NMR Assignments

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

 Use
13C & 15N isotope enrichment to simplify the NMR spectra need to assign these NMR
resonances
1
 a typical protein will have hundreds of H,
13C and 15N NMR resonances to assign
Model 1
ATOM 1 N ASP A 1 20.897 9.212 4.618 1.00 0.00
ATOM 2 CA ASP A 1 20.874 8.930 3.154 1.00 0.00
ATOM 3 C ASP A 1 20.456 7.476 2.921 1.00 0.00
ATOM 4 O ASP A 1 21.196 6.691 2.361 1.00 0.00
ATOM 5 CB ASP A 1 22.271 9.155 2.570 1.00 0.00
ATOM 6 CG ASP A 1 22.154 9.514 1.088 1.00 0.00
ATOM 7 OD1 ASP A 1 22.132 8.602 0.278 1.00 0.00
ATOM 8 OD2 ASP A 1 22.088 10.695 0.789 1.00 0.00
ATOM 9 1H ASP A 1 21.578 8.579 5.083 1.00 0.00
ATOM 10 2H ASP A 1 19.948 9.056 5.016 1.00 0.00
ATOM 11 3H ASP A 1 21.182 10.199 4.777 1.00 0.00
ATOM 12 HA ASP A 1 20.170 9.590 2.670 1.00 0.00
ATOM 13 1HB ASP A 1 22.757 9.961 3.100 1.00 0.00
ATOM 14 2HB ASP A 1 22.854 8.252 2.674 1.00 0.00
ATOM 15 N SER A 2 19.279 7.108 3.349 1.00 0.00
ATOM 16 CA SER A 2 18.826 5.701 3.152 1.00 0.00
ATOM 17 C SER A 2 17.435 5.682 2.511 1.00 0.00
ATOM 18 O SER A 2 16.466 6.118 3.095 1.00 0.00
ATOM 19 CB SER A 2 18.770 4.994 4.507 1.00 0.00
ATOM 20 OG SER A 2 18.165 5.857 5.461 1.00 0.00
1H NMR Spectra Protein PDB File

NMR Assignments
... Ala 70 Ser 71 Leu 72 ...
O O O
... H2N CH C
H
N CH C
H
N CH C OH ...
CH3 CH2 CH2
OH CH CH3
Again, as illustrated here, the goal is to explicitly
assign each H, C, & N in the protein’s primary CH3
sequence with its corresponding NMR resonance
... Ala 70 Ser 71 Leu 72 ...
15 15N 13Ca
55.5 ppm O N 114.8 ppm O O
15N 13Ca 125.6 ppm 58.6 ppm
119.3 ppm
HN 7.76 ppm Ha 3.76 ppm HN 7.08 ppm HN 8.20 ppm Ha 4.09 ppm
... H2N CH C
H
N CH C
H
N CH C OH ...
13CO 171.9 ppm 13CO 178.1 ppm 13CO 170.9 ppm
13Cb17.5 ppm CH3 CH2 CH2 13Cb42.9 ppm
13Cb64.8 ppm 13Cg27.9 ppm
Hb 1.45 ppm 13Ca59.9 ppm Hb 1.52 ppm
Hb 3.73 ppm Hg 1.65 ppm
Ha 4.35 ppm
OH CH CH3
13Cd25.4 ppm; 25.7 ppm
CH3 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 N CO Ca Cb Ot her s
D1 120.1 (8.08) 179.1 53.8 (4.37) 39.9 (3.00,0.58)
E2 128.9 (9.93) 176.6 56.0 (4.55) 30.9 (2.11,1.78) Cg 36.2(2.75,2.41)
D3 116.1 (8.90) 176.5 56.2 (4.73) 38.9 (2.70,2.34)
E4 113.3 (7.45) 175.6 52.9 (4.71) 27.2 (1.23,0.63) Cg 34.8(2.57,1.79)
R5 123.8 (7.97) 173.1 54.3 (4.43) 28.9 (1.84,1.47) Cg 26.2(1.59,1.16);Cd 42.6(3.09,3.02)
W6 127.8 (9.27) 177.1 55.4 (5.50) 30.8 (3.11)
T7 109.9 (9.32) 174.8 59.6 (4.85) 71.3 (4.34) Cg 20.2(0.73)
N8 115.2 (8.42) 174.5 50.9 (5.06) 38.7 (3.13,2.70)
N9 116.7 (7.88) 173.3 52.1 (4.94) 38.0 (3.33,3.01)
F10 110.1 (7.57) 177.2 58.2 (4.46) 38.5 (2.63,2.67)
R11 121.2 (8.03) 175.0 55.8 (4.06) 29.7 (1.83,1.67) Cg 27.4(1.45,1.35);Cd 43.1(3.13)
E12 119.0 (8.56) 177.9 52.3 (3.79) 27.4 (1.16,-0.05) Cg 36.6(2.19,1.97)
Y13 122.1 (8.28) 173.2 61.9 (3.80) 41.2 (2.99,2.52)
N14 121.5 (8.45) 175.7 54.7 (4.89) 39.2 (2.26)
L15 127.7 (9.02) 176.9 58.0 (4.48) 41.2 (1.96,1.64) 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
• 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
Processing
steps for
X,Y,Z Y
dimensions of
3D spectra
Z
NMR Assignments
• 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

• 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

• Some common window functions with the corresponding NMRPipe command
NMR Assignments
• Want to maximize digital resolution, number of data points in each dimension
 time constraints are a practical limitation for nD NMR data
NMR Assignments
• 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 zero-fill
8K FID 16K FID
No zero-filling 8K zero-filling
NMR Assignments
• 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
• 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
• Effects of Combining Linear Prediction with Zero Filling
 significant improvement in resolution for nD NMR data collected with minimal data points
NMR Assignments
• 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
• 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 Exponential in both

in t2 t1 and t2
randomly sampled from an Random in t1 and t2.

exponential distribution in
t1 and t2
Graham A. Webb (ed.), Modern Magnetic Resonance, 1305–1311.

NMR Assignments

 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 NUS NMR
much of the FID can be collected FID is under-sampled, but the
entire FID is sampled.
NMR Assignments

 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
 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
 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
 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
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.

 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
• 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

• Solvent Removal (SOL)
with Solvent Subtraction without Solvent Subtraction

NMR Assignments
• 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
 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
 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:
 STATES – phase cycles the 90 -pulses prior to t1 incrimination by 90

o 0
 TPPI – phase cycles both the receiver and the 90 -pulses prior to t1 by 90 for each t1
o o
increment
 States-TPPI – phase cycles both the receiver and the 90 -pulses prior to t1 by 180 for
o o
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
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
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
• 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 128 \
-xT 1024 -yT 20 -zT 64 \
-xMODE Complex -yMODE Echo-AntiEcho -zMODE STATES-TPPI\
-xSW 8928.571 -ySW 2189.142 -zSW 3333.333 \
-xOBS 600.182 -yOBS 60.823 -zOBS 150.942 \
-xCAR 4.773 -yCAR 117.086 -zCAR 179.715 \
-xLAB 1H -yLAB 15N -zLAB CO \
-ndim 3 -aq2D States \
-out 1/FID/HNCO%03d.fid -verb -ov
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 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

15 1
Peak# N (ppm) H (ppm)
1 127.747 9.537
2 127.803 9.405
3 114.644 9.312
4 121.299 9.287
5 119.425 9.225
6 126.940 9.181
7 121.296 9.107
8 122.376 9.090
9 133.054 8.983
10 127.974 8.934
11 122.890 8.944
12 117.582 8.928
.
.
.
NMR Assignments
 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
 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
• 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 CH3
Takes advantage of short
sequential distances between CbiH O
CaiH, CbiH and NHi+1
Ni Ci a Ci Ni+1
dbN dbN
daN
daN H H H
dNN
dNN
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
 Correlates H resonances that close in space (≤5Å)
1
 also contains COSY peaks
 NOE intensity builds up during mixing time (t m), ususally 100-150 ms
 Correlates NH
i+1 resonances with CaHi resonances
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
NHi-Cai
D26
NHi-Cai NHi+1-Cai
D26 A24 F25 T27 Y28
NH Chemical Shifts (ppm) 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
Leu - expected
Cb Cg Cd
Ca
Leu - actual
Cb Cb/Cg Cd
Ca
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
 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
Ni-1 Cai-1 Ci-1 Ni Cai Ci
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
 correlates NH to C
i-1 (carbonyl carbon, CO or 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 Cbi O
1J
NC’
i-1 i-1 i-1
N Ca C Ni Cai Ci
1J
NH
H H H H
NMR Assignments
3D NMR Experiments
NMR Assignments
slice through
3D NMR Experiments 3D cube
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.
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
 match NH -CO (HN(CA)CO with NH -CO
i i-1 (HNCO)
 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 O
1J 1J
NCa CaC’
1J
NH
H H H H
NMR Assignments
3D NMR Experiments
NMR Assignments
3D NMR Experiments
Connects HNi-COi HNCO and HN(CA)CO

with HNi-COi-1 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
 typically the intensity of NH -Ca > NH -Ca , JNCa > JNCa
2
i
 NH -Ca
i-1 correlation not always seen
 could be weak or degenerate with NH -Ca

i i
 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
Cbi-1 O Cbi O
2J 1J
NCa NCa

1J
NH
H H H H
NMR Assignments
3D NMR Experiments
• 3D HNCA Experiment
NMR Assignments
3D NMR Experiments Amide “Strips” from the 3D
• 3D HNCA Experiment HNCA experiment arranged in
sequential order
Correlation of the Cai and Cai-1 Cai-1

sequentially aligns the two NHs
in the protein’s sequence. 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
 NH -Ca
i-1 correlation is more sensitive than HNCA experiment
 unambiguous NH -Ca
i i-1 assignments
 avoids possible overlap in HNCA experiment
 companion experiment to HNCA
 provides a means to sequential connect NH and Ca chemical shifts

i i
 NH -Ca (HNCA) matches with NH -Ca
i i-1 (HN(CO)CA)
not sufficient to complete backbone assignments because of overlap
 need 2-3 connecting inter and intra correlations
Cbi-1 O Cbi O
1J 1J
C’Ca NC’

1J
NH
H H H H
NMR Assignments
3D NMR Experiments
NMR Assignments
3D NMR Experiments one residues NH
HN(CO)CA HNCA
NHi-Cai-1 NHi-Cai

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
 typically the intensity of NH -Ca & NH -Cb > NH -Ca
i i i-1 & NHi-Cbi-1
1 2
 JNCa > JNCa
 can usually distinguish Ca from Cb from chemical shift difference

i i
 NH -Ca & NH -Ca
i i-1 are opposite sign of NH-Cbi & NH-Cai-1
– one set of peaks are positive intensity and the other set is negative
 only Gly NH -Ca

i i-1 & NHi-Cai correlations are seen
 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 Cbi O
1J 1J
2J NCa NCb
NCa
1J
NH
H H H H
NMR Assignments
3D NMR Experiments
• 3D CBCANH Experiment
NMR Assignments
Amide “Strips” from the 3D
3D NMR Experiments CBCANH experiment arranged
• 3D CBCANH Experiment in sequential order
Correlation of the Cbi and Cbi-1

in the protein’s sequence.
Correlation of the Cai and Cai-1

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
 sometimes NH -Ca
i i-1 and NHi-Cbi-1 may be oppositely phased
– one peak positive intensity the other negative
 only Gly NH -Ca

i i-1 correlations are seen
 no peaks for proline (no NH) breaks assignment chain
 transfer is started on simultaneously on Ha , Hb , relayed through CO without

i-1 i-1
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
 companion experiments would provide NH -Ca (HNCA) and NH -Cb
i i
(CBCANH)
 the 2 connections of inter and intra correlations may be sufficient to
unambiguously assign the backbone
Cbi-1 O Cbi O
1J
C’Cb
i-1 i-1 i-1
N Ca C Ni Cai Ci
1J 1J
CaC’ NH
H H H H
NMR Assignments
3D NMR Experiments
NMR Assignments
3D NMR Experiments
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
 Ca  Ca
i i-1
 Cb  Cb
i i-1
 CO  CO
i i-1
 Ha  Ha
i i-1

NMR Assignments
3D NMR Experiments
 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 NH N15 Ca Cb Cai-1 Cbi-1
2.00 8.58 129.50 60.65 38.63 64.84 69.56
3.00 8.68 128.63 53.65 18.58 53.27 43.21
4.00 8.98 128.56 53.07 45.72 60.66 32.82
5.00 8.93 127.98 61.03 40.67 60.58 34.68
6.00 9.15 127.45 60.20 32.32 61.13 40.71
7.00 9.38 126.47 53.76 44.74 61.70 69.26
8.00 9.38 126.46 54.26 44.74 61.70 69.26
9.00 8.63 125.79 60.91 29.76 57.23 30.09
10.00 8.79 125.73 60.61 34.73 54.47 35.21
11.00 8.19 125.61 58.67 42.86 61.38 62.40
12.00 8.21 125.51 57.15 **** 61.31 62.40
13.00 8.11 125.59 60.76 32.89 61.17 36.07
14.00 9.01 125.50 59.76 41.21 57.95 35.22
15.00 8.22 125.40 57.22 **** 55.69 29.56
16.00 8.22 125.40 55.83 **** 55.69 29.56
17.00 9.04 125.12 54.75 39.51 58.80 33.07
18.00 7.82 124.78 54.62 32.46 62.56 33.07
19.00 8.57 124.32 57.99 35.22 59.26 36.57
20.00 9.05 123.83 64.05 31.96 53.90 42.80
.
.
.
NMR Assignments
3D NMR Experiments
(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 8.58 129.49 60.61 38.63 64.82 69.56

202.00 8.55 116.39 62.15 69.49 60.62 38.62
3.00 8.68 128.63 53.65 18.58 53.27 43.21

230.00 8.78 105.35 45.64 **** 53.72 18.60
4.00 8.98 128.57 52.96 45.72 60.64 32.82

193.00 8.22 117.39 54.54 36.27 52.95 45.73
5.00 8.93 127.98 60.90 40.67 60.57 34.68

6.00 9.16 127.45 60.14 32.32 61.10 40.71
6.00 9.16 127.45 60.14 32.32 61.10 40.71

108.00 8.78 119.65 58.97 34.36 60.16 32.27
7.00 9.38 126.46 54.17 44.74 61.65 69.26

197.00 8.95 117.12 55.46 37.23 54.14 44.78
8.00 8.64 125.80 60.88 29.76 57.16 30.09

206.00 8.85 116.15 58.95 **** 60.86 29.65
9.00 8.79 125.73 60.59 34.73 54.37 35.21

5.00 8.93 127.98 60.90 40.67 60.57 34.68
10.00 8.19 125.62 58.60 42.86 61.31 62.40

203.00 8.55 116.32 62.15 69.49 58.61 42.85
.
.
.
NMR Assignments
3D NMR Experiments
(3) Extend pairs of NH roots and match to protein primary sequence

.
.
.
Identify 5.00 8.93 127.98 60.90 40.67 60.57 34.68
overlapping 6.00 9.16 127.45 60.14 32.32 61.10 40.71
spin-system 6.00 9.16 127.45 60.14 32.32 61.10 40.71

108.00 8.78 119.65 58.97 34.36 60.16 32.27
pairs .
.
.
connect spin- 5.00 8.93 127.98 60.90 40.67 60.57 34.68

6.00 9.16 127.45 60.14 32.32 61.10 40.71
system pairs 108.00 8.78 119.65 58.97 34.36 60.16 32.27
NMR Assignments
3D NMR Experiments
Identify possible residue types by

chemical shift ranges
NMR Assignments
3D NMR Experiments

Y,F,I,C 5.00 8.93 127.98 60.90 40.67 60.57 34.68
V, W, C 6.00 9.16 127.45 60.14 32.32 61.10 40.71
V, W, C 108.00 8.78 119.65 58.97 34.36 60.16 32.27
Find potential match in sequence
MTLKQVIVVRDDLKLSRGKLAVQVAHAAIIGYLKSDSSLRRKWLDEGQKKVVLKVKS
LEELLGIKHKAESLGLVTGLVQDAGLTEVPPGTITAVVIGPDEERKIDKVTGNLPLLKLE
HHHHHH
Make assignment
I 7 5.00 8.93 127.98 60.90 40.67 60.57 34.68

V 8 6.00 9.16 127.45 60.14 32.32 61.10 40.71
V 9 108.00 8.78 119.65 58.97 34.36 60.16 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 HCC(CO)NH CC(CO)NH
the C’s?
3D NMR Experiments d1
g2
 CC(CO)NH & HCC(CO)NH
 Can assign residue type by the number of g1
observed resonances and the chemical shift
ranges
 may be able to assign Cg, Cd, Ce from
b
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
a
carbon and hydrogen chemical shifts.
Biochemistry, Vol. 34, No. 42, 1995

NMR Assignments
3D NMR Experiments
 HCCH-TOCSY & HCCH-COSY
1
 relays magnetization from side-chain and backbone H &
13C via coupling
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 triple-
resonance experiments
– sample can be collected in D2O
NMR Assignments
3D NMR Experiments
 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?
4D HNCA
NMR Assignments
4D NMR Experiments
Correlates 1HCai
with NHi & NHi+1
Correlates NHi with

1HCai & 1HCai+1
NMR Assignments
4D NMR Experiments
 Quality improves with
deuterium labeling
TROSY
specific labeling
J. AM. CHEM. SOC. 9 VOL. 124, NO. 34, 2002

NMR Assignments

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NMR Assignments

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NMR Assignments

What is the NMR Assignment Issue?

number of residues of the protein

1H NMR Spectra Protein PDB File

... Ala 70 Ser 71 Leu 72 ...

J. Biomol. NMR 2010 48(1):13.

 not “100%” efficient but significantly aids in the manual assignment

• NMRPipe, Felix, ACD and others

 all handle common instrument data formats (Bruker, Varian)

 choice is primarily based on personal preference

- uses UNIX pipe

 1D NMR 8-32K pts  2D 2K x 512 pts  3D 2K x 128 x 80 pts

• Two major impacts:

 FIDs in indirect dimension have very low resolution

• These issues are addressed in processing the data

NMR Data Processing Software

Truncated FID with spectra “wiggles”

Apodized FID removes

NMR Data Processing Software

8K FID 16K FID

 works best for data without significant relaxation

 assumes sinusoid shape

next point in the series.

 forward-backward linear prediction - combines results from separate forward- and

backward-linear prediction calculations.

 process can be continued indefinitely

 becomes unstable when group contains all synthetic points

 LP order (number of coefficients) must be as large as the number of signals to extract,

but smaller than half the original data size.

complex conjugate for the LP calculation and then discard it.

Progress in Nuclear Magnetic Resonance Spectroscopy (1988), 20(6),515-626

The Nyquist theorem

Need to sample twice as fast (DW)as the fastest frequency

Traditional NMR acquires EVERY

Reason why nD NMR experiments

exponential in t1 and linear Exponential in both

randomly sampled from an Random in t1 and t2.

Graham A. Webb (ed.), Modern Magnetic Resonance, 1305–1311.

NMR Data Processing Software

 Avoid FID truncation, maximize resolution

NMR Data Processing Software

Magn. Reson. Chem. 2011, 49, 483–491

 Highly resolved spectra is pT2

TSMP – time constant for the exponential

Magn. Reson. Chem. 2011, 49, 483–491

Sampling Density/LW = TSMP/T2

Magn. Reson. Chem. 2011, 49, 483–491

particularly at the beginning and end of the FID

Top Curr Chem. 2012 ; 316: 125–148

J Biomol NMR. 2009 November; 45(3): 283–294.

 maximum entropy reconstruction is a common approach

 Carrier frequency is usually centered on the water signal

time domain of the FID

NMR Data Processing Software

with Solvent Subtraction without Solvent Subtraction

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

between real (sine) and imaginary (cosine)

 Some Common Phase Cycle Schemes:

 STATES – phase cycles the 90 -pulses prior to t1 incrimination by 90

combines N (echo) and P(antiecho) coherence selection

Experiment Increment Pulse Phase Receiver Phase