what is the nmr assignment issue?

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 resonances to assign

NMR Assignments

1H NMR Spectra Protein PDB File

H2N CH C

CH3

O

HN CH C

CH2

O

OH

HN CH C

CH2

OH

O

CH CH3

CH3

... ...

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

H2N CH C

CH3

O

HN CH C

CH2

O

OH

HN CH C

CH2

OH

O

CH CH3

CH3

... ...

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

15N 119.3 ppmHN 7.76 ppm

13C17.5 ppmH 1.45 ppm

13C55.5 ppmH 3.76 ppm

13CO171.9 ppm

15N 114.8 ppmHN 7.08 ppm

13CO178.1 ppm

13C64.8 ppmH 3.73 ppm

13C59.9 ppmH 4.35 ppm

15N 125.6 ppmHN 8.20 ppm

13CO170.9 ppm

13C42.9 ppmH 1.52 ppm

13C58.6 ppmH 4.09 ppm

13C27.9 ppmH 1.65 ppm

13C25.4 ppm; 25.7 ppmH 0.82 ppm; 0.98 ppm

NMR Assignments

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

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

NMR Assignments

J. Biomol. NMR 2010 48(1):13.J. Biomol. NMR 2012 54(3):257

http://casegroup.rutgers.edu/qshifts/about.htm

http://www.shiftx2.ca/

http://spin.niddk.nih.gov/bax/software/SPARTA+/

http://www-vendruscolo.ch.cam.ac.uk/camshift/camshift.php

http://spin.magnet.fsu.edu./

http://www.uef.fi/en/4dspot

http://www.bionmr.chem.au.dk/bionmr/software/shaic.php

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

Residue N CO Ca Cb OthersD1 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)

.

.

.

Assignment Table

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

X

Y

Z

Standard Processing Script for 3D NMR Data

Processing steps for X,Y,Z dimensions of 3D spectra

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

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


• 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

231.40 231.39 231.38 231.37 231.36 231.35 231.34 231.33 231.32 231.31 231.30 231.29 231.28 231.27 231.26 231.25 231.24f1 ppm

231.42 231.40 231.38 231.36 231.34 231.32 231.30 231.28 231.26 231.24 231.22 231.20f1 ppm

0 0.20 0.40 0.60 0.80 1.00 1.2 1.4 1.6 1.8 2.0 2.2t1 sec

8K data 8K zero-fill

8K FID 16K FID

No zero-filling 8K zero-filling


• 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

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

LP

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


• 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

time

voltage

Traditional NMRFID is truncated because number of points and DW determine how much of the FID can be collected

NUS NMRFID is under-sampled, but the entire FID is sampled.

Magn. Reson. Chem. 2011, 49, 483–491

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


• Non-uniform data sampling What is the optimal sampling density? Increase enhancement by increase exponential bias, eventually regenerate truncated FID Highly resolved spectra is T2

TSMP – time constant for the exponentialweighting of the sampling.- enhancementlw – line width

Magn. Reson. Chem. 2011, 49, 483–491


• 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

Sampling Density/LW = TSMP/T2

Truncated FID

Magn. Reson. Chem. 2011, 49, 483–491


• 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


Dramatic gain in resolution for 48 kDa

protein with only 3% sampling of the

Nyquist matrixSame 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.htmlNewton: http://newton.nmrfam.wisc.edu/newton/static_web/index.htmlRNMRTK: http://rnmrtk.uchc.edu/rnmrtk/RNMRTK.htmlmpiPipe: Available by contacting the Wagner Group

http://www.enmr.eu/webportal/mdd.html

http://newton.nmrfam.wisc.edu/newton/static_web/index.html

http://rnmrtk.uchc.edu/rnmrtk/RNMRTK.html

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


• 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(i)) to set 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)


• 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:

STATES – phase cycles the 90o-pulses prior to 1 incrimination by 900

TPPI – phase cycles both the receiver and the 90o-pulses prior to 1 by 90o for each 1 increment States-TPPI – phase cycles both the receiver and the 90o-pulses prior to 1 by 180o for

each 1 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) x x

(4k + 2) t1(0) + (4k + 1) y x

(4k + 3) t1(0) + (4k + 2) -x x

(4k + 4) t1(0) + (4k + 3) -y x

STATES

(4k + 1) t1(0) + (4k)2 x x

(4k + 2) t1(0) + (4k)2 y x

(4k + 3) t1(0) + (4k + 1)2 x x

(4k + 4) t1(0) + (4k + 1)2 y x

States-TPPI

(4k + 1) t1(0) + (4k)2 x x

(4k + 2) t1(0) + (4k)2 y x

(4k + 3) t1(0) + (4k + 1)2 -x -x

(4k + 4) t1(0) + (4k + 1)2 -y -x

The phase introduced by a gradient of duration τG to coherence of order p which involves kspins 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 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 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

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 4Echo-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

Phase cycling determines how the data is stored and retrieved

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


• 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– crowded overlap regions and noise regions (solvent, T2 ridges) cause problems– noise peaks and missing real peaks cause problems in automated assignment software

J. OF MAG. RES. 135, 288–297 (1998)

NMR Assignments

NMR Data Processing Software• NMR data analysis/visualization

What is the Statistical likelihood that a signal is a peak?

J Biomol NMR (2013) 55:167–178.

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.


• 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)

Ni

H

Ci

H

Ci

O

Ni+1

H

CiH

H3C CH3

dNN

dN

dN

dNN

dN

dN

dNN

Takes advantage of short sequential distances between CiH, CiH and NHi+1

NMR Assignments 2D NMR Experiments

• 2D COSY Correlation Spectroscopy Correlates 1H resonances that are scalar coupled (3J) Identifies which NHi resonances are bonded to CHi resonances

separated by three-bonds chemical shift evolution based on J occurs during t1

requires the sample be in H2O (90/10 H2O/D2O) to observe NH

all three-bond couplings observed, not just NH-Cspectra is symmetricstrength 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 1H resonances that close in space (≤5Å)

also contains COSY peaks NOE intensity builds up during mixing time (tm), ususally 100-150 ms

Correlates NHi+1 resonances with CHi resonances

NMR Assignments

2D NMR Experiments• 2D NOESY

Typical Protein NOESY (Lysozyme)

Both NHi-Ci and NHi+1-Ci are present

Biochemistry 1989, 28, 1048-1054


• Making the Sequential Assignments Connecting COSY (NHi-Ci) peaks with NOESY (NHi+1-Ci)

COSY experiment allows you to identify the NHi-Ci cross peaks in the NOESY experiment N-terminal amino acid only has one cross peak associated with its NH chemical shift

COSY cross peak

NOESY cross peak

The Backbone Walk

NHi-Ci NHi+1-Ci

NHi+1-Ci

NHi+1-Ci

NHi+1-Ci

T27D26 F25A24 Y28

NH Chemical Shifts (ppm)

T27

D26

F25

A24

Y28NHi-Ci

NHi-Ci

NHi-Ci

NHi-Ci

NMR Assignments

2D NMR Experiments• Verifying the Sequential Assignments and Side-Chain Assignments

The accuracy of the backbone assignments from connecting COSY (NH i-Ci) peaks with NOESY (NHi+1-Ci) 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 chainAla – single methyl (1.39 ppm)Val – two methlys (0.97 & 0.94 ppm)

one H(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

CC C C

Leu - actual

CC/C CC

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)


• 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

Spin-Lock Pulse (~14ms)

COSY TOCSY

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 and 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

Bull. Korean Chem. Soc. 2001, Vol. 22, No. 5 507

2D NMR Experiments• 2D TOCSY

Typical Small Protein TOCSY Side-chain spin systems are correlated with NH resonance

NMR Assignments

Boxed regions indicate side-chain spin systems for His and Ile, respectively


• 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

Primarily uses one-bond heteronuclear coupling (1H-13C, 1H-15N) 1J generally stronger than 3J

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


• 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 NH (Trp) position in HSQC depends on local structure and sequence no peaks for proline (no NH)

Side-chain NH2

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

• 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

Ni-1

H

Ci-1

H

Ci-1

O

Ni

H

Ci

Ci-1

H

Ci

Ci

O

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 correlates NHi to Ci-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

identifies potential overlap in 2D 1H-15N HSQC spectra, especially for larger MW proteins most sensitive 3D triple resonsnce experiment may observe side-chain correlations

1JNC’

1JNH

Ni-1

H

Ci-1

H

Ci-1

O

Ni

H

Ci

Ci-1

H

Ci

Ci

O

NMR Assignments


NMR Assignments


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


• 3D HN(CA)CO Experiment correlates NHi to Ci

relays the transfer through Ci without chemical shift evolutionuses stronger one-bond coupling

contains only intra correlation provides a means to sequential connect NH and C chemical shifts

match NHi-COi (HN(CA)CO with NHi-COi-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

1JCC’

1JNH

1JNC

Ni-1

H

Ci-1

H

Ci-1

O

Ni

H

Ci

Ci-1

H

Ci

Ci

O

NMR Assignments

3D NMR Experiments• 3D HN(CA)CO Experiment

NMR Assignments

3D NMR Experiments• 3D HN(CA)CO Experiment

Amide “Strips” from the 3D HNCO and HN(CA)CO experiments arranged in sequential order

HNCO and HN(CA)CO pair for one residues NH

Connects HNi-COi with HNi-COi-1

Journal of Biomolecular NMR, 9 (1997) 11–24


• 3D HNCA Experiment correlates NHi to Ci-1 and Ci

typically the intensity of NHi-Ci > NHi-Ci-1, 1JNC > 2JNC

NHi-Ci-1 correlation not always seen

could be weak or degenerate with NHi-Ci

contains both inter and intra correlations provides a means to sequential connect NH and C chemical shifts

not sufficient to complete backbone assignments because of overlap need 2-3 connecting inter and intra correlations


1JNH

1JNC2JNC

Ni-1

H

Ci-1

H

Ci-1

O

Ni

H

Ci

Ci-1

H

Ci

Ci

O

NMR Assignments

3D NMR Experiments• 3D HNCA Experiment

NMR Assignments

3D NMR Experiments• 3D HNCA Experiment

J. of Biomol. NMR, 14: 85–88, 1999.

Amide “Strips” from the 3D HNCA experiment arranged in sequential order

Each strip corresponds to one NH resonance in a given 15N plane

Ci-1

Ci

Correlation of the Ci and Ci-1 sequentially aligns the two NHs in the protein’s sequence.

NMR Assignments

3D NMR Experiments• 3D HN(CO)CA Experiment

correlates NHi to Ci-1

relays through 1JNC’ without chemical shift evolutionNHi-Ci-1 correlation is more sensitive than HNCA experiment

unambiguous NHi-Ci-1 assignments avoids possible overlap in HNCA experiment

companion experiment to HNCA provides a means to sequential connect NH and C chemical shifts

NHi-Ci (HNCA) matches with NHi-Ci-1 (HN(CO)CA) not sufficient to complete backbone assignments because of overlap need 2-3 connecting inter and intra correlations


1JNH

1JC’C1JNC’

Ni-1

H

Ci-1

H

Ci-1

O

Ni

H

Ci

Ci-1

H

Ci

Ci

O

NMR Assignments


NMR Assignments



HNCA HN(CO)CA NHi-Ci-1 NHi-Ci

one residues NH


• 3D CBCANH Experiment correlates NHi to Ci, Ci-1 and Ci, Ci-1

transfer is simultaneously started on H & Hboth i and i-1 typically the intensity of NHi-Ci & NHi-Ci > NHi-Ci-1 & NHi-Ci-1

1JNC > 2JNC can usually distinguish Ca from Cb from chemical shift difference NHi-Ci & NHi-Ci-1 are opposite sign of NH-Ci & NH-Ci-1

– one set of peaks are positive intensity and the other set is negative only Gly NHi-Ci-1 & NHi-Ci correlations are seen

contains both intra and inter correlations provides a means to sequential connect NH, C and C 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

Ni-1

H

Ci-1

H

Ci-1

O

Ni

H

Ci

Ci-1

H

Ci

Ci

O

1JNH

1JNC2JNC1JNC

Match-up the intra and inter correlations

NMR Assignments

3D NMR Experiments• 3D CBCANH Experiment

Amide “Strips” from the 3D CBCANH experiment arranged in sequential order



Note: contours of opposite intensity are shown in different colors

NMR Assignments

3D NMR Experiments• 3D CBCANH Experiment

IUBMB Life, 52: 291–302, 2001


• 3D CBCA(CO)NH Experiment correlates NHi to Ci-1 and Ci-1

can usually distinguish C from C from chemical shift difference sometimes NHi-Ci-1 and NHi-Ci-1 may be oppositely phased

– one peak positive intensity the other negative only Gly NHi-Ci-1 correlations are seen no peaks for proline (no NH) breaks assignment chain

transfer is started on simultaneously on Hi-1, Hi-1relayed through CO without chemical shift evolution (1JCC’,

1JC’N) contains only inter correlations provides a means to sequential connect NH, C and C chemical shifts with a companion experiment (s)

companion experiments would provide NHi-Ci (HNCA) and NHi-Ci

(CBCANH) the 2 connections of inter and intra correlations may be sufficient to unambiguously assign the backbone

Ni-1

H

Ci-1

H

Ci-1

O

Ni

H

Ci

Ci-1

H

Ci

Ci

O

1JCC’

1JC’C

1JNH

NMR Assignments

3D NMR Experiments• 3D CBCA(CO)NH Experiment

NMR Assignments

3D NMR Experiments• 3D CBCA(CO)NH Experiment


Amide “Strips” from the 3D CBCANH (right) and CBCA(CO)NH (left) experiment arranged in sequential order

Correlation of the Ci & Ci-1

and Ci & Ci-1 sequentially aligns each pair of NHs in the protein’s sequence.

NMR Assignments


• 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

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


NMR Assignments

3D NMR Experiments• Backbone Assignments

Need to correlate all the information from all the available experiments

Ci Ci-1

Ci Ci-1

COi COi-1

Hi Hi-1

NMR 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

NMR 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 C C Ci-1 Ci-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 . . .


• Backbone Assignments The process is a multi-step approach:

(3) Extend pairs of NH roots and match to protein primary sequence

.

.

. 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 . . .

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 40.71 108.00 8.78 119.65 58.97 34.36 60.16 32.27

Identify overlapping spin-system pairs

connect spin-system pairs




Identify possible residue types by chemical shift ranges

MTLKQVIVVRDDLKLSRGKLAVQVAHAAIIGYLKSDSSLRRKWLDEGQKKVVLKVKSLEELLGIKHKAESLGLVTGLVQDAGLTEVPPGTITAVVIGPDEERKIDKVTGNLPLLKLEHHHHHH




Find potential match in sequence

Make assignment


• 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 C and C chemical shifts from other triple-resonance experiments to confirm side-chain assignments

Biochemistry, Vol. 34, No. 42, 1995

NMR Assignments

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 C, C, C from chemical shift values and from previously assigned C and C less likely to assign H, H and H, unless unique chemical shift need companion experiments to connect carbon and hydrogen chemical shifts.

CC(CO)NHHCC(CO)NH

Which H’s match the C’s?


• Side-chain Assignments HCCH-TOCSY & HCCH-COSY

relays magnetization from side-chain and backbone 1H & 13C via coupling constants Experiments have symmetry

–1H-13C diagonal shows cross peak to 1HAND

– 1H-13C diagonal shows cross peak to 1H 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• 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 HC shows a cross peak to H and the HC shows a crosspeak to both HC

Note: Symmetry peaks may not always be present (separate pathways, separate relative sensitivity). Presence of a symmetry peak increase the likelihood of correct assignment


• 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


Correlates 1HCi with NHi & NHi+1

Correlates NHi with 1HCi & 1HCi+1

4D HNCA

J. AM. CHEM. SOC. 9 VOL. 124, NO. 34, 2002


Quality improves with deuterium labeling TROSYspecific labeling

NMR Assignments

what is the nmr assignment issue?

Documents

c atom

h atom

n atom

o atom

cg asp

n nmr resonances

d nmr spectra

cb asp