NMR data processing &
peak assignments
nmrPipe
nmrDraw
nmrView
Contents
1. nmrPipe
2. nmrDraw
3. nmrview
nmrPipe
nmrPipe:
Spectral Processing as a UNIX Pipeline
http://spin.niddk.nih.gov/NMRPipe/
☺ Software system for processing, analyzing, and exploiting NMR spectroscopic data
☺ One of the most popular software packages for NMR Data Processing in part due to
its efficiency
☺ NMRPipe consists of a series of "functions" which can be applied to a FID data file in
any sequence, by using UNIX pipes
☺ Processed data can be used with well-known spectral analysis programs such as
nmrview and sparky etc…
F. Delaglio, S. Grzesiek, G. W. Vuister, G. Zhu, J. Pfeifer and A. Bax: NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J. Biomol. NMR. 6, 277-293 (1995)
nmrPipe stream
Zero-filling
Phase correction
Zero-Filling
Input data
(ser file)
SP
ZF
FT
PS
Transpose
TP
Window function
Fourier Transformation
Processed
output
stream
FID (Free Induction Decay)
Biological NMR lecture, Korea Magnetic resonance society
Zero-Fill
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Improve digital resolution by adding zero data points at end of FID
raw data
No zero-filling
zero-fill
zero-filling
FT (Fourier Transform)
Pulse
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1D Spectrum
FID
FT
time
frequency
A pulse creates magnetization in the XY plane, which is ultimately detected by the coil in the
NMR probe
The RF signal detected by the coil, consisting of a superposition of many sinusoidal waves of
differing frequencies, amplitudes and phases, is termed “free induction decay”, or FID
FID dies out with time in an exponential manner and is converted to the frequency domain by a
function called “Fourier transformation”, or FT:
f(ω) = ∫f(t) eiωt dt
from trigonometry
eiωt = cosωt + isinωt
FID thus consists of real and imaginary components:
Re(f(ω)) = ∫f(t)cosωt dt
and
Im(f(ω)) = ∫f(t)sinωt dt
Both the real and the imaginary components are Fourier transformed to generate a spectrum in
the frequency domain
Transpose
☺
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Ft.com file
XYZ→X axis Processing→TP→
YXZ→Y axis Processing->ZTP →
ZXY→Z축 Processing→TP →
XZY-> result file
# TP:
Transpose 1 and 2
# ZTP: Transpose 1 and 3
C
XYZ
N
→ 측정 시 HNC
H-N 2D plane을 C domain
축을 따라 가는 방식으로 분석
→
N
H
XZY
C
H
측정 시 HCN
H-C2D plane을 N domain축을
따라 가는 방식으로 분석
nmrPipe
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Frank Delaglio www.nmrscience.com/embo
nmrPipe
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2D experiment
3D experiment
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nmrPipe
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Conversion Script (Fid.com): nmrdata를 nmrPipe로 인식할 수 있게 하는 format conversion
C-shell script
#! /bin/csh
Read raw file
Digital filter
Bruk2pipe –in ./ser
\
-bad 0.0 –noswap -DMX -decim 16 -dspfvs 12 -grpdly 0 \
-xN
-xT
-xMODE
-xSW
-xOBS
-xCAR
-xLAB
- ndim
2048
1024
DQD
8992.806
500.132
4.70
1H
2
- out ./test.fid –verb -ov
-yN
256
-yT
128
-yMODE Echo-Antiecho
-ySW
-yOBS
-yCAR
-yLAB
-aq2D
1875.293
50.684
117.359
15N
States
\
\
\
Data point
Time domain
Acquisition mode
\
\
\
\
\
Sweep width (Spectral width)
Base frequency
Carrier frequency (O1/BF1)
Label
Dimension number ,
2D plane acquisition mode
Save the test.fid file in
this directory
NMR data size
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NMR data size
Increase in data points, resolution and acquisition time
→ Increase in the number of data points  increase in resolution→ Increases acquisition time
Protein NMR lecture , Amjad Farooq, University of Miami
SW (Sweep Width)
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Sweep Width
(range of radio-frequencies monitored for nuclei absorptions)
Ft.com (Fourier Transform script)
#! /bin/csh
nmrPipe –in test.fid
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\
SOL
\
POLY time
\
SP -off 0.3 -end 0.99 -pow 2 -c 0.5 \
ZF -size -auto \
FT -auto \
PS -p0 -31 -p1 0 -di \
EXT -left -sw -di \
TP \
SP -off 0.3 -end 0.99 -pow 1 -c 0.5 \
ZF -size -auto \
FT -auto \
PS -p0 -93 -p1 0 -di \
TP \
HSQC.ft -ov -verb \
nmrPipe –in HSQC.ft
│ pipe2xyz
-out SAV0816_HSQC.nv -nv -ov -verb
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Solvent Removal
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• Solvent Removal (SOL)
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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
Without Solvent Subtraction
With Solvent Subtraction
Linear Prediction
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FID의 초기 감소 경향으로부터 예측하여 FID를 완전한 형태로 만들어 주는 조작
(apply complex linear prediction to predict or replace data at the head, tail, or
interior of data vectors. Forward, backward, and mixed modes are supported
along with mirror image modes)
│ nmrPipe -fn LP
LP전
Before
LP
-> 일반적인 LP를 가하는 함수를 적용하라
│ nmrPipe -fn LP -ps0-0
-> Mirror image processing. 32포인트 이하의
data에 효과적임
│ nmrPipe -fn LP –fb –auto
-> Forward-back 양 방향으로 LP하여
나머지는 자동으로 처리하라
After
LP후LP
Linear Prediction
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☺ 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
Progress in Nuclear Magnetic Resonance Spectroscopy (1988), 20(6),515-626
Linear Prediction
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Frank Delaglio www.nmrscience.com/embo
Linear Prediction
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LP
Frank Delaglio www.nmrscience.com/embo
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nmrDraw
nmrDraw
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☺ NMRDraw is the companion graphical interface for NMRPipe
☺ Real-time phasing of one or more vectors for any dimension
nmrDraw (general example)
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Phase
correction OK…
nmrDraw
nmrDraw (SAV0816)
P0
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P1
Vertical mode: Click ‘V’
P0,P1 값 조절
nmrDraw (SAV0816)
P0
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P1
Horizontal mode: Click ‘H’
P0,P1 값 조절
nmrDraw (SAV0816)
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Phasing and Zero-Filling
#! /bin/csh
nmrPipe –in test.fid
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\
SOL
\
Zerofilling
POLY time
\
SP -off 0.3 -end 0.99 -pow 2 -c 0.5 \
ZF -size -auto \
Phasing
FT -auto \
PS -p0 -31 -p1 0 -di \
EXT -left -sw -di \
TP \
SP -off 0.3 -end 0.99 -pow 1 -c 0.5 \
ZF -size -auto \
FT -auto \
PS -p0 -93 -p1 0 -di \
TP \
HSQC.ft -ov -verb \
nmrPipe –in HSQC.ft
│ pipe2xyz
-out SAV0816_HSQC.nv -nv -ov -verb
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Parameter Identifier in acqu file
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Essential Parameters
Identifier
Time domain (data points)
TD
Sweep Width in ppm
SW
Sweep Width in Hertz
SW_h
Pulse program (Excitation
sequence)
PULPROG
Type of Nucleus
NUC
Carrier Frequency
O1, O2, O3
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Assignment
NMR Assignment
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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
☺ A typical protein will have hundreds of 1H,
13C
and
15N
NMR resonance to assign
NMR Assignment
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... Ala 70
Ser 71
O
... H2N
CH
O
H
N
C
CH
CH3
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
119.3 ppm
HN 7.76 ppm
13Ca
55.5 ppm
Ha 3.76 ppm
... H2N
CH
13CO
CH3
13Cb
17.5 ppm
Hb 1.45 ppm
O
H
N
C
CH
CH2
CH2
OH
CH
C
OH
...
CH3
CH3
... Ala 70
15N
Leu 72 ...
Ser 71
O
15N
114.8 ppm
HN 7.08 ppm
C
H
N
CH
Leu 72 ...
O
C
13CO
171.9 ppm
125.6 ppm
HN 8.20 ppm
H
N
178.1 ppm
64.8 ppm CH2 13Ca 59.9 ppm
Ha 4.35 ppm
Hb 3.73 ppm
13Cb
OH
O
15N
CH
CH
58.6 ppm
Ha 4.09 ppm
C
13CO
CH2
13Ca
...
170.9 ppm
13Cb
42.9 ppm
Hb 1.52 ppm
CH3
13Cd
CH3
OH
13Cg
27.9 ppm
Hg 1.65 ppm
25.4 ppm; 25.7 ppm
Hd 0.82 ppm; 0.98 ppm
NMR Assignment
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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
☺ 1D NMR ~few mins.  2D ~few hours  3D ~ few days
Kurt Wüthrich
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NMR Assignment Protocol (peptide)
☺ 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
1. Applicable for proteins of <100 amino acids
2. Primarily dependent on three 2D experiments: NOESY, COSY, TOCSY
3. Sequence-Specific Resonance Assignments in Proteins (Backbone Assignemnts)
H3C
CiH O
Takes advantage of short sequential
distances between CaiH, CbiH and NHi+1
CH3
Ni
Ci
H
H
Ci
Ni+1
H
3D NMR Experiments
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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 tripleresonance (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

Frank Delaglio www.nmrscience.com/embo
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nmrview
NMRView
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☺ NMRView version 1 was written by Bruce A. Johnson and Richard A
☺ NMRViewJ, released in the first useful version at the time of the NMRView 5
release, is written in the Java programming language.
☺ NMRView is a computer program that is designed to be useful in visualizing
and analyzing these data. NMRView works with various types of NMR datasets
and can have multiple datasets and display windows opened simultaneously.
Virtually all actions of the program can be controlled through the Tcl scripting
language, and new graphical user interface components can be added with
the Tk toolkit. NMR spectral peaks can be analyzed and assigned.
A suite of tools exists within NMRView for assigning the data from
triple-resonance experiments.
http://www.onemoonscientific.com/index.html
NMRView
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NMRView is a program for the visualization and analysis of
NMR datasets.
 Multiple views on one or more NMR spectra.
 Corresponding cursors in different windows track each other automatically.
 Spectral displays may be transferred from one window to another using a
Copy/Paste protocol.
 Automatic peak picking.
 Peak searching.
 Facilitated peak analysis and interactive peak editing.
 Comprehensive NOE constraint generation and analysis.
NMR Assignments
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Correlates backbone amide 15N through one-bond coupling to amide
In principal, each amino acid in the protein sequence will
exhibit one peak in the 1H-15N
2D
F2 (NH)
☺ side-chain NH2s (ASN,GLN) and NeH (Trp)
☺ position in HSQC depends on local structure and sequence
☺ no peaks for proline (no NH)
3D experiment as a collection of 2D experiments
F1 (H)
2D-HSQC
3D
The backbone assignments are then obtained by piecing together
all the“jigsaw” puzzles pieces from the various NMR experiments
to reassemble the backbone
F2 (Ca)
F1 (H)
F3 (NH)
3D-HNCA
HSQC (NMRView)
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HSQC (NMRView)
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HSQC (NMRView)
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①
③
0827.Seq (file format)
Met
Arg
Asn
Ile
Tyr
Val
④
1
2
3
4
5
6
②
Read Topology
Sequence File
HSQC (NMRView)
①
②
③
Add
Ph.D Jang Sun Bok
HSQC (NMRView)
Ph.D Jang Sun Bok
①
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Open Datasets
③
HSQC (NMRView)
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File
Misc
Data Set
Auto Lvl
HSQC (NMRView)
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Level up
Before
After
HSQC (NMRView)
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②
③
①
Before
After
HSQC (NMRView)
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①
②
⑥
Peaks
⑤
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hsqc ④
Write List
⑦
Navigator (nav.tcl)
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Pulse sequence
90º
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180º
Delay time t1
Delay time t2
A pulse sequence is a computer program, like a FPLC program, that runs the NMR spectrometer
with pre-defined instructions to collect NMR data on a sample of interest
It is essentially a serious of sequential and parallel pulses, separated by delays, which excite
the specific nuclei and allow them to transfer their magnetization – a necessity for probing
all the 3D information on the positioning of nuclei relative to each other
Each pulse is denoted by a rectangular block, the width of the block corresponding to the
size of the pulse – for example, a 180 pulse is twice the width of a 90 pulse
The angle of the pulse dictates the tilt of the magnetization vector
Why delays?
The delays allow the evolution of resonances and allow spin-spin interactions before they can be
further excited by a second or subsequent pulse
Protein NMR lecture , Amjad Farooq, University of Miami
1H/15N-HSQC
– the most popular NMR experiment
Well-folded globular protein
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Unfolded or unstructured protein
NMR Titration
113
HA
S154
S154
N
CA
C
115
HN
R
Q148
O
Q148
T123
117
9.5
9.3
9.1
☺The quality of HSQC spectrum provides a footprint for the state of protein:
Well-folded globular protein  Good chemical shift dispersion & sharp peaks
Unfolded or unstructured protein  Poor chemical shift dispersion & peak broadening
☺The quality of HSQC spectrum dictates whether structural studies would be feasible
3D HNCA Experiment
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HNCA
Pulse name derives from magnetization transfer….
Amide proton (H) → Amide Notrogen (N) → α carbon (Cα)
both the starting residue(i) and the previous residue(i-1) in the protein's amino acid sequence
1. correlates NHi to Cαi-1 and Cαi
2. typically the intensity of NHi-Cai > NHi-Cai-1, 1JNCa > 2JNCa
3. Provides a means to sequential connect NH and Ca chemical shifts
4. No peaks for proline (no NH) breaks assignment chain
HNCACB similar to HNCA : correlates NHi to Cβi-1 and Cβi
Bruker AVANCE 3D / Triple resonance Manual
3D HNCA Experiment
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HN(CO)CA
Pulse name derives from magnetization transfer….
Amide proton (H) → Amide Notrogen (N) → α carbon (Cα)
alpha carbon of the previous residue(i-1) in the protein's amino acid sequence
1. correlates NHi to Cai-1
2. Companion experiment to HNCA
3. No peaks for proline (no NH) breaks assignment chain
HN(CO)CACB similar to HN(CO)CA : correlates NHi to Cβi-1
Bruker AVANCE 3D / Triple resonance Manual
3D HN(CA)CO Experiment
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HN(CA)CO
Amide proton (H) → Amide Notrogen (N) → Carbonyl carbon (CO)
both the starting residue(i) and the previous residue(i-1) in the protein's amino acid sequence
1. correlates NHi to Coi
2. provides a means to sequential connect NH and CO chemical shift
→ match NHi-COi [(HN(CA)CO with NHi-COi-1 (HNCO)]
3. No peaks for proline (no NH) breaks assignment chain
Bruker AVANCE 3D / Triple resonance Manual
3D HNCO Experiment
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HNCO
Amide proton (H) → Amide Notrogen (N) → Carbonyl carbon (CO)
alpha carbon of the previous residue(i-1) in the protein's amino acid sequence
1. correlates NHi to Ci-1 (carbonyl carbon, CO or C’)
HN(CA)CO
2. Most sensitive 3D triple resonsnce experiment
3. Identifies potential overlap in 2D 1H-15N HSQC spectra,
especially for larger MW proteins
HNCO
Bruker AVANCE 3D / Triple resonance Manual
Backbone Assignment
— HNCA and HN(CO)CA experiments
HA
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HA
N
CA
C
N
CA
C
HN
R
O
HN
R
O
HNCA
CAi
CAj
CAi-1
i
i-1
CAj-1
13C
15N
i
HNCA correlates HN and N of residue i to CA of residues i and i-1
HA
j
HA
N
CA
C
N
CA
C
HN
R
O
HN
R
O
i-1
1HN
HN(CO)CA
CAj-1
13C
CAi-1
i
15N
i
j
1HN
HN(CO)CA correlates HN and N of residue i to CA of residue i-1
Protein NMR lecture , Amjad Farooq, University of Miami
Backbone assignments
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Backbone assignments
i-1
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i-1
HN(CO)CA
HNCA
i
HNCACB
i-1
i
i-1
HN(CO)CACB
HNCA and HN(CO)CA exercise
HNCA
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HN(CO)CA
15N
15N
i-1
i-1
15N
i
15N
i
15N
15N
i+1
i+1
13CA
i+1
13CA
i
13CA
i-1
13CA
i-2
1HN
1
i-1 HNi-1
Scan through
15N
1HN
i
1HN
i
1HN
1
i+1 HNi+1
dimension of HNCA until a peak matching the resonance of CAi-1 is detected
Strip plotting (HNCA)
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Strip Plot
Strip plot of HP0827
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HNCA
HNCACB
The top is 3D HNCA and bottom is 3D HNCACB using NMRView
HP0827
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S.B Jang et al .J. Biochem 2009;146(5)667-674
15N-TOCSY
experiment
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CE HE1
15N-TOCSY
CD HD1
CG HG1
CB
HA
CA
R
C
O
N
HB1
CA HA
HN C
O
i
N
HN
HB1j
HB1i
1H
HA1i
HA1j
HNi
HNj
15N
j
i
1HN
i-1
15N-TOCSY correlates HN and N resonances to sidechain protons within the same residue
Experiment is run on 15N-labeled protein in H2O
In combination with HNCA/HN(CO)CA and HN(CA)CB/HN(CO)CACB, resonance assignments
of backbone and sidechain H atoms can be made
Assignment for sidechain H atoms for aromatic residues cannot be obtained from the above
experiments but alternative experiments are available
Protein NMR lecture , Amjad Farooq, University of Miami
HBHA(CO)NH experiment
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HBHA(CO)NH
R
HB1 CB
HA
CA
C
N
N
O
HN
i-1
CA
C
O
i
HB1j-1
HB1i-1
1H
HA1i-1
HA1j-1
15N
j
i
1HN
HBHA(CO)NH correlates HN and N resonances to sidechain protons at previous
residue
Protein NMR lecture , Amjad Farooq, University of Miami
15N-TOCSY, HBHA(CO)NH (HP0827)
15N-TOCSY (i)
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HBHA(CO)NH (i-1)
Sidechain Assignment (HP0827)
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7 Gly
7Gly-8Asn
8 Asn
15N-TOCSY
HBHA(CO)NH
15N-TOCSY
HBHA(CO)NH
15N-TOCSY
HBHA(CO)NH
N:113.062
HN:9.20
N:116.453
HN:8.24
Ha: 4.75
Ha: 3.65
N:116.453
HN:8.21
N:116.234
HN:7.90
Hα: 4.50
Hα: 3.83
N:116.234
HN:7.92
N:113.062
HN:9.20
Hα: 4.31
Hα: 1.24
Hα: 4.77
Hα: 3.66
Hα: 4.50
Hα: 3.80
8Asn-9Leu
9 Leu
Hα: 4.32
Hα: 1.25
9Leu-10Val
CCCONH
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☺ confirm the previous sequential assignment
specifically designed to correlate the 1H and 15N amide resonances of one residue with
and all other 13C side-chain resonances of its preceding residue via the intervening
13CO spin
(i-1)
(i)
13CA
CCCONH
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6Cγ2
6Cγ1
19Cγ
19Cδ
19Cβ
6Cβ
(weak)
C
C
7Cα
C
19Cε
6Cα
6Val
19Cα (weak)
19Lys ←20Glu
←7Gly
N:112.753
HN:9.20
HN
N:120.750
HN:8.26
HN
HN
7Gly ←8Asn
N:116.463
HN:8.28
Sidechain Assignment — HCCH-TOCSY experiment
CE
CD
HA
CA
R
C
O
N
HE1
Experimen
Acceptor
t
Donor
HD1
3D H(C)CH
C-H
-TOCSY
H
3D (H)CCH
C-H
-TOCSY
C
CG
HG1
CB
HB1
CA
HA
HN C
i
1H
j
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HCCH-TOCSY
HAi HB1i
HAj HB1j HG1j
HG1i
HD1j
13C
N
1H
O
HN
i
i-1
HCCH-TOCSY correlates H and C resonances within the same residue
Experiment is run on
15N/13C-labeled
protein in D2O
In combination with HNCA/HN(CO)CA & HN(CA)CB/HN(CO)CACB and C(CO)NH &
resonance assignments of backbone and sidechain H atoms can be made
15N-TCOSY,
Assignment for sidechain H and C atoms for aromatic residues can also be obtained from the
above experiment run in the aromatic region
Protein NMR lecture , Amjad Farooq, University of Miami
Sidechain Assignment — HCCH-TOCSY exercise
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Diagonal Peak
Cross Peak
Open up 3-5 windows aligned up vertically above each other in NMRView
Scan through
13C
dimension of HCCH-TOCSY to display the CA-plane of a spin system
Repeat the above step for other C atoms, such as CB and CG, in other planes
Match up all the spin systems across all C planes
Protein NMR lecture , Amjad Farooq, University of Miami
HCCH-TOCSY exercise 1(HP0827)
Hβ1
Hβ2
2.12
2.01
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Cβ
30.06
Cγ
34.75
Cx
Cx
Cx
Ca
52.91
Hγ1
Hγ2
2.13
2.02
Ha
5.24
1H
49Glu
(Ca:52.91)
1H
49Glu
(Cβ: 30.06)
1H
49Glu
(Cγ:34.75)
HCCH-TOCSY exercise 2(HP0827)
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Cγ2
20.23
Cγ1
21.07
Cβ
29.31
Hγ1
Hβ1
Cx
Cx
Cx
Cx
Hγ21
Hγ22
Ca
64.47
Ha
1H
55Val
(Ca:64.47)
1H
55Val
(Cβ:29.31)
1H
55Val
(Cγ1:21.07)
1H
55Val
(Cγ2:20.23)
NOE Assignment —
15N-NOESY
experiment
CE HE1
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15N-NOESY
CD HD1
CG HG1
CB
HA
CA HA
N
CA
C
N
HN
R
O
HN C
i-1
HB1
i
N
1H
HB1i
HB1i-1
HA1i-1
HA1i-1
HNi-1
HNi
HNi-1
HNi
HA1i
i-1
i
15N
1HN
HN
Protein NMR lecture , Amjad Farooq, University of Miami
What is NOE?~~~
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Frank Delaglio www.nmrscience.com/embo
NOE Assignment —
15N-NOESY
experiment
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15N-NOESY
correlates through-space HN and N resonances of residue i to any
protons < 5Å of backbone HN of residue i
Experiment is run on
15N-labeled
protein in H2O
15N-NOESY
provides information on NOEs between HN and all other protons in
the protein but, in practice, only local short range NOEs can be assigned
with certainty (due to lack of asymmetry)
15N-NOESY
is particularly useful for identification of local secondary
structure elements:
-helices  strong dHN,HN(i, i+1) and dHA,HN(i, i+3)
-strands  strong dHA,HN(i, i+1)
NOE Assignment —
13C-NOESY
experiment
CE HE1
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13C-NOESY
CD HD1
CG HG1
CB
HA
HB1i
HB1
HA
CA HA
CA
C
N
R
O
HN C
i-1
1H
O
i
N
N
CA
HN R
j
HAj
C
HAi HB1i
HAj HB1j
HG1i
HG1j
HD1j
13C
1H
O
Protein NMR lecture , Amjad Farooq, University of Miami
NOE Assignment —
13C-NOESY
experiment
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13C-NOESY
correlates through-space C and H resonances of residue i to any
protons < 5Å of HC, not HN, of residue i
Experiment is run on
13C-labeled
protein in D2O
13C-NOESY
provides information on NOEs between i and j pairs of protons in
the protein and, because of its symmetrical nature, it is the only means with
which long range NOEs can be assigned with certainty
13C-NOESY
is also useful for identification of local secondary structure elements:
-helices  strong dHA,HB(i, i+3)
NOE Assignment —
13C-NOESY
exercise
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In 13C-NOESY, NOEs appear as cross-peaks with a symmetrical relationship
The symmetry of NOE cross-peaks avoids ambiguity in assignment and thus
plays a crucial role in the assignment of long-range NOEs
Protein NMR lecture , Amjad Farooq, University of Miami
characteristic chemical shifts
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Knowledge of protein sequence coupled with
characteristic chemical shifts of specific spin
systems (specific atoms within amino acids) is
sufficient to provide near-complete backbone
connectivities for small to medium-sized proteins
(< 300 residues)
Chemical shift table
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Random coil 1H and 13C chemical shifts in the natural amino acids
Ph.D Jang Sun Bok
10
Aβ
Carbon-13 Chemical Shift
20
Hβ
Q,Eβ
Cβ
Rβ
Wβ
30
Kβ
F,Yβ
40
Gα
Nβ
Dβ
Iβ
N,D,Hα
Lβ
Aα
50
M,R,Q,E,K,Lα
60
C,F,W,Yα
70
P,M,Vβ
5
T,Iα
Sα
Pα
Vα
Tβ
4
Sβ
3
2
1
0
Proton Chemical Shift
Bruker AVANCE 3D / Triple resonance Manual
Random coil 1H and 13C chemical shifts in the natural amino acids
10
Mε
Carbon-13 Chemical Shift
20
Pγ
Mγ
30
Qγ
Eγ
40
Rδ
Lγ
Rγ
Kδ
Ph.D Jang Sun Bok
Iδ
IγCH3
Tγ
Vγ
Kγ
Lδ
IγCH2
Kε
Pδ
50
60
70
5
4
3
2
1
0
Proton Chemical Shift
Bruker AVANCE 3D / Triple resonance Manual
NOE
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1H-1H
Distances From NOEs
Long-range
(tertiary structure)
Sequential
Intraresidue
A
B
C
D
••••
Z
Medium-range
(helices)
Challenge is to assign all peaks in NOESY spectra
Good luck to you~~^^*