 relays the transfer through Ca without chemical shift evolution
 uses stronger one-bond coupling
 contains only intra correlation
 provides a means to sequential connect NH and CO chemical shifts
i
i
i
i-1 (HNCO)
 match NH -CO (HN(CA)CO with NH -CO
 not sufficient to complete backbone assignments because of overlap and
missing information
 every possible correlation is not observed
 need 2-3 connecting inter and intra correlations for unambiguous
assignments
 no peaks for proline (no NH) breaks assignment chain
 but can identify residues i-1to prolines

Cbi-1 O
Cbi
1J
1J
NCa
Ni-1
Cai-1 Ci-1 Ni
1J
NH
Cai
H
H
H
H
O
CaC’
Ci
NMR Assignments
3D NMR Experiments
• 3D HN(CA)CO Experiment
NMR Assignments
3D NMR Experiments
• 3D HN(CA)CO Experiment
Connects HNi-COi
with HNi-COi-1
HNCO and HN(CA)CO
pair for one residues NH
Amide “Strips” from the 3D HNCO and HN(CA)CO
experiments arranged in sequential order
Journal of Biomolecular NMR, 9 (1997) 11–24
NMR Assignments
3D NMR Experiments
• 3D HNCA Experiment
correlates NHi to Cai-1 and Cai
i
i
i
i-1 1
2
 typically the intensity of NH -Ca > NH -Ca , JNCa > JNCa
i
i-1 correlation not always seen
 NH -Ca
i
i
 could be weak or degenerate with NH -Ca
 contains both inter and intra correlations
 provides a means to sequential connect NH and Ca chemical shifts
 not sufficient to complete backbone assignments because of overlap
 need 2-3 connecting inter and intra correlations
 no peaks for proline (no NH) breaks assignment chain
 but can identify residues i-1to prolines

Cbi-1 O
2J
NCa
Ni-1
Cai-1 Ci-1
Cbi
1J
NCa
Ni
Cai
1J
H
H
H
O
NH
H
Ci
NMR Assignments
3D NMR Experiments
• 3D HNCA Experiment
NMR Assignments
Amide “Strips” from the 3D
HNCA experiment arranged in
sequential order
3D NMR Experiments
• 3D HNCA Experiment
Correlation of the Cai and Cai-1
sequentially aligns the two NHs
in the protein’s sequence.
Cai-1
Cai
Each strip corresponds to one NH
resonance in a given 15N plane
J. of Biomol. NMR, 14: 85–88, 1999.
NMR Assignments
3D NMR Experiments
• 3D HN(CO)CA Experiment
correlates NHi to Cai-1
1
 relays through JNC’ without chemical shift evolution
i
i-1 correlation is more sensitive than HNCA experiment
 NH -Ca
i
i-1 assignments
 unambiguous NH -Ca
 avoids possible overlap in HNCA experiment
 companion experiment to HNCA
 provides a means to sequential connect NH and Ca chemical shifts
i
i
i
i-1 (HN(CO)CA)
 NH -Ca (HNCA) matches with NH -Ca
not sufficient to complete backbone assignments because of overlap
 need 2-3 connecting inter and intra correlations
 no peaks for proline (no NH) breaks assignment chain
 but can identify residues i-1to prolines

Ni-1
Cbi-1 O
1J
1J
C’Ca
NC’
Cbi
O
Cai-1 Ci-1
Cai
Ci
Ni
1J
H
H
H
NH
H
NMR Assignments
3D NMR Experiments
• 3D HN(CO)CA Experiment
NMR Assignments
3D NMR Experiments
• 3D HN(CO)CA Experiment
one residues NH
HN(CO)CA
NHi-Cai-1
HNCA
NHi-Cai
Journal of Biomolecular NMR, 9 (1997) 167–180
NMR Assignments
3D NMR Experiments
• 3D CBCANH Experiment
correlates NHi to Cai, Cai-1 and Cbi, Cbi-1
 transfer is simultaneously started on Ha & Hb (both i and i-1)
i
i
i
i
i
i-1 & NHi-Cbi-1
 typically the intensity of NH -Ca & NH -Cb > NH -Ca
1
2
 JNCa > JNCa
 can usually distinguish Ca from Cb from chemical shift difference
i
i
i
i-1 are opposite sign of NH-Cbi & NH-Cai-1
 NH -Ca & NH -Ca
– one set of peaks are positive intensity and the other set is negative
i
i-1 & NHi-Cai correlations are seen
 only Gly NH -Ca
 contains both intra and inter correlations
 provides a means to sequential connect NH, Ca and Cb chemical shifts
 the 2 connections of inter and intra correlations may be sufficient to unambiguously
assign the backbone
 weakest experiment, so all the necessary data is usually not present and the single
experiment is typically inadequate to assign the complete backbone

Match-up the intra
and inter correlations
Cbi-1 O
2J
Ni-1
NCa
Cai-1 Ci-1
Cbi
1J
Cai
1J
H
H
H
1J
NCa
Ni
O
NH
H
NCb
Ci
NMR Assignments
3D NMR Experiments
• 3D CBCANH Experiment
NMR Assignments
3D NMR Experiments
• 3D CBCANH Experiment
Amide “Strips” from the 3D
CBCANH experiment arranged
in sequential order
Correlation of the Cbi and Cbi-1
sequentially aligns the two NHs
in the protein’s sequence.
Correlation of the Cai and Cai-1
sequentially aligns the two NHs
in the protein’s sequence.
Note: contours of opposite intensity
are shown in different colors
IUBMB Life, 52: 291–302, 2001
NMR Assignments
3D NMR Experiments
• 3D CBCA(CO)NH Experiment
correlates NHi to Cai-1 and Cbi-1
 can usually distinguish Ca from Cb from chemical shift difference
i
i-1 and NHi-Cbi-1 may be oppositely phased
 sometimes NH -Ca
– one peak positive intensity the other negative
i
i-1 correlations are seen
 only Gly NH -Ca
 no peaks for proline (no NH) breaks assignment chain
i-1
i-1
 transfer is started on simultaneously on Ha , Hb , relayed through CO without
chemical shift evolution (1JCaC’, 1JC’N)
 contains only inter correlations
 provides a means to sequential connect NH, Ca and Cb chemical shifts with a
companion experiment (s)
i
i
i
i
 companion experiments would provide NH -Ca (HNCA) and NH -Cb
(CBCANH)
 the 2 connections of inter and intra correlations may be sufficient to
unambiguously assign the backbone

Cbi-1 O
1J
Ni-1
H
Cbi
O
Cai
Ci
C’Cb
Cai-1 Ci-1
1J
CaC’
Ni
H
H
1J
NH
H
NMR Assignments
3D NMR Experiments
• 3D CBCA(CO)NH Experiment
NMR Assignments
3D NMR Experiments
• 3D CBCA(CO)NH Experiment
Correlation of the Cai & Cai-1
and Cbi & Cbi-1 sequentially
aligns each pair of NHs in the
protein’s sequence.
Amide “Strips” from the 3D
CBCANH (right) and CBCA(CO)NH
(left) experiment arranged in
sequential order
Journal of Biomolecular NMR, 10 (1997) 77–88
NMR Assignments
NMR Assignments
3D NMR Experiments
• Typically collect 1024 x 64 x 40 complex points in
each dimension
• Typical digital resolution is 0.02ppm (1H) x 0.15
ppm (13C) x 0.28 ppm (15N)
 resolution is better in some experiments that require
smaller sweep-width.
 need to allow for significant error when comparing
chemical shift values from different NMR experiments
 conservative use twice digital resolution
• Typical experiment time is 2.5 days
NMR Assignments
NMR Assignments
3D NMR Experiments
• Large Variety of Experiments Based on These 3D Triple Resonance Experiments
Proton Versions of the Experiments
 CBCA(CO)NH  HBHA(CO)NH
 HNCA  HNHA
 CBCANH  HBHANH
 provides even more possible i & i-1 types of correlations
– more confirmed observed correlations more definitive the assignment
 Modifications are constantly being made and new versions or variations are
constantly described in the literature to improve sensitivity and eliminate artifacts
 constant time, gradient enhancements, out-and-back, cryoprobe versions, etc
 Specific modifications to handle larger molecular-weight proteins
 deuterium decoupling  deuterated proteins
 TROSY versions

NMR Assignments
3D NMR Experiments
• Backbone Assignments
Need to correlate all the information
from all the available experiments
i
i-1
 Ca  Ca
i
i-1
 Cb  Cb
i
i-1
 CO  CO
i
i-1
 Ha  Ha

Journal of Biomolecular NMR, 9 (1997) 167–180
NMR Assignments
3D NMR Experiments
• Backbone Assignments

The process is a multi-step approach:
(1) correlate all the experimental data with each NH root observed in the 2D 1H-15N HSQC spectra
Pk-ID
2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00
10.00
11.00
12.00
13.00
14.00
15.00
16.00
17.00
18.00
19.00
20.00
.
.
.
NH
8.58
8.68
8.98
8.93
9.15
9.38
9.38
8.63
8.79
8.19
8.21
8.11
9.01
8.22
8.22
9.04
7.82
8.57
9.05
N15
129.50
128.63
128.56
127.98
127.45
126.47
126.46
125.79
125.73
125.61
125.51
125.59
125.50
125.40
125.40
125.12
124.78
124.32
123.83
Ca
60.65
53.65
53.07
61.03
60.20
53.76
54.26
60.91
60.61
58.67
57.15
60.76
59.76
57.22
55.83
54.75
54.62
57.99
64.05
Cb
38.63
18.58
45.72
40.67
32.32
44.74
44.74
29.76
34.73
42.86
****
32.89
41.21
****
****
39.51
32.46
35.22
31.96
Cai-1
64.84
53.27
60.66
60.58
61.13
61.70
61.70
57.23
54.47
61.38
61.31
61.17
57.95
55.69
55.69
58.80
62.56
59.26
53.90
Cbi-1
69.56
43.21
32.82
34.68
40.71
69.26
69.26
30.09
35.21
62.40
62.40
36.07
35.22
29.56
29.56
33.07
33.07
36.57
42.80
NMR Assignments
3D NMR Experiments
• Backbone Assignments

The process is a multi-step approach:
(2) Match pairs of NH roots based on i and i-1 correlations
Pk-ID
NH
N15
Ca
Cb
Cai-1
Cbi-1
2.00
202.00
8.58
8.55
129.49
116.39
60.61
62.15
38.63
69.49
64.82
60.62
69.56
38.62
3.00
230.00
8.68
8.78
128.63
105.35
53.65
45.64
18.58
****
53.27
53.72
43.21
18.60
4.00
193.00
8.98
8.22
128.57
117.39
52.96
54.54
45.72
36.27
60.64
52.95
32.82
45.73
5.00
6.00
8.93
9.16
127.98
127.45
60.90
60.14
40.67
32.32
60.57
61.10
34.68
40.71
6.00
108.00
9.16
8.78
127.45
119.65
60.14
58.97
32.32
34.36
61.10
60.16
40.71
32.27
7.00
197.00
9.38
8.95
126.46
117.12
54.17
55.46
44.74
37.23
61.65
54.14
69.26
44.78
8.00
206.00
8.64
8.85
125.80
116.15
60.88
58.95
29.76
****
57.16
60.86
30.09
29.65
9.00
5.00
8.79
8.93
125.73
127.98
60.59
60.90
34.73
40.67
54.37
60.57
35.21
34.68
10.00
203.00
.
.
.
8.19
8.55
125.62
116.32
58.60
62.15
42.86
69.49
61.31
58.61
62.40
42.85
NMR Assignments
3D NMR Experiments
• Backbone Assignments

The process is a multi-step approach:
(3) Extend pairs of NH roots and match to protein primary sequence
Identify
overlapping
spin-system
pairs
connect spinsystem pairs
.
.
.
5.00
6.00
6.00
108.00
.
.
.
5.00
6.00
108.00
8.93
9.16
127.98
127.45
60.90
60.14
40.67
32.32
60.57
61.10
34.68
40.71
9.16
8.78
127.45
119.65
60.14
58.97
32.32
34.36
61.10
60.16
40.71
32.27
8.93
9.16
8.78
127.98
127.45
119.65
60.90
60.14
58.97
40.67
32.32
34.36
60.57
61.10
60.16
34.68
40.71
32.27
NMR Assignments
3D NMR Experiments
• Backbone Assignments

The process is a multi-step approach:
(3) Extend pairs of NH roots and match to protein primary sequence
Identify possible residue types by
chemical shift ranges
NMR Assignments
3D NMR Experiments
• Backbone Assignments

The process is a multi-step approach:
(3) Extend pairs of NH roots and match to protein primary sequence
Y,F,I,C
V, W, C
V, W, C
5.00
6.00
108.00
8.93
9.16
8.78
127.98
127.45
119.65
60.90
60.14
58.97
40.67
32.32
34.36
60.57
61.10
60.16
34.68
40.71
32.27
Find potential match in sequence
MTLKQVIVVRDDLKLSRGKLAVQVAHAAIIGYLKSDSSLRRKWLDEGQKKVVLKVKS
LEELLGIKHKAESLGLVTGLVQDAGLTEVPPGTITAVVIGPDEERKIDKVTGNLPLLKLE
HHHHHH
Make assignment
I 7
V 8
V 9
5.00
6.00
108.00
8.93
9.16
8.78
127.98
127.45
119.65
60.90
60.14
58.97
40.67
32.32
34.36
60.57
61.10
60.16
34.68
40.71
32.27
NMR Assignments
3D NMR Experiments
• Side-chain Assignments
Help confirm the backbone assignment
 Similar in principal to 2D assignment approach
 Correlate entire spin-system with NH backbone
 Use TOCSY to observe entire spin-system
 CC(CO)NH & HCC(CO)NH
– Relay magnetization from NH through side-chain carbon or hydrogen chemical
shifts
– Start simultaneously on all side-chain hydrogens
– Also, overlap with Ca and Cb chemical shifts from other triple-resonance
experiments to confirm side-chain assignments

NMR Assignments
Which H’s match
the C’s?
3D NMR Experiments
• Side-chain Assignments
CC(CO)NH & HCC(CO)NH
 Can assign residue type by the number of
observed resonances and the chemical shift
ranges
 may be able to assign Cg, Cd, Ce from
chemical shift values and from
previously assigned Ca and Cb
 less likely to assign Hg, Hd and He,
unless unique chemical shift
 need companion experiments to connect
carbon and hydrogen chemical shifts.

HCC(CO)NH
CC(CO)NH
d1
g2
g1
b
a
Biochemistry, Vol. 34, No. 42, 1995
NMR Assignments
3D NMR Experiments
• Side-chain Assignments

HCCH-TOCSY & HCCH-COSY
1
13C via coupling
 relays magnetization from side-chain and backbone H &
constants
 Experiments have symmetry
– 1Ha-13Ca diagonal shows cross peak to 1Hb
AND
– 1Hb-13Cb diagonal shows cross peak to 1Ha
 does not correlate to backbone NH no direct connection with other tripleresonance experiments
– sample can be collected in D2O
NMR Assignments
3D NMR Experiments
• Side-chain Assignments
HCCH-TOCSY
 HCCH-COSY

Slices taken from different 13C
chemical shift planes at different 1H
chemical shifts illustrates the entire
spin system for a single side-chain
Symmetry – each HCd shows a
cross peak to Ha and the HCa
shows a crosspeak to both HCd
Note: Symmetry peaks may not always be
present (separate pathways, separate relative
sensitivity). Presence of a symmetry peak
increase the likelihood of correct assignment
Journal of Biomolecular NMR, 9 (1997) 445–446
NMR Assignments
4D NMR Experiments
• Consider a 4D NMR experiment as a
collection of 3D NMR experiments
 still some ambiguities present when
correlating multiple 3D triple-resonance
experiments
 4D NMR experiments make definitive
sequential correlations
 increase in spectral resolution
– Overlap is unlikely
 loss of digital resolution
– need to collect less data points for
the 3D experiment
– If 3D experiment took 2.5 days,
then each 4D time point would be a
multiple of 2.5 days i.e. 32 complex
points in A-dimension would require
an 80 day experiment
 loss of sensitivity
– an additional transfer step is
required
– relaxation takes place during each
transfer
Get less data that is less ambiguous?
NMR Assignments
4D NMR Experiments
• Backbone Assignments
Correlates 1HCai
with NHi & NHi+1
Correlates NHi with
1HCai & 1HCai+1
4D HNCA
NMR Assignments
4D NMR Experiments
• Backbone Assignments
Quality improves with
deuterium labeling
TROSY
specific labeling

J. AM. CHEM. SOC. 9 VOL. 124, NO. 34, 2002