Rapid acquisition of multidimensional NMR data June 21 … · Rapid acquisition of multidimensional...

GRC 6/22/04© Thomas Szyperski

Rapid acquisition Rapid acquisition of multidimensional of multidimensional

NMR dataNMR data

‘‘The NMR sampling problemThe NMR sampling problem’’

LetLet’’s take stocks take stock……

Conventional, Conventional, multidimensional FT NMR multidimensional FT NMR spectroscopyspectroscopy…………

Measurement times scale with Measurement times scale with ΠΠnnjj

1 scan / second (16 complex / dimension) =>1 scan / second (16 complex / dimension) =>1D 1D --> 1 second> 1 second2D 2D --> ~0.5 minutes> ~0.5 minutes3D 3D --> ~0.25 hours> ~0.25 hours4D 4D --> ~8 hours> ~8 hours5D 5D --> ~12 days> ~12 days6D 6D --> ~1.1 years> ~1.1 years

Drawbacks of Multidimensional FT NMRDrawbacks of Multidimensional FT NMR

The ‘NMR sampling problem’:The ‘NMR sampling problem’:

minimal measurement minimal measurement ‘explodes’ with dimensionality‘explodes’ with dimensionality

SamplingSampling versus versus SensitivitySensitivity LimitationLimitation

t3 FTFT

Time DomainTime Domain Frequency DomainFrequency Domain

3D and 4D:3D and 4D:

5D +:5D +:Sampling LimitationSampling Limitation

600 MHz Cryogenic Probe at UB

First FID of 3D HCCH-COSYConventional Cryogenic probe

Drawbacks of Multidimensional FT NMRDrawbacks of Multidimensional FT NMR

Measurement times scale with Measurement times scale with ΠΠnnjj

Low precision of chemical shift Low precision of chemical shift measurement in indirect dimensionsmeasurement in indirect dimensions

The The ‘‘Gordian knotGordian knot’’::Speeding up Speeding up multidimensional multidimensional

NMR data acquisition, NMR data acquisition,

while while increasingincreasing the the accuracy accuracy ofof

the measurement of NMR parametersthe measurement of NMR parameters

RD/GFT NMR SpectroscopyRD/GFT NMR Spectroscopy

Reduced dimensionality (RD)Reduced dimensionality (RD)

NMR SpectroscopyNMR Spectroscopy

33--dimensionaldimensional 22--dimensionaldimensional

2∆Ω3

Challenge: keep information upon projection

‘‘projectedprojected’’chemical shiftchemical shift

2∆Ω3

Challenge: keep information upon projection

‘‘projectedprojected’’chemical shiftchemical shift

ΩΩKK

ΩΩSS

ΩΩcarriercarrier∆Ω∆ΩSS

ΩΩKK

Structural genomics Structural genomics lead to increased interest lead to increased interest

in RD NMRin RD NMR

RDpackRDpack

Total Measurement time Total Measurement time for RD NMR spectra: for RD NMR spectra:

44.5 hours44.5 hours3D 3D HCHCCHCH--COSY: 8.9 hoursCOSY: 8.9 hours

RD NMR Data for NESGCRD NMR Data for NESGCProteins from TorontoProteins from Toronto

MT1362 (8 kDa)MT1362 (8 kDa)SH3SH3--peptide complex peptide complex (12.5 kDa)(12.5 kDa)SRm160 domain (13 kDa)SRm160 domain (13 kDa)MT1598 (15 kDa)MT1598 (15 kDa)VT1 (17 kDa)VT1 (17 kDa)TT212 (14 kDa)TT212 (14 kDa)

Proteins from RutgersProteins from RutgersWR4 (14 kDa)WR4 (14 kDa)WR33 (21 kDa)WR33 (21 kDa)MT467 (13 kDa)MT467 (13 kDa)SR17 (18 kDa)SR17 (18 kDa)ER14 (12 kDa)ER14 (12 kDa)ER75 (17 kDa)ER75 (17 kDa)QR6 (14 kDa)QR6 (14 kDa)GR2 (7 kDa)GR2 (7 kDa)MR19 (15 kDa)MR19 (15 kDa)SR64 (17 kDa)SR64 (17 kDa)HR41 (22 kDa)HR41 (22 kDa)PfR13 (12 kDa)PfR13 (12 kDa)CCR19 (16 kDa)CCR19 (16 kDa)HR532 (13 kDa)HR532 (13 kDa)HR2106 (11 kDa)HR2106 (11 kDa)

[Structures determinedin Szyperski Lab]

Why not leaning back?Why not leaning back?

From conventional 600 MHz to From conventional 600 MHz to 800 MHz spectrometer w/cryoprobe800 MHz spectrometer w/cryoprobe

•• Conventional 600: Conventional 600: S/N 1,200:1S/N 1,200:1

•• 800 w/cryoprobe: 800 w/cryoprobe: S/N 7,700:1S/N 7,700:1

•• Reduction in measurement time: ~20+Reduction in measurement time: ~20+•• (RD) DR/TR data for 17 KDa ER75 in ~12 h(RD) DR/TR data for 17 KDa ER75 in ~12 h•• Simultaneously: All spectral widths increase Simultaneously: All spectral widths increase

by 1.33 (~ 2.3by 1.33 (~ 2.3--fold increased sampling fold increased sampling demand for 3D)demand for 3D)

RD NMR spectroscopy not RD NMR spectroscopy not fast enoughfast enough

33--dim.dim.

33-->2>2--dim.dim.

44-->2>2--dim.dim.

K=1K=1 K=2K=2

‘chemical shift multiplets’

33--dim.dim.

33-->2>2--dim.dim.

44-->2>2--dim.dim.

K=1K=1 K=2K=2

Challenges: -keep information of conventional experiment

-avoid spectral crowding

GFT NMRGFT NMR-- SpeedSpeed: Phase: Phase--sensitive joint sampling sensitive joint sampling of of K+1K+1 dimensions and dimensions and ‘‘recursive recursive central peak detectioncentral peak detection’’

-- Alternative data processingAlternative data processing: Editing of : Editing of resulting resulting ‘‘chemical shift multipletschemical shift multiplets’’((‘‘GG--matrixmatrix’’) and ) and FourierFourierTransformation Transformation

-- PrecisionPrecision: Least squares fit to obtain : Least squares fit to obtain shifts from edited multipletsshifts from edited multiplets

A pictorial approach toA pictorial approach toGFT NMR….GFT NMR….

((NN,,NN--2)D2)DGFT NMR GFT NMR

GFT GFT ==Combined Combined

GG--matrix andmatrix andFFourierourier

TTransformationransformation

K=2K=2

Joint Sampling Joint Sampling of of 3D3D Subspace Subspace of an of an NDND FT FT

NMR NMR Experiment Experiment

cos(Ω0t)

ωdirect

‘‘ExhaustiveExhaustive’’ samplingsamplingof linear combinations of linear combinations of chemical shifts:of chemical shifts:

Basic spectraBasic spectra

11stst order central peaksorder central peaks

22ndnd order central peaksorder central peaks

Reduction of minimal measurement timeReduction of minimal measurement time

NDND (N,N(N,N--1)D1)D (N,N(N,N--2)D2)D (N,N(N,N--3)D3)D

100100

200200

300300

400400

50050012

An example…..An example…..

(5,2)D (5,2)D HACACONHACACONHNHN

8 8 BasicBasic SpectraSpectra

4 4 First Order First Order Central Peak SpectraCentral Peak Spectra

2 2 Second Order Second Order Central Peak SpectraCentral Peak Spectra

1 1 Third Order Third Order Central Peak SpectraCentral Peak Spectra

K = 3K = 3

complexcomplex GG--matrixmatrix

realreal GG--matrix for matrix for KK = 3= 3

2D 2D InformationInformation13.8 min13.8 min

3D 3D Information Information +25.2 min+25.2 min

4D 4D Information Information +52.8 min+52.8 min

5D 5D Information Information +108 min+108 min

How about the increased How about the increased precision of the shift precision of the shift

measurements?measurements?

GFT NMR: Increased precision GFT NMR: Increased precision

-- OverdeterminationOverdeterminationσσGFTGFT((ΩΩjj) = [1/) = [1/√√nn ] ] σσeditededited (..(..ΩΩjj ±± ΩΩk k ±± .... ) )

-- ConstantConstant--time chemical shift evolutiontime chemical shift evolutionσσeditededited = = σσFTFT((ΩΩjj) )

-->> σσGFTGFT((ΩΩjj) = [1/) = [1/√√nn ] ] σσFTFT ((ΩΩjj))

(5,2)D (5,2)D HACACONHACACONHNHN

-- 15*53(15*53(tt11)*512()*512(tt22))

-- 15*512(15*512(ωω11)*512()*512(ωω22) ) [16 [16 MbyteMbyte]]

-- Minimal measurement Minimal measurement time: 33 mintime: 33 min

-- Precision of chemical Precision of chemical shift measurement: shift measurement: ~2~2--3 fold increased3 fold increased

5D 5D HACACONHNHACACONHN

-- 10(10(tt11)*11()*11(tt22)*13()*13(tt33)*13()*13(tt44)*512()*512(tt55))

-- 32(32(ωω11)*32()*32(ωω22)*32()*32(ωω33)*32()*32(ωω44)*512()*512(ωω55))2.1 2.1 GbyteGbyte

-- 96(96(ωω11)*96()*96(ωω22)*256()*256(ωω33)*128()*128(ωω44)*512()*512(ωω55))[618 [618 GbyteGbyte]]

-- Minimal measurement time: 5.8 daysMinimal measurement time: 5.8 days

GFTGFT FTFT

Features of GFT NMRFeatures of GFT NMR•• Generally applicable acquisition schemeGenerally applicable acquisition scheme•• Adaptation of measurement times to Adaptation of measurement times to

sensitivity requirement sensitivity requirement withoutwithoutsacrificing digital resolution or high sacrificing digital resolution or high dimensional correlationdimensional correlation

•• Realize Realize 5+D5+D•• High Precision of Shift MeasurementsHigh Precision of Shift Measurements

--> Systems with high shift degeneracy (RNA, > Systems with high shift degeneracy (RNA, Lipids)Lipids)

Features of GFT NMR cont..Features of GFT NMR cont..•• No additional hardware requiredNo additional hardware required•• Data size reductionData size reduction•• Greatly accelerated processing speedGreatly accelerated processing speed•• Robustness of data analysisRobustness of data analysis•• Combine with other approaches to reduce Combine with other approaches to reduce

the ‘sampling demand’:the ‘sampling demand’:-- NonNon--linear sampling (Threelinear sampling (Three--way decomposition) and MEM, way decomposition) and MEM,

Filter Filter diagonalizationdiagonalization, , -- HadamardHadamard NMR, SingleNMR, Single--scan scan NDND acquisition, acquisition,

•• ‘Orthogonal’ to TROSY:‘Orthogonal’ to TROSY: GFTGFT--TROSYTROSY

Major developments 1997-20031. TROSY and large systems 2. Residual dipolar couplings3. Rapid data collection4. Functional protein dynamics

SizeSize

Measurement timeMeasurement time

Sensitivity limitedSensitivity limitedSampling limitedSampling limited

Side chain assignmentSide chain assignment

t1,GFT (1H(1),13C(1);13C(2))

t2(13C(2))

t3(1H(2))

3D HCC-CH COSY

Higher PrecisionHigher Precisionfor Shift Measurementsfor Shift Measurements

Gain is precision of shift measurementsGain is precision of shift measurementsin (5,3)D in (5,3)D HCCHCC--CH CH versusversus3D (H)CCH and 3D H(C)CH3D (H)CCH and 3D H(C)CH

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