EMBO Practical Course - Sept. 12-19, 2001 Structure ... · - LP = linear prediction (-ps0-0 for mirror-image LP with the first time increment set to 0 and -ord is the LP order ≈

EMBO Practical Course - Sept. 12-19, 2001

Structure Determination of Biological Macromolecules by Solution NMR

Florence CordierSebastian Meier

Stephan GrzesiekBiozentrum der Universität Basel

Klingelbergstr. 50-70CH-4056 Basel

e-mail: [email protected], [email protected]. ++41 61 267 2100 or -2080

9/3/01 7:26 PM

1 OVERVIEW OF PRACTICALS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

2 PRACTICAL: NMRPIPE DATA PROCESSING (P4) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

2.1 EXERCISE 3D PROCESSING WITH DOUBLE LP............................................................................... 32.2 QUESTIONS: ............................................................................................................................ 42.3 NMRPIPE SCRIPTS................................................................................................................... 42.4 EXERCISE STRIP PLOTS............................................................................................................. 62.5 REFERENCES........................................................................................................................... 8

3 PRACTICAL: RESIDUAL DIPOLAR COUPLINGS (P7). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

3.1 MOTIVATION........................................................................................................................... 93.2 SAMPLE PREPARATION........................................................................................................... 10

3.2.1 Bicelles ......................................................................................................................... 103.2.2 Filamentous phage Pf1 .................................................................................................... 113.2.3 Purple membranes........................................................................................................... 113.2.4 Mechanically stressed polyacrylamide gels ........................................................................... 12

3.3 RESIDUAL DIPOLAR COUPLING MEASUREMENT.......................................................................... 123.4 NMRPIPE SCRIPTS................................................................................................................. 13

3.4.1 Exercise ........................................................................................................................ 133.5 FITTING OF THE ALIGNMENT TENSOR......................................................................................... 17

3.5.1 Exercise ........................................................................................................................ 173.5.2 Matlab Script ................................................................................................................. 17

3.6 REFERENCES......................................................................................................................... 19

4 PRACTICAL: RELAXATION (EVENTUALLY AS PART OF P7). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 0

4.1 PROTON T2 MEASUREMENT WITH THE ONEONE ECHO EXPERIMENT (WATER SUPPRESSION) ............... 204.1.1 Pulse Program................................................................................................................ 234.1.2 Demonstration of the T2-Measurement with the Oneone Echo Sequence.................................... 23

4.2 15N T2 1D ............................................................................................................................ 24

4.2.1 Pulse Program................................................................................................................ 244.3 15

N T2 2D ............................................................................................................................ 284.3.1 NMRPipe scripts (conv.com, p.com).................................................................................. 28

2

1 Overview of practicals

NMRPIPE DATA PROCESSING (P4)

• The NMRPipe program is a multidimensional NMR data processing system which is based onUNIX pipelines. The UNIX pipe “ | “ approach allows commands to be connected together in aseries where the output of one command is used directly as the input to the next command. Asan application we will show 3D data processing using Linear Prediction (LP) in two dimensions(15N and 13CACB) on a CBCA(CO)NH experiment.

RESIDUAL DIPOLAR COUPLINGS SAMPLE PREPARATION AND NMR (P7)

• We will show the anisotropic solute alignment by a number of different media such as bicelles,filamentous phage Pf1, purple membrane and mechanically stressed polyacrylamide gels.

• The HDO-quadrupolar splitting, which is a – though not directly scaleable – measure for theanisotropy of the solutes in a weakly aligning phase, is determined on a prepared sample oforienting medium via a simple 1D-experiment.

• With a water-flipback 2D-experiment of HSQC-type the dipolar couplings of protein-amidesare determined. The DSSE (doublet separated sensitivity enhanced HSQC) experiment is notproton-decoupled during nitrogen evolution and therefore yields the NH-splitting. To decreasethe spectral overlap the two components of the doublets are separated into two differentspectra. In the appearence of the subspectra the DSSE-experiment therefore resembles theIPAP-experiment, but with a sensitivity-enhancement of √2 and a signal to noise loss comparedto a HSQC singlet of only √2. While in principle similar to TROSY-type experiments,decoupling of the X-nucleus is used during detection. From the orientation data the molecularalignment tensor can be evaluated.

RELAXATION (EVENTUALLY AS PART OF P7)

• The HN-T2-relaxation time can be determined in echo-type 1D-oneone experiments with variousrelaxation delays while the proton magnetization is transverse. During this delay the transverseHN-magnetization relaxes exponentially. From the ratios of the HN-intensities at different delaysthe HN- T2-time for the sample of ubiquitin is immediately gained (Sklenar and Bax, 1987).

• T2-times for 15NH-resonances are determined with both 1- and 2-D-spectra. Multiples of 8 msare used as relaxation delays for the transverse nitrogen-magnetization. A series of 1D-spectra isrecorded, a prerecorded 2D-spectrum on NS3 is processed. The spectrum was recorded with avclist for the variable loop counter (lo to n times c) definig the number of 8 msrelaxation delays. Nitrogen- T2-measurements are one basis of dynamics measurements inbiomolecules.

3

2 Practical: NMRPipe Data Processing (P4)

• Useful literature to study in advance:

About NMRPipe:

1. Delaglio, F., Grzesiek, S., Vuister, G.W., Zhu, G., Pfeifer, J., and Bax, A., J. Biomol. NMR,

6,277-293 (1995).

About linear prediction:

2. Press, W.H., Flannery, B.P., Teukolsky, S.A., and Vetterling, W.T., “Numerical Recipes: The

Art of Scientific Computing” (chapt. 13.6), Cambridge Univ. Press, Cambridge (1986).

3. Stephenson, D.S., Prog. NMR Spectrosc., 20, 515-626 (1988).

A 3D-CBCA(CO)NH experiment has been previously recorded on a sample of the NS3 protease of

the Hepatitis C Virus (21.6 kDa). The time domain data (ser file) is located in the directory

XXXX/BRUKER_DATA/cbcaco/36/ser.Z.

2.1 Exercise 3D Processing with double LP

The NMRPipe scripts used to process the 3D-CBCA(CO)NH experiment use linear prediction in

both indirect dimensions. The scripts are given below with a description in italic and have to be run

like this: conv_xy.com, z_lp.com, y_lp.com.

(You can get help from nmrPipe –help, nmrPipe -help -fn FT, xyz2pipe –help,

man nmrPipe, man xyz2pipe, etc.)

1. Try to understand the parameters for the conversion (use the command bruk2pipe -help

for help).

2. Convert to nmrPipe time domain data and look at it in nmrDraw.

3. Do stepwise processing until the interferogram is made (until “TP”). Try to understand the

parameters.

4. Process the whole first plane, look at it. Try phasing.

5. Do stepwise processing for the rest of the scripts. Look at the 15N interferogram before and

after LP. Try 15N processing without LP.

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2.2 Questions:

1. How do you calculate the phase parameters in the indirect dimension from the pulse sequence?

2. When you linear predict data in one dimension, in what domain (time or frequency) should be all

the other dimensions of the data? What about imaginaries in the other dimensions?

3. Why is the constant phase correction applied on the time domain data before the mirror image

linear prediction?

2.3 NMRPipe Scripts

• conv_xy.com (Fourier transform in the 1st and 2nd dimensions {1H} and {13CA,13CB}):- bruk2pipe = conversion of bruker ser file to NMRPipe format- xLAB, xN, ..., aqN, ... = all needed parameters of the experiment for each dimension

Acquisition dimension {1H}:- POLY = polynomial baseline correction (time domain) to subtract low-frequency solventsignal in the FID

- SP = window function (sine-bell)- ZF = zero-filling- FT = Fourier transform

- PS = phase correction (see Bax,A. et al., J. Magn. Reson., 91, 174-178 (1991))- EXT = extract a region from the 1H dimension

2nd dimension {13CA,13CB}:- TP = exchange vectors from X- to Y-axis of the data stream to process the 2nd dimension asX-vectors

- SP = window function (sine-bell)- ZF = zero-filling- FT = Fourier transform- PS = phase correction- pipe2xyz = write NMRPipe frequency domain data into xyz format (“pipe2xyz -out”) tohave independent 2D planes for each point acquired in the 3rd dimension

conv_xy.com##bruk2pipe -in ../ser \zcat ../ser.Z | bruk2pipe \-xLAB HN \-xN 1024 \-xT 512 \-xCAR 4.658 \-xOBS 600.14076701 \-xSW 9259.2592593 \-xMODE Complex \\-yLAB CACB \-yN 114 \-yT 57 \-yCAR 46 \

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-yOBS 150.911337 \-ySW 8445.94595 \-yMODE Complex \\-zLAB N \-zN 76 \-zT 38 \-zCAR 116.48 \-zOBS 60.81855 \-zSW 1457.72595 \-zMODE Complex \\-aqN 512 -noswap -DMX -decim 16 -dspfvs 12 \-ndim 3 -verb \| nmrPipe -fn POLY -time \| nmrPipe -fn SP -off 0.35 -end 0.95 -pow 2 -c 0.5 \| nmrPipe -fn ZF -size 1024 \| nmrPipe -fn FT \| nmrPipe -fn PS -p0 33.4 -p1 0.0 -di \| nmrPipe -fn EXT -x1 6.2ppm -xn 11.3ppm -sw \| nmrPipe -fn TP \| nmrPipe -fn SP -off 0.3333 -end 0.95 -pow 1 -c 0.5 \| nmrPipe -fn ZF -size 128 \| nmrPipe -fn FT \| nmrPipe -fn PS -p0 17.0 -p1 0.0 -xi \| pipe2xyz -out B%03d.DAT -ov -nofsexit 0

• z_lp.com (Fourier transform and linear prediction in the 3rd dimension):3rd dimension {15N}:- xyz2pipe -z = read Z-axis vectors- LP = linear prediction (-ps0-0 for mirror-image LP with the first time increment set to 0and -ord is the LP order ≈ TD(complex)/3 or at least (number of signals in a row + 2))

- SP = window function (sine-bell)- ZF = zero-filling- SIGN -i = reverse frequency domain by negating the imaginary data (only if needed)- FT = Fourier transform

- PS = phase correction- pipe2xyz = write NMRPipe frequency domain data into xyz format

z_lp.com#xyz2pipe -in B%03d.DAT -z -verb \| nmrPipe -fn LP -ord 9 -ps0-0 \| nmrPipe -fn SP -off 0.5 -end 0.99 -pow 2 -c 0.5 \| nmrPipe -fn ZF -size 152 \| nmrPipe -fn SIGN -i \| nmrPipe -fn FT \| nmrPipe -fn PS -p0 0 -p1 0 -xi \| pipe2xyz -out C%03d.DAT -z -ov -nofsexit 0

6

• y_lp.com (Linear prediction in the 2nd dimension {13CA,13CB})- xyz2pipe -x = read X-axis vectors- HT = Hilbert transform to reconstruct imaginary data- PS -inv = remove the phase correction- FT -inv = inverse Fourier transform- ZF -inv = remove the zero-filling- SP -inv = remove the window function- PS = phase correction- LP = linear prediction (-ps0-0 for mirror-image LP with the first time increment set to 0and -ord is the LP order ≈ TD(complex)/3 or at least (number of signals in a row + 2))

- SP = window function (sine-bell)- ZF = zero-filling- FT = Fourier transform- pipe2xyz = write NMRPipe frequency domain data into xyz format

y_lp.com#xyz2pipe -in C%03d.DAT -x -verb \| nmrPipe -fn HT -auto \| nmrPipe -fn PS -inv -hdr \| nmrPipe -fn FT -inv \| nmrPipe -fn ZF -inv \| nmrPipe -fn SP -inv -hdr \| nmrPipe -fn PS -p0 17 -p1 0 \| nmrPipe -fn LP -ord 9 -ps0-0 \| nmrPipe -fn SP -off 0.5 -end 0.99 -pow 2 -c 0.5 \| nmrPipe -fn ZF -size 228 \| nmrPipe -fn FT -xi \| pipe2xyz -out D%03d.DAT -ov -nofsexit 0

2.4 Exercise Strip Plots

The following step is to create strip plots of the 3D-CBCA(CO)NH experiment for all H,N-

frequencies that are contained in a file (see figure joined “strip.DAT”).

• Files needed:cbcaconh.par = parameters of the experiment.strip.com = script creating strip plots at the H,N-frequencies that are written in this file(the corresponding peaks are in the region of the 1H-15N-HSQC spectrum joined to thisdocument).

• Command to create strip plots:

plotpseq cbcaconh.par

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• Exercise: Run the command plotpseq cbcaconh.par to create strip plots with the

given cbcaconh.par and strip.com files. This will create a file strip.DAT. Look at it in

nmrDraw.

cbcaconh.par#DATABASE_NAME /XXX/cbcaconh/36/nmr_p/DX_OFFSET 0.000000Y_OFFSET 0.000000Z_OFFSET 0.000000Z_PHASE_PAR 0Y_LINE_WIDTH 0.02Z_LINE_WIDTH 0.6Y_WIDTH_P 0.1Z_WIDTH_P 1DISPLAY_FILTER 1#DATA#COMMANDSread_command_file strip.com#COMMANDS

strip.com#COMMANDSopen strip.DAT 1get_strip_p 122 8.053581 107.47117get_strip_p 31 8.137132 108.05358get_strip_p 66 7.899154 109.11642get_strip_p 120 8.086138 109.15849get_strip_p 55 8.192581 110.13718get_strip_p 53 8.436196 109.71749get_strip_p 90 8.577157 110.14014get_strip_p 147 7.772433 110.58064get_strip_p 60 8.764078 107.41756get_strip_p 141 9.116767 106.60326get_strip_p 124 9.146719 108.92209get_strip_p 37 8.771285 112.11298get_strip_p 93 8.056153 111.78489close#COMMANDS

8

2.5 References

NMRPipe:

1. Delaglio, F., Grzesiek, S., Vuister, G.W., Zhu, G., Pfeifer, J., and Bax, A., J. Biomol. NMR,

6,277-293 (1995).

Linear prediction:

1. Kumaresan, R., and Tufts, D.W., IEEE Trans. Acoust. Speech Signal Process., 30, 833-840

(1982)

2. Barkhuijsen, H., De Beer, R., Bovée, W.M.M., and van Ormondt, D., J. Magn. Reson., 61,

465-481 (1985)

3. Barkhuijsen, H., De Beer, and van Ormondt, D., J. Magn. Reson., 73, 553-557 (1987)

4. Stephenson, D.S., Prog. NMR Spectrosc., 20, 515-626 (1988)

5. Hoch, J.C., Methods Enzymol., 176, 216-241 (1989)

6. Olejniczak, E.T., and Eaten, H.L., J. Magn. Reson., 87, 628-632 (1990)

7. Zhu, G., and Bax, A., J. Magn. Reson., 98, 192-199 (1992)

Forward-backward LP:

8. Delsuc, M.A., Ni, F., and Levy, G.C., J. Magn. Reson., 73, 548-552 (1987)


Mirror image LP:


Phasing:

1. Bax, A., Mitsuhiko, I., Kay, L.E., and Zhu, G., J. Magn. Reson., 91, 174-178 (1991)

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3 Practical: Residual Dipolar Couplings (P7)


About residual dipolar couplings1. Tjandra, N., and Bax. A., Science 278, 1111–1114 (1997).

About the 15N-1H dipolar splitting experiments:2. Ottiger, M., Delaglio, F., and Bax, A., J. Magn. Reson. 131, 373–378 (1998).3. Cordier, F., Dingley, A.J., and Grzesiek, S., J. Biomol. NMR, 13, 175-180 (1999).

3.1 Motivation

Residual dipolar coupling arise from the partial alignment of molecules in orienting media or via an

intrinsic anisotropic magnetic susceptibility of the molecule itself (predominantly the magnetic

susceptibility of aromatic groups in nucleic acids and protein side chains, paramagnetic ligands, or

to a weaker extent of the peptide group (helices!)) when samples are placed in a magnetic field.

Practically useful alignment leaves residual (to a large extent averaged) dipolar couplings of up to ca.

30 Hz from the several-kHz-couplings observed in solids where no averaging occurs. Molecules of

the solution in an anisotropic medium are oriented by steric clashing, electrostatic interactions

and/or weak transient binding. Dipolar couplings are mostly determined for C-H and N-H-groups in

non-decoupled, spin-state separated HSQC-like experiments (IPAP, DSSE) and for H-H-couplings

in COSY-type experiments. The value is determined by comparison of the splitting in an aligned

state with a reference spectrum in isotropic phase where only the J-splitting is detected. Dipolar

couplings serve as angular restraints in the structure determination process.

Dipolar couplings yield immediate long-range information on the relative orientation of internuclear

vectors to the molecular alignment tensor and thereby to each other. Via its quadratic angular

dependence the dipolar coupling yields the orientation of these vectors in the form of two cone-like-

surfaces. Determining the orientation in two suffienctly different aligned states restricts the

orientation of a given internuclear vector to no more than two points in space via the intersection of

the two cone-like-surfaces restraining the orientation. Therefore it is highly desireable to determine

the orientation of biomacromolecules in different media in order to strongly enhance the structural

information gained from NMR-data. Prerequisite to the measurements is of course the assignment

of the observed residues.

10

Aligning media are for example

- bicelles consisting of various charged or uncharged lipids

- filamentous phage Pf1,

- purple membrane of Halobacterium salinarum with bacteriorhodopsin in two-dimensional

crystalline arrangement and

- mechanically stressed polyacrylamide gels.

Dipolar couplings can improve local and global accuracy of the geometry and can be used for

structure validation. They can be especially useful for nucleic acid NMR-structure determination,

where comparably few NOEs are observed. Recently, various approaches have been described to

use dipolar couplings for the determination of multidomain organization and of protein folds (at low

resolution, by data base search) to generate starting models for homology modelling to speed up the

structure determination process.

3.2 Sample Preparation

3.2.1 Bicelles

Bicelles are two-dimensional, flat lipid bilayer assemblies produced by the mixing of a lipid with

larger hydrophobic chain (e.g. DMPC=dimyristoyl-phosphatidylcholine) and a lipid with smaller

sidechain occupying the edges of the discs (e.g. DHPC=dihexanoyl-phosphatidylcholine). Bicelles

orient themselves in the magnetic field by their intrinsic magnetic susceptibility. The typical

thickness of the lipid bilayers is between 40 and 50 Å. The polar lipid bilayer faces can be

differently charged by different additives resulting in different interactions with the macromolecule

and therefore different alignment. Bicelles are stable only for defined temperature and salt

concentration ranges.

Bicelle stock solutions of 15% w/v lipid are prepared using either pure water or a predefined buffer

solution such as 5-10 mM phosphate buffer, pH 6.6, 0.15 mM sodium azide, 93% H2O, 7% D2O.

DHPC (being hygroscobic and instable in presence of water) is weighed in a dry atmosphere and

dissolved in cold buffer or water (typically 4 °C, e.g. in a cold room). The cold DHPC-solution is

added to the solid DMPC to give a predetermined molar ratio (DHPC:DMPC ≈ 1:3) and a total

lipid concentration of 150 mg/ml. This mixture is incubated at maximally 18 °C for ca. 10 hours.

Incubation in a refrigerator or in a cold room at 4 °C also yields good results. Occasional vortexing

can be used to better dissolve the DMPC. The lipid-esters are prone to acid and base catalyzed

hydrolysis, so that the pH during sample preparation should be kept in the pH 6-7 range. Bicelles

11

can be stored frozen. The NMR-samples are prepared by diluting the 15% bicelle stock solutions

with the buffered protein sample to the desired bicelle concentration. This mixing should also be

performed well below the phase transition temperature of the DMPC (ca. 23 °C).

Above the phase transition temperature, the fluid sample adopts a solid consistency. In this solid

state, the bicelles can be magnetically aligned. To get the best alignments, the cold sample is put into

the preheated magnet at temperatures above 28 °C such that the phase transition temperature is

passed very quickly within the magnetic field. Above a certain temperature (depending on salt, pH,

lipid, and protein conditions) this magnetically aligned liquid crystalline state becomes unstable.

Usable temperature ranges are typically between 30 and 40 °C.

3.2.2 Filamentous phage Pf1

is a 7,349-nucleotide DNA-phage where the circular DNA is packaged with coat protein at a 1:1

nucleotide: coat protein-ratio. The assembly forms rods of ca 20,000 Å length and 60 Å diameter

and spontaneously aligns in the magnetic field. Pf1 has a negative surface charge. The observed

deuterium quadrupolar splitting in deuterated water scales up with the phage concentration and

seems to also be temperature dependent. PH-values recommended in the original publication are

6.5-8.0 and NaCl-concentrations below 100 mM. Pf1-Phages can be grown in Pseudomonas

aeruginosa or are comercially available. Phages are rebuffered by washing with the desired buffer

and centrifuging at 95,000 rpm (320,000 g) in a table ultracentrifuge for one hour. Supernatant is

discarded and phage resuspended preferably with a teflon tube. Washing is repeated twice. The

sample volume is adjusted to the desired phage concentration (30 mg/ml in this case).

3.2.3 Purple membranes

Purple membranes (PM) are bacterial membranes containing bacteriorhodopsin as a sole protein.

Typical sizes of PM patches are a few microns in diameter and 45 Å in thickness. PM is isolated

from Halobacterium salinarum as described in the literature. PMs are rather stable with respect to

temperature, pH, and other conditions. PMs align themselves in the magnetic field by the intrinsic

magnetic susceptibility of the seven trans-membrane alpha helices of bacteriorhodopsin. The

alignment is such that the direction of the membrane normal is parallel to the magnetic field. PMs

are highly negatively charged.

Sample preparation for alignment of biomacromolecules is performed by simply titrating suitable

amounts of purple membranes to the biomolecular solution. Due to the negative charge of the PM,

solute-membrane interactions are usually too strong for positively charged biomolecules (i.e. below

the pI). Good results were obtained for the proteins ubiquitin and p53 at pH 7.6 and 1-3 mg/ml

PM. The aligment of the PM suspension can easily be checked by measuring the deuterium

12

splitting of the H2O/D2O solvent (typically several Hz). Alignment is temperature independent

over a wide range and scaleable by the addition of more PM.

Above certain salt (70 mM NaCl) and PM concentrations, PM suspensions undergo a transition

from a fluid to a highly viscous state. In this state, the single PM patches form aggregates due to

van der Waals interactions. When the transition to this “salt frozen” state is performed in the

magnetic field, alignment of embedded proteins can also be observed.

3.2.4 Mechanically stressed polyacrylamide gels

The pore size and diffusion properties of polyacrylamide gels can be tuned by adjusting the

arcylamide and N,N’-methylenbiscacrylamide concentration from stocks of 29.2% w/v and 0.78%

w/v respectively. A certain mechanical stability of the gels is required for the orientation

experiments. Good results were obtained at concentrations of ≥ 4% (w/v) acrylamide.

Polymerization is started in 3.5 mm to 8 mm tubes sealed with parafilm on one side by the addition

of 0.1% w/v ammonium persulfate and 0.5 % w/v TEMED.

The gels are pushed out from these tubes and washed for 5 hours at 37 °C with water and dried in a

drying oven at 37 °C for several hours (over night) which yields the gels dehydrated and completely

solid. The gel is reswollen in a NMR sample tube with the desired biomacromolecule solution in

buffer. Mechanical stress can be applied vertically by pushing the plunger of a Shigemi tube onto

the gel at the end of the reswelling process or radially if the gel is originally polymerized in a tube

of larger diameter than the sample tube. For these two cases, the alignment tensors of embedded

protein are exactly opposite. In contrast to common intuition this does not yield new information.

3.3 Residual Dipolar Coupling Measurement

• Exercise 1: Measure the HDO-splitting in an oriented sample. When not locking on deuterium

in the XWINNMR-lock display, you can see two resonances. They arise from the orientation

of deuterated water at the interface to the orienting medium giving rise to a deuterium

quadrupolar splitting. After locking first on the one maximum and then on the other one, record

two simple 1-D experiments in proton dimension and determine the difference in Hz for the

water signal when locking on the different signals. From that, immediately determine deuterium-

splitting (the gyromagnetic ratios of proton and deuteron relate ca. like 6.5:1). The HDO-

splitting is not necessarily proportional to the protein alignment (see Ottiger and Bax, JBNMR,

12, 361 (1998)).

• Exercise 2: Measure the 15N-1H dipolar coupling by recording the DSSE-15N-1H-HSQC

experiments on one of the samples provided.

13

As a backup, a DSSE-15N-1H-HSQC experiment has been recorded previously on a sample of

ubiquitin and a sample of ubiquitin + 1 mg/ml of Purple Membrane. The time domain data (ser files)

are located in the directory XXX/BRUKER_DATA/water_nh/854 (ubiquitin ref.) and ../856

(ubiquitin + 1mg/ml PM).

3.4 NMRPipe Scripts

The NMRPipe scripts used to process the DSSE-15N-1H-HSQC experiment allow the separation

of the upfield and downfield components in the indirect dimension into different subspectra. The

scripts are given below with a description in italic and have to be run like this:

conv.com 1

xy.com 1 >> create a spectrum C1.DAT for the upfield component

conv.com 2

xy.com 2 >> create a spectrum C2.DAT for the downfield component

3.4.1 Exercise

1. Try to understand the parameters for the conversion (use the command bruk2pipe -help

for help).

2. Run the conversion script on the reference data (not oriented) in order to create the upfield

(conv.com 1) and then the downfield (conv.com 2) components. This will produce 2

datasets that will be processed independently in the following steps. Look at the result in

nmrDraw.

3. Process the 1H and 15N dimension with the script xy.com (1 or 2) and visualize the 2 spectra

in nmrDraw.

4. Repeat the whole procedure for the oriented data.

5. Visualize or quantify changes in the J-couplings.

conv.com and xy.com:- “bruk2pipe” = conversion of bruker ser file to NMRPipe format- enter all needed parameters of the experiment for each dimension##### Acquisition dimension {1H}:- MAC = run a macro to separate the upfield and downfield components (see below (*))- POLY = polynomial baseline correction (time domain) to subtract low-frequency solventsignal in the FID

- SP = window function (sine-bell)- ZF = zero-filling- FT = Fourier transform

14

- PS = phase correction- EXT = extract a region from the 1H dimension##### 2nd dimension {15N}:- TP = exchange vectors from X- to Y-axis of the data stream to process the 2nd dimension asX-vectors

- SP = window function (sine-bell “SP”)- ZF = zero-filling- FT = Fourier transform- PS = phase correction- TP, POLY, TP = add a polynomial baseline correction (frequency domain) in the 1Hdimension

- “-out” = write data into a file

conv.com#bruk2pipe -in ../ser -DMX -decim 16 -dspfvs 10 \

-xN 1024 -yN 400 -zN 0 \-xT 512 -yT 100 -zT 0 \-xMODE Complex -yMODE Complex -zMODE Complex \-xSW 9259.25926 -ySW 1666.6667 -zSW 0 \-xOBS 600.14076018 -yOBS 60.81855 -zOBS 0 \-xCAR 4.63 -yCAR 116.50 -zCAR 0 \-ndim 2 -aq2D States -verb -noswap -ov \

| nmrPipe -fn MAC -macro kwrk$1.M -noRd -noWr -verb –all \-out B$1.DAT -ovexit 0

xy.com#nmrPipe -in B$1.DAT -fn POLY -time \| nmrPipe -fn SP -off 0.33 -end 0.95 -pow 2 -c 0.5 \| nmrPipe -fn ZF -size 2048 \| nmrPipe -fn FT \| nmrPipe -fn PS -p0 -212.8 -p1 224 -di \| nmrPipe -fn EXT -x1 6.0ppm -xn 10.3ppm -sw \| nmrPipe -fn TP \| nmrPipe -fn SP -off 0.333333 -end 0.9 -pow 1 -c 1.0 \| nmrPipe -fn ZF -size 2048 \| nmrPipe -fn FT -verb \| nmrPipe -fn PS -p0 -90 -p1 180 -di \| nmrPipe -fn TP \| nmrPipe -fn POLY -ord 1 -auto \| nmrPipe -fn TP \-out C$1.DAT -ovexit 0

(*) The macro to separate the upfield (kwrk1.M) and downfield (kwrk2.M) components combines

the first 4 fids in the following way:

{1} + {2} + {3} - {4}

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{1} + {2} - {3} + {4}, negate ℑm

• kwrk1.M

/****************************************************************//* kwrk1.M: *//****************************************************************/

if (sliceCode == CODE_INIT) { (void) printf( "ySize %3.0f \n", ySize ); (void) setParm( fdata, NDSIZE, ySize/2, CUR_YDIM ); };

if (sliceCode % 4 ) { exit( 0 ); };if (sliceCode < 0 ) { exit( 0 ); };

float rdata1[wordLen*size], idata1[wordLen*size],rdata2[wordLen*size], idata2[wordLen*size],rdata3[wordLen*size], idata3[wordLen*size],rdata4[wordLen*size], idata4[wordLen*size],sumR[wordLen*size], sumI[wordLen*size],sumR1[wordLen*size], sumI1[wordLen*size],sumR2[wordLen*size], sumI2[wordLen*size];

(void) dReadB( inUnit, rdata1, wordLen*size );(void) dReadB( inUnit, idata1, wordLen*size );(void) dReadB( inUnit, rdata2, wordLen*size );(void) dReadB( inUnit, idata2, wordLen*size );


(void) vvCopy( sumR1, rdata1, size );(void) vvCopy( sumI1, idata1, size );(void) vvAdd( sumR1, rdata2, size );(void) vvAdd( sumI1, idata2, size );

(void) vvCopy( sumR2, rdata3, size );(void) vvCopy( sumI2, idata3, size );(void) vvSub( sumR2, rdata4, size );(void) vvSub( sumI2, idata4, size );

(void) vvCopy( sumR, sumR1, size );(void) vvCopy( sumI, sumI1, size );

16

(void) vvAdd( sumR, sumR2, size );(void) vvAdd( sumI, sumI2, size );

(void) vvSub( sumR2, sumR1, size );(void) vvSub( sumI2, sumI1, size );

(void) vNeg( sumI2, size );

(void) dWrite( outUnit, sumR, wordLen*size );(void) dWrite( outUnit, sumI, wordLen*size );(void) dWrite( outUnit, sumI2, wordLen*size );(void) dWrite( outUnit, sumR2, wordLen*size );

• kwrk2.M

/****************************************************************//* kwrk2.M: *//****************************************************************/

if (sliceCode == CODE_INIT) { (void) printf( "ySize %3.0f \n", ySize ); (void) setParm( fdata, NDSIZE, ySize/2, CUR_YDIM ); };

if (sliceCode % 4 ) { exit( 0 ); };if (sliceCode < 0 ) { exit( 0 ); };

float rdata1[wordLen*size], idata1[wordLen*size],rdata2[wordLen*size], idata2[wordLen*size],rdata3[wordLen*size], idata3[wordLen*size],rdata4[wordLen*size], idata4[wordLen*size],sumR[wordLen*size], sumI[wordLen*size],sumR1[wordLen*size], sumI1[wordLen*size],sumR2[wordLen*size], sumI2[wordLen*size];



(void) vvCopy( sumR1, rdata1, size );(void) vvCopy( sumI1, idata1, size );(void) vvSub( sumR1, rdata2, size );

17

(void) vvSub( sumI1, idata2, size );

(void) vvCopy( sumR2, rdata3, size );(void) vvCopy( sumI2, idata3, size );(void) vvAdd( sumR2, rdata4, size );(void) vvAdd( sumI2, idata4, size );

(void) vvCopy( sumR, sumR1, size );(void) vvCopy( sumI, sumI1, size );(void) vvAdd( sumR, sumR2, size );(void) vvAdd( sumI, sumI2, size );

(void) vvSub( sumR2, sumR1, size );(void) vvSub( sumI2, sumI1, size );

(void) vNeg( sumR, size );(void) vNeg( sumI, size );(void) vNeg( sumI2, size );

(void) dWrite( outUnit, sumR, wordLen*size );(void) dWrite( outUnit, sumI, wordLen*size );(void) dWrite( outUnit, sumI2, wordLen*size );(void) dWrite( outUnit, sumR2, wordLen*size );

3.5 Fitting of the alignment tensor

3.5.1 Exercise

The following Matlab script (linearfit.m) allows the determination of the alignment tensor

(Az, rhombicity, ...) from measured dipolar coupling data and a known structure. The fitting routine

is a linear algorithm exploiting the fact that the couplings depend in a linear way on the alignment

tensor. This is described in some detail in ‘Moltke, S. and S. Grzesiek, Structural constraints from

residual tensorial couplings in high resolution NMR without an explicit term for the alignment

tensor. J Biomol. NMR, 1999. 15(1): p. 77-82.’

Try to understand the matlab script and run this script on suitable data.

3.5.2 Matlab Script

• linearfit.m

clear allcosfile = ['coord.f'];jfile = ['dipolar.f'];

18

valjerri=1;global j_res xyzi jmesi jerri jnb nfree_ax nfree_asym ;declare(cosfile,jfile,valjerri)

for ii = 1:jnb pol = cart2pol( xyzi(ii,:)); xdata(ii,1:5) = y2( pol(2), pol(3));end

yvec = xdata'*jmesi; % linear fitxmatrix = xdata'*xdata;avec = xmatrix\yvec;ytheo = real(xdata*avec)';

yerrs = ones(size(jmesi))*valjerri;diff=(ytheo'-jmesi)./yerrs;chisq = diff'*diff/nfree_asym;

stt = clebshg5to3( avec ); % 5*1 vector to 3*3 matrix

[v,d] = eig(stt); % matrix diagonalisation to get Axx,Ayy,Azzv = real(v);d = diag(real(d));[dd i] = sort(abs(d));d = d(i);v = v(:,i);

Az = d(3);Rhomb = (Ax-Ay)/Az;

19

3.6 References

Residual dipolar couplings:

1. Tjandra, N., and Bax. A., Science 278, 1111–1114 (1997).2. Saupe, A. and Englert, G., Phys. Rev. Lett. 11, 462–465 (1963).3. Bothner-By, A.A., Domaille, P.J. and Gayathri, C., J. Am. Chem. Soc. 103, 5602–5603 (1981).4. Tolman JR, Flanagan JM, Kennedy MA, Prestegard JH., Proc Natl Acad Sci U S A;

92(20):9279-83 (1995).5. Tjandra N, Grzesiek S, and Bax A, J. Am. Chem. Soc. 118; 6264-6272 (1996).6. Moltke, S. and S. Grzesiek, J Biomol. NMR, 1999. 15(1): p. 77-82.

15N-1H dipolar splitting experiments:

1. Ottiger, M., Delaglio, F., and Bax, A., J. Magn. Reson. 131, 373–378 (1998).2. Cordier, F., Dingley, A.J., and Grzesiek, S., J. Biomol. NMR, 13, 175-180 (1999).

Aligning media:

• purple membrane alignment:

1. Koenig BW. Hu JS., Ottiger, M., Bose S., Hendler RW. and Bax A., J. Am. Chem. Soc., 121;1385-1386 (1999)

2. Sass, J., Cordier, F., Hoffmann, A., Rogowski, M., Cousin, A., Omichinski, J., Löwen, H., andGrzesiek, S., J. Am. Chem. Soc., 121, 2047-2055 (1999).

• filamentous phage alignment:

3. Hansen MR, Mueller L and Pardi A., Nat Struct Biol, Dec; 5(12):1065-74 (1998).4. Clore GM, Starich MR, and Gronenborn AM., 120; J. Am. Chem. Soc., 10571-10572 (1998).5. Zweckstetter M, Bax A., J Biomol NMR, 18(4):365-377 (2001)

• stressed Polyacrylamide-gel alignment:

6. Tycko R. , Blanco F., and Ishii Y., J. Am. Chem. Soc., 122(38); 9340-9341 (2000).7. Sass HJ., Musco G., Stahl SJ., Wingfield PT. and Grzesiek S., J Biomol NMR, Dec;18(4):303-9

(2000).

• bicelle alignment:

8. Tjandra, N., and Bax. A., Science 278, 1111–1114 (1997).9. Ottiger M., Bax A. J Biomol NMR, Feb;13(2):187-91 (1999).10. Ottiger M., Bax A. J Biomol NMR, Oct;12(3):361-72 (1998).

20

4 Practical: Relaxation (eventually as part of P7)


1. Sklenar, V.; Bax, A.J. Magn. Reson. 1987, 74, 469-479.

2. Kay, LE; Nicholson, LK; Delaglio, F; Bax, A; Torchia, D; J. Magn. Reson. 1992, 97, 359-375.

4.1 Proton T2 measurement with the oneone echo experiment (water suppression)

In a normal proton 1D experiment, the protein signals (~ 1mM) are completely obscured by the

water signal (concentration of water protons 2* 95% *(1 kg/l) / (18 g/mol) = 105 mol/l = 105 M).

This is 105 times more than the concentration of a single protein proton. In order to see the protein

signals we have to suppress the water signal. One of these methods, is the so-called jump and return

or oneone sequence. In its simplest form it consists of two pulses and the aquisition:

90˚x - delay (d7) - 90˚-x - acquire

The delay d7 is set to ~100 µs. The carrier (reference) frequency is set to the water frequency. The

first 90˚x pulse flips the water signal from the z-axis to the -y axis. During the delay d7 nothing

happens for the water spins in the rotating (carrier) frame, because this frame rotates at exactly the

same speed as the water around the z-axis. Therefore, in this frame the water signal stays along the -

y-axis. The second 90˚-x-pulse flips this magnetization back to the z-axis. No water signal should be

observed in the receiver.

Now consider the fate of a proton spin whose frequency is off by 2.5 kHz (i.e. 2500/600 = 4.16

ppm) from the water (typically the water resonance is close to 4.7 ppm). After the 90˚x-pulse, this

spin starts to rotate around the rotating reference frame with an angular speed of 2500 revolutions

per second. After d7 = 100 µs, this spin has rotated by 100 µs * 2500 Hz = 0.25, i.e. a quarter of a

full circle around the z-axis. This means that the spin has moved from the -y-axis to the x-axis. The

second 90˚-x does not change the direction of this spin. Therefore its signal is observed in the

receiver. For spins with other offset frequencies only some part of the magnetization is along the x-

axis before the second pulse. Therefore only part of their signal is observed. The excitation profile

looks like a sine function with a maximum at an offset of 2.5 kHz and a zero crossing at an offset of

0 Hz, i.e. at the water frequency. By applying this sequence, we can strongly suppress the water

signal, but keep parts of the protein signals, e.g. the amide protons at 8-9 ppm.

The oneone experiment can be extended to a oneone echo sequence

21

90˚x-delay (d7)-90˚-x-delay (d4) 90˚x-delay (2*d7)-90˚-x delay (d4) acquire

Here the second set of 90˚ pulses sandwiches a delay 2*d7. This pulse-delay-pulse combination

acts like a selective 180˚ pulse (sine excitation profile) with no excitation of the water. The pulse

sequence therefore acts like

90˚(selective) delay (d4) 180˚(selective) delay (d4) acquire

The selective 180˚ pulse reverses the protein spins which have precessed during the first delay d4,

such that at the end of the second delay d4 all the selectively excited magnetization is back along the

-y axis. This is the general principle of the spin echo (just with suppression of the water

resonance):

Fig.: Harris p 83.

An initial 90˚ pulse turns the equilibrium magnetization into the xy-plane. The magnetization

vectors from different nuclei then begin to fan out (b) and the signal decays. After a time delay d4

(time point c), a 180˚y pulse is applied which has the effect of rotating all the magnetization vectors

about the y-axis, or in other words reflecting them in the zy-plane. After this the spins continue to

move in the same direction (d), and after a further time d4 (e) they are again pointing in the y-

direction, and the signal in the receiver coil is maximal.

The signal in the receiver is proportional to exp(-d4*2/T2). Therefore the oneone echo sequence can

be used to measure T2, by aquiring two data sets with different settings for d4 (typically 100 µs

22

and 2.9 ms). The ratio of the signals in the two experiments I1/I2 is then given by exp(-2*100 µs

/T2)/ exp(-2*2.9 ms /T2) = exp( 5.6 ms/T2). T2 is calculated as 5.6 ms/ln(I1/I2).

For typical macromolecules (MWT > 6 kDa) in aqueous solution, the slow tumbling limit is a good

approximation and all T2 are inversely proportional to the molecular tumbling time τc (in the

absence of chemical exchange broadening). This means that T2s of various backbone nuclei should be

proportional to each other. Therefore, an estimate for these T2s can be derived if only the T2 of the

amide proton is known:

N

H

C

OH

γ

C C

H C

α

β

68

N

H OH

C

H

αC C N

H

C

α

β

14

8

5242

13

6

H H

slow tumbling limit:

1/T2 ~ J(0) ~ τc ~ MWT

Typical T2-values (ms) for a (τc ~ 15 ns) protein(MWT ~ 30 kDa)

140

35

1192 55 15 140

-7

11

3 - 10

J-values (Hz) of a protein

J T2 << 0.5 => no efficient transfer

1JNCα T2(Cα) = 0.15 !! for 30 kDa

H

Fig.: T2-values for a 30 kDa protein and J-couplings of a protein.

23

4.1.1 Pulse Program

11ee:#include "bits.sg"

#define PROTEIN#ifdef PROTEIN;"d7=85u"#else;"d7=50u"#endif

1 ze 10u pl31:N2 10u LOCK_ON d1 pl1:H 10u 10u p1 ph1 ;first 90 degree pulse d 7 p2 ph2:r ;second 90-degree pulse, slightly different length for optimal water

;suppression d8 ;unimportant small delay 98u ;compensates for 10+88u on the other side of the 180 degree pulse d 4 ;first spin echo delay d4 p1 ph3 ;combination of pulse delay pulse for selective 180 degree pulse d 7 d 7 p2 ph4:r 10u 88u LOCK_OFF d4 ph0 ;second spin echo delay d4 go=2 ph31 cpd2:N ;acquisition 10u do:N wr #010u LOCK_ONexit

ph0=0ph1=0 1 2 3ph2=2 3 0 1ph3=0 0 0 0 1 1 1 1 2 2 2 2 3 3 3 3ph4=2 2 2 2 3 3 3 3 0 0 0 0 1 1 1 1ph31=0 3 2 1 2 1 0 3

4.1.2 Demonstration of the T2-Measurement with the Oneone Echo Sequence

1. We set the length of the proton pulses to the measured values for the 90˚ pulse and d4 to 100 µs,

acquire, and transform the signal.

p1 xx

p2 xx

d4 100u

zg

qfp

24

We see a protein spectrum with a sine modulated excitation. We keep these data for the next

experiment.

2. We make a copy of the data set, set the length of d4 to 2.9ms, acquire, and transform the signal.

d4 2.9m

zg

qfp

3. We compare the intensity ratios of the first and second experiment and calculate T2 for the amide

protons (should be between 15 and 20 ms).

4.2 15N T2 1D

Exercise: Estimate the 15N T2 of the sample of ubiquitin with a series of 1D by changing the loop

counter in the relaxation delay T=d22*16*loop, with d22=0.5m [use the pulse program “t2n15.sg”

(Kay et al., 1992) and try to understand it].

4.2.1 Pulse Program

t2n15.sg:

#include "bits.sg"

#define ONE_D;#define TWO_D;#define FLIP_TEST#define FLIP_BACK#define CARBON_LABEL#define INTERLEAVED

;Gradient pulses"p10=2.5m" ;at gp0=+50%"p11=1m" ;at gp1=-50%"p13=400u" ;at gp0=+50%

;p1 proton 90 at pl1, 9u;p2 1ms proton 90 at pl2 ;sklenar

"p5=53u";carbon pulse at pl5

;p7 high power n15 90 on N pl7

25

"p8=500u" ;high power n15 SL on N pl8;p31 low power n15 90 (160us) on N at pl31

;nitrogen evolution:"d9=4u""d10=2.7m""d0=d9+d10+p7*0.637"

;in0=d0/(l3+1);in9=in10-in0;in10=1/(2sw)

"d4=2.25m" ;hsq h to n15

"d7=p7*0.637""d17=p1*2.0"

"d11=50m""d12=10m""d22=500u""d23=d22-p7""d24=d23-p1""d26=p7-p1""d27=p7-p23""d28=p5"

#define ON#undef OFF

1 ze2 d11*2 1m d12#ifdef INTERLEAVED21 d12#endif3 d12*5.04 10u do:C1 do:N 10u pl5:C1 10u pl7:N 10u pl1:H#ifdef ON d1 LOCK_ON 1m LOCK_OFF;***** start 90-degree on h-n ***** (p1 ph0) d4 (p7*2 ph6):N (d26 p1*2 ph4) d4;***** hsqc to nitrogen ***** (p1 ph6) 8u p10:gp0 2.5m pl1:H (p7 ph3):N 2.7m (p7*2 ph0):N (d26 p1*2 ph4) 2.7m

26

d7;***** n15 relaxation delay *******70 d23 (p7*2 ph8):N d23 d23 (p7*2 ph8):N d23 d23 (p7*2 ph8):N d23 d23 (p7*2 ph8):N d24 (p1*2 ph4) d24 (p7*2 ph8):N d23 d23 (p7*2 ph8):N d23 d23 (p7*2 ph8):N d23 d23 (p7*2 ph8):N d23#ifdef INTERLEAVED lo to 70 times c#else lo to 70 times 5#endif;rel. delay = d22*16*c; (p8 ph4):N;***** n15 evolution delay ******* d0#ifdef CARBON_LABEL 4u 20u d28*2.0 4u 20u d28*2.0#else d17#endif (p7*2 ph10):N d9#ifdef CARBON_LABEL 4u 20u fq2:C1 (p5*2 ph10):C1 (p1*2 ph0) 4u 20u fq2:C1 ;jump to 177ppm (p5*2 ph10):C1#else (p1*2 ph0)#endif

27

d10 (p7 ph7):N 2u p11:gp1 2m (p1 ph0) p13:gp0 950u pl2:H (p2 ph14:r) 2u 5u pl1:H (p1*2 ph15) 2u 5u pl2:H (p7*2 ph10):N (p2 ph14:r) 2u p13:gp0 (2u ph0) 877u pl31:N#endif#ifdef ONE_D go=2 ph31 cpd2:N 1m LOCK_ON d11 wr #0 d11 do:N#endif#ifdef TWO_D go=2 ph31 cpd2:N 1m LOCK_ON d11 wr #0 if #0 zd d11 do:N#ifdef INTERLEAVED d12 ivc lo to 21 times 6;number of entries in vc list#endif d12 ip7 ;nitrogens lo to 3 times 2 d12 dd0 d12 id9 d12 id10 d12 ip31 d12 ip31lo to 4 times l3#endif#ifdef FLIP_TEST 1m LOCK_ON; lo to 2 times 20 d1

10u pl1:H p1 ph31

(2u ph0)go=1 ph31d11 wr #0#endif

28

d12 do:C1d12 do:Nexit

ph0=0ph1=0ph3=0 2ph4=1ph6=1ph7=0ph8=PHASE_4( 0 ) PHASE_4( 2 )ph10=0ph14=2 2 0 0 ; adjust phcor14ph15=0 0 2 2ph31=0 2

4.3 15N T2 2D

A 15N T2 experiment (2D) has been recorded previously on a sample of the NS3 protease of the

Hepatitis C Virus (21.6 kDa), using the pulse program “t2n15.sg” with the flag TWO_D instead of

ONE_D. The experiments have been recorded in an interleaved manner with 6 relaxation delays.

The time domain data (ser file) is located in the directory XXXXX. The NMRPipe scripts

(conv.com, p.com) are given below with a description in italic (only p.com has to be run). Try

to understand those scripts. Do the processing stepwise and look at the result in nmrDraw.

4.3.1 NMRPipe scripts (conv.com, p.com)

- bruk2pipe = conversion of bruker ser file to NMRPipe format-xN, ..., aq2D, ... = all needed parameters of the experiment for each dimension

• Acquisition dimension {1H}:- COADD -cList $argv[2-7] = select 1 row over 6 to process the 2D for one relaxation delay(i.e conv.com 1 1 0 0 0 0 0 for the 1st relaxation delay, conv.com 2 0 1 0 0 0 0 for the 2nd, ...)

- POLY = polynomial baseline correction (time domain) to subtract low-frequency solventsignal in the FID

- SP = window function (sine-bell)- ZF = zero-filling- FT = Fourier transform- PS = phase correction- EXT = extract a region from the 1H dimension

• 2nd {13CA,13CB}:

29

- TP = exchange vectors from X- to Y-axis of the data stream to process the 2nd dimension asX-vectors

- SP = window function (sine-bell “SP”)- ZF = zero-filling- SIGN -i = reverse frequency domain by negating the imaginary data (only if needed)- FT = Fourier transform- PS = phase correction- “-out C$argv[1].DAT” = write data into a file for the current relaxation delay (indicated byargv[1])

conv.com#t2n15 interleaved#bruk2pipe -in ../ser -DMX -decim 16 -dspfvs 12 \ -xN 1536 -yN 1200 -zN 0 \ -xT 768 -yT 100 -zT 0 \ -xMODE Complex -yMODE Complex -zMODE Complex \

-xSW 9259.2592 -ySW 1666.6667 -zSW 0 \ -xOBS 600.14076753-yOBS 60.81855 -zOBS 0 \ -xCAR 4.658 -yCAR 116.48 -zCAR 0 \ -ndim 2 -aq2D States -verb -noswap -ov \| nmrPipe -fn COADD -cList $argv[2-7] -axis Y -time \| nmrPipe -fn POLY -time \| nmrPipe -fn SP -off 0.33 -end 0.95 -pow 2 -c 0.5 \| nmrPipe -fn ZF -size 1536 \| nmrPipe -fn FT \| nmrPipe -fn PS -p0 -86.2 -p1 0.0 -di \| nmrPipe -fn EXT -x1 6.2ppm -xn 11.3ppm -sw \| nmrPipe -fn TP \| nmrPipe -fn SIGN -i \| nmrPipe -fn SP -off 0.35 -end 0.95 -pow 1 -size 100 -c 0.5 \| nmrPipe -fn ZF -size 512 \| nmrPipe -fn FT \| nmrPipe -fn PS -p0 4.0 -p1 0.0 -di \-out C$argv[1].DAT -ovexit 0

p.comcsh conv.com 1 1 0 0 0 0 0csh conv.com 2 0 1 0 0 0 0csh conv.com 3 0 0 1 0 0 0csh conv.com 4 0 0 0 1 0 0csh conv.com 5 0 0 0 0 1 0csh conv.com 6 0 0 0 0 0 1

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