P1&P2

EMBO Practical Course:
Structure, Dynamics and function of biomacromolecules by solution NMR

Biozentrum, Basel, July 20-27 2013
Practical: Heteronuclear NMR
Pulse and parameter calibration

Basic heteronuclear NMR experiments
Bernd Simon & Michael Sattler

EMBL Heidelberg
Helmholtz Zentrum & Technische Universitt Mnchen
EMBO Practical Course: Structure determination of biological macromolecules by solution NMR
Heteronuclear NMR Practical
Table of contents:
1. Introduction ............................................................................................................................ 3
2. Bruker software commands and our pulse program conventions .......................................... 4
Pulse phases ........................................................................................................................ 5
Shaped pulses ...................................................................................................................... 6
Delays ................................................................................................................................. 6
Gradients ............................................................................................................................. 6
Example: 1D pulse program ............................................................................................... 7
Processing ........................................................................................................................... 8
3. Calibration of frequency offset, pulses, gradients etc. ........................................................... 9
Pulse calibrations .............................................................................................................. 10
Calibration of the frequency offset and the power level for the proton pulse .................. 10
Calibration of nitrogen pulse ............................................................................................ 12
Calibration of carbon pulses ............................................................................................. 13
Calibration of deuterium pulse ........................................................................................ 16
Calibration of water-flip-back pulse ................................................................................. 16
Calibration of soft pulse water-gate .................................................................................. 17
Echo/Antiecho gradient calibration .................................................................................. 18
Calibration of adiabatic pulses .......................................................................................... 19
4. 1D & basic 2D pulse sequences (heteronuclear correlations) .............................................. 20
1D experiments ................................................................................................................. 23
1H,15N HSQC (Sensitivity-enhanced, watergate, SOFAST) ............................................ 23
1H, 15N TROSY: ............................................................................................................... 24
1H,13C correlations ............................................................................................................ 28
Isotope Filters ................................................................................................................... 31
Estimation of amide proton T2 times ............................................................................... 31
Bruker standard pulse programs (3D pp) .......................................................................... 31
2
1. Introduction
The practical will be done during two sessions, 2h30 each. The first part concerns basic
steps for heteronuclear triple resonance experiments, e.g. determination of pulses (rectangular &
shapes), phases, gradients, etc. During the second part, some basic heteronuclear 2D
experiments will be set up and compared (e.g. HSQC, HMQC, TROSY).
The different NMR experiments and pulse programs are stored in the data sets indicated
below. For each data set, you have a brief description of the pulse program showing what the
pulse program does and which are the important parameters to be set up and checked. Copies of
the pulse programs are included. Graphical representations for many of the pulse programs can
be found in the lecture notes.
Note that the experiments together with the discussions easily go over the time limit
of the practical session. The intention is to guide you towards a critical evaluation of
elements of a pulse sequence rather than to quickly cover all experiments in the handout.
So take your time and discuss!
NOTE:
A very brief overview of the most important parameters and commands used on Bruker
spectrometers is provided in the next section and should be read before the practical.
3
2. Bruker software commands and our pulse program conventions
There are 4 channels (f1, f2, f3 and f4) on the NMR spectrometers: f1 for protons, f2 for
carbon, f3 for nitrogen and f4 for deuterium (it can be checked with the command edasp in
TOPSPIN).
Basically, the pulse program is executed line by line. When a line is finished the
program continues on the next line. When two commands are written on one line they start both
at the same time. The line lasts as long as the longest command on that line. The pulse program
text file is visible when clicking on the PulProg tab. A more graphical representation is obtained
by clicking on the pulse icon in the PulProg window, executing spdisp in TOPSPIN or running
nmrsim showp (name) in a shell.
Power levels
Power levels are either specified directly in Watt plw[0-64] or as attenuation of the
maximum power for a given channel pldb[0-64] (in dB). (note that topspin is not case
sensitive, e.g. Plw1,PLW1,pLw1 are all the same)
Example: pl1:f1 sets up the power level to the value pl1 on channel 1 (1H).
Pulse flip angles are proportional to the B1 field strength as given below:
= 1 p= B1 p
Radio frequency power levels are adjusted by attenuating the maximum amplifier power,
typically 100W/300Wor500W for 1H/X nuclei. On Bruker spectrometers power levels P are
therefore measured directly in Watt or as attenuation in the logarithmic decibel (dB) scale:
P = Pref 10dB/10
The attenuation difference between two power levels (P1, P2) is then given by:
dB= 10 log10(P1/P2)
Note, that rf power (and sample heating!!) is proportional to the square of the voltage:
P = V2/R
with R being the resistance, usually 50 in a probe.

Therefore, the voltage, B1 and the pulse flip angles scale as:
dB = 20 log10(V1/V2)
Thus, attenuation by 6.02 dB reduces the power to and the pulse flip angle by 2; or requires
doubling of the pulse width ( p) to obtain the same flip angle. At the same time this corresponds
to depositing of rf power (and thus heating) in the sample.
4
Shaped pulses are scaled in a similar way; however, the amplitude scales with the integral over
the rf shape. This can be conveniently calculated with the TOPSPIN module called stdisp (which
starts the shape tool).
Usually the amplifiers are linearized during the installation of the spectrometer. The real output
for each channel is measured and the phase and power are corrected using the cortab table to
produce the theoretical output. Thus power levels of all hard and shaped pulses can be
calculated if the pulse length at one power level is known. From this one can use the automation
calcpowlev in TOPSPSIN or the shell script db in a Unix Shell.
Remarks:
- On Bruker spectrometers the lower the dB, the higher the power (this is
opposite to the Varian convention!).
- Starting with topspin3 the power and attenuation levels are defined
independent of the routing. So if the cortab is set correctly, the physical
attenuation on the corresponding channel (SGU) is set automatically. The
attenuation on the input level is defined by setting 0 dB = 1 W (topspin 3), while
in earlier topspin versions the attenuation specifies the attenuation on the
corresponding SGU (with -6 dB corresponding to full power or 1Vpp on the
SGU output).
ALWAYS CHECK THE POWER LEVEL LIMITS, THE PULSE DURATION AND THE
ACQUISITION TIME ON THE SPECTROMETERS, DONT BURN THE PROBE!!
If you have activated the Powercheck in topspin (see e.g. POWCHK in the status line
of topspin) the program checks if the maximum allowed power on any channel
exceeds the specification of the probe and does not start acquisition if any power is out
of limit. However, the power deposition is not calculated, so you still can burn the
probe by giving full power for long times.
Pulses
Rectangular pulses are p[0-31]
We use the following nomenclature:
1
p1 H 90
1
p2 H 180
13
p3 C 90
13
p4 C 180
15
p5 N 90
15
p6 N 180
1
p12 H 90 selective shape
p16 and p19 Pulsed field gradient
1
p29 H presaturation
Example: p1:f1 (means a 90 deg pulse on protons)
Pulse phases
Pulses can be given a certain phase (ph) (if nothing is specified, x is used)
5
Example: p2 ph7:f1 (means a 180 deg hard pulse on protons with phase 7)
Phases are defined at the bottom of the pulse program.

Example: ph7 = 0 2 (means ph7 is cycled between 0 and 2)
where: 0 = x, 1 = y, 2 = -x, 3 = -y
Phases can be incremented during the pulse program with ip

Example: 10m ip3 increases phase3 by 90 deg at the start of a 10 ms delay
Shaped pulses
Shaped pulses power levels are indicated with sp# (there is sp0 to sp31) and come with
some variables including:
spnam = the name of the shaped pulse

spoffs = the offset of the pulse
We use the following convention:
p13:sp1 90 on resonance
p13:sp2 90 time reverse on resonance
p14:sp3 180 on resonance
p14:sp4 180 off resonance
p13:sp5 90 off resonance
p13:sp6 90 time reverse off resonance
(NOTE: normally on resonance is on C and off resonance is on C)
In the previous table, the pulse length is defined by p# and the power level is defined by sp#
Additionally, the phase needs to be defined: e.g (p14:sp4 ph0):f2 is a shaped pulse with name
spnam4, offset spoffs4, power level sp4 and length p14, having a phase of ph0 (a 180 deg on the
C' when o2p is at 40ppm and spoffs is set accordingly)
Delays
Delays are indicated by d# (there is d0 to d63) and can either be given in the dataset (in
XWINNMR) or calculated on top of the pulse program.
Examples: "d6=9.0m" or "d30=d6+6u-d7+p13*2"
A delay can also be written directly in the pulse program (e.g 20u or 10m) and can be
incremented or decremented during the pulse program with id# and dd# e.g. id10 means
the delay d10 is incremented with value in10.
Gradients
Gradients can be rectangular or shaped. A rectangular gradient has a constant strength
during its execution whereas a shaped gradient has a variable length.
Rectangular gradients are created with the gron# statement; the groff statement switches
off all gradients that were switched on by a gron# command.
6
Example: 200u gron0

100u groff
The command lines above switch on the gradient defined by gpx0, gpy0 and gpz0, at the
beginning of a 200 s delay and leave it active for a period of 200 s. It is switched off
with the beginning of the line containing the command groff.
Shaped gradients are called gp# and come with gpnam# (the name of the gradient) and
with p# which gives the length of the gradient. The strength of the rectangular and
shaped gradients is given by gpx#, gpy# and gpz# for the three dimensions (gp# goes
from 0 to 100%)
Example: p16:gp3 (is a gradient of length p16, with name gpnam3 and strength
gpx3,gpy3 and gpz3)
Gradients can be inverted using the following trick:

p16:gp1*EA
igrad EA
where EA is a list containing 1 and 1. The command igrad gives EA the next value in
the list. (Used for echo/antiecho type of experiments)
Example: 1D pulse program

The simplest case: a 1D 1H spectrum without solvent presaturation.
PULSE PROGRAM |EXPLANATION:

;EMzg Some comments
;avance-version
;1D sequence with f1 presaturation
#include <Avance.incl> Import nomenclature and definitions from file Avance.incl
1 ze Start (zero buffer)

2 30m 30ms delay
d1 pl1:f1 d1 delay (1s); power level for 1H to pl1 on f1 channel
p1 ph1 Pulse on 1H of length p1*2 and phase ph1 (see below)
go=2 ph31 go is a macro command for acquisition, receiver opening with phase ph31, jump to label 2
d11 wr #0 d11 delay and write fid to disk
exit end
ph1=0 2 2 0 1 3 3 1 ph1 is cycled as: x,-x,-x,x,y,-y,-y,y1 (incremented every scan)

ph31=0 2 2 0 1 3 3 1 ph31 receiver phase
;set Some remarks

;pl1 : f1 channel power level for pulse
;p1 : f1 channel high power pulse
;d1 : relaxation delay; 1-5 * T1
;NS: number of scans
What also has to be set up in the dataset (in TOPSPIN) in this case?
1. offset for protons: o1 (in Hz) or o1p (in ppm) (e.g. on resonance at solvent signal!)
2. number of scans: ns (multiple of the phase cycle)
3. number of dummy scans: ds
4. receiver gain: rg (the higher the number the more sensitive)
1
The CYCLOPS phase cycling was designed to cancel out imperfections associated with errors in the x and y
phase detectors.
7
Exercise:
Get an overview of essential parameters that have to be set from ased and eda in TOPSPIN.
Processing
An outline of the processing parameters can be found with edp.
1D spectra can be processed using the following commands:
ft = Fourier transform
fp = ft + use predefined phases (see edp)
efp = fp + exponential window function (line broadening is defined by lb)
gfp = fp + Lorentzian-to-Gaussian window function (defined by lb and gb)
2D spectra are processed with the statements below:

xfb = process both dimensions
xfb n = process in both dimensions and keep only real data
xf2 = process dimension F2
xf1 = process dimension F1
abs1 = baseline correction in F1
abs2 = baseline correction in F2
rser # = extract 1d number # from a 2D, can be further processed as a 1D
8
3. Calibration of frequency offset, pulses, gradients etc.
During this session the calibration of 1H, 15N, 13C and 2H pulses will be explained.
Change to directory "cal 1 1 embo nmr" by typing emcal in TOPSPIN (emcal is a user defined
TOPSPIN macro; it can be displayed with edmac emcal)
Standard procedures before you can start: Temperature, Wobble, Lock, Shim
Before we can start the experiment we have to put the sample in the magnet and perform some
standard procedures. These standard settings are very important to get high quality spectra.
First check the sample temperature using the command edte. This opens a window with several
icons to define different parameters to stabilize the sample temperature during the experiment.
The adjustments are achieved by a feedback loop, which measures the Temperature in the
sample chamber and regulates the power of a heater to warm up the cold nitrogen gas that flows
at a constant defined flow rate through the probe chamber. The reaction time and amplitude of
the heater change upon a change of temperature is determined by additional parameters, which
are automatically determined by the self-tune procedure. These parameters have to be adjusted if
you change the Temperature by more than ~5deg, change the flow rate or turn the BCU on or
off. On cryoprobes it is important to keep the flow rate relatively high and avoid heater power
above 15%. The absolute Temperature of the sample can be calibrated using a chemical shift
thermometer. (for cryoprobes use very diluted samples to avoid radiation damping, e.g. 99.8%
Methanol d4)
After inserting the sample, we have to tune and match the resonance circuit. The tune and
match should be checked for all channels in use, starting from the least sensitive nucleus. The
procedure is started with the command wobb. We see the rf reflection profile of the channel that
is selected in edasp. We start the adjustment always with the match, then continue with tune and
iterate until we have reach the optimum. The match should move the minimum of the reflection
curve to the x-axis and the tune should move the minimum to the center y-axis of the screen. Be
careful not to overwind the screws!!
The lock is a feedback loop that controls the slow frequency drift of the magnet by readjusting
the field strength if the resonance frequency of a NMR experiment changes. The lock
experiment is running constantly in the background and operates in our cases on the signal of
deuterated water. As for the temperature control, the feedback loop has certain parameters that
control how fast the correction follows a change in frequency. The lock is started by typing lock
and choosing the corresponding solvent. The command reads standard parameters for the chosen
solvent which are stored in a table accessible by the command edlock. There are parameters for
the lock power, lock phase and the loop time, loop filter and loop gain. The optimal lock power
can be determined via an automatic adjustment or manually by just checking at which power the
lock starts to saturate. For a given lock power the other relevant parameters are set by the
automation loopadj. If one uses regularly different H2O/D2O or other mixtures, the edlock table
can be extended to store the corresponding solvent.
The last procedure before starting an experiment is the homogenization of the magnetic field
across the sample by adjusting the current through the shim coils. This procedure is automated
by the program topshim, which you can start by topshim gui or the corresponding macro tg. The
program sets the axial shims in 1D mode and for water sample also all shims in an automatic
way. You can check the adjustments by clicking on the Report in the graphical user interface.
9
Pulse calibrations
Calibration of the frequency offset and power level for the proton pulse.
1 EMzg (see pulse program above)
Set p1 to 0.5 s and record a single scan experiment. Use H2O signal for calibrating
the 1H pulse and make sure the carrier is set up on resonance (o1) on the water
signal.
[1H pulse (p1, pl1) detection]
Use larger flip angles and try to determine 180 and 360 pulses.
Why does the spectrum look so funny for larger flip angles? Look at the FID of
the 180 pulse: How can this be used to calibrate the pulse?
2 EMzgpr
Calibration of the offset. (make sure water is on resonance)
gs modify o1
Is the calibrated o1 the same as in cal/1?
;EMzgpr Name of the pulse program

;avance-version
#include <Avance.incl> Import nomenclature and definitions from Bruker file Avance.incl
"d12=20u" Set up d12 to 20us
1 ze Start zero buffer

2 d12 pl7:f1 d12 delay to set up power level for 1H to pl7 (presaturation)
(p29 ph0):f1 ; presaturation at pldb7 +55 dB p29 1H pulse of phase ph0 (1s, it is also the relaxation delay)
d12 pl1:f1 d12 delay, set up power level on 1H channel to pl1
p1 ph1 Pulse p1 (90 on 1H) with phase ph1
go=2 ph31
d11 wr #0
exit
ph0=0
ph1=0 2 2 0 1 3 3 1
ph31=0 2 2 0 1 3 3 1
[presat (p29, pl7) 1H pulse (p1, pl1) detection]
Advanced procedure for quantitative 1D spectroscopy:
- Make sure that the pre saturation is in a steady state (generally: first nutation, then
saturation): depends on the relaxation properties of the water ~ 4-10 s
- Run the experiment with DS=0, NS=1, set phc1 = 0 and phase the right hand side of
the spectrum using phc0 only
- If the rest water resonance is dispersive with a dip on the right, the o1 is too small,
otherwise it is too large
- The residual water origins from the edges of the sample. Sequences with several
pulses and gradients can achieve better water suppression (e.g. a 1D noesy pr
sequence)
10
3 EMzgproff
Calibration of the 1H pulse
[off resonance presat (p18*10, sp0, spoffs0) 1H pulse (p1, pl1) detection]
Optimize p1 and pl1 for zero signal (180 pulse or 360 pulse?)
What do we change p1 or pl1 ?
Why do we use off-resonance presaturation? Which offset do we use?

;EMzgproff
;avance-version
; off resonance presat
#include <Avance.incl>
"d12=20u" Set up delays and/or pulses.

"p18=100m"
1 ze
2 d1 Delay of length d1 for interscan T1 relaxation
d12 pl7:f1 Set power level on 1H channel to pl7
3u
18 (p18:sp0 ph0):f1 ; squ.1000, 100m, 55 db Off-resonance presaturation
2u Check the spnam0, spoffs0, also coroffs (!) for processing
lo to 18 times 10 Loop (presat is p18*l0)
3u
10u pl1:f1
p1 ph1
go=2 ph31
50m wr #0
exit
ph0=0
ph1=0 2 2 0 1 3 3 1
ph31=0 2 2 0 1 3 3 1
In general you can optimize a parameter by running a series of experiments,

changing one parameter step by step. The command for this is paropt or the more
general popt. Before starting the optimization, you must run one spectrum, select a
spectral range for the optimization by zooming in the spectrum and store the region
with the command dpl1.
Calibrate the proton pulse using the automation pulsecal
How does the value compare to the value that you obtained before? The automation
creates a dataset 99999 where it executes the pulse program pulsecal. The
calibration is done in a single scan nutation experiment. Have a look at the pulse
program and the spectrum. How is the pulse length determined here?
11
Calibration of nitrogen pulse
4 EMinvp90f3
Calibration of 15N hard pulse
[Off resonance presat 90 1H delay of 1/(2JHN) 180o 1H 90 15
N 90 /180 on 15N delay
1/(2JHN) detection]
Optimize p4 and pl4 for pulse calibration.
Unclear? - make a little drawing of the pulse program and use the product operators.
Why use d22 (which is < d2)???

; EMinvp90f3
; for calibration of rectangular pulse on F3 (15N)
; p3 pl3: ~ 90deg
; p4 pl4: 1) 90deg: phase spectrum
; 2) 180/360deg: optimize for zero signal
; F1 1H 4.7ppm (H2O)
; F2 15N ~118ppm (or select amide with unique 1H shift in 1D)
"d2=5.4m" ; 1/2J(N,H)
"d22=d2-p3-p4-16u"
"p18=100m" ; for off resonance presaturation set SPOFFS0
"p2=p1*2"
1 ze
2 d1 do:f3
d12 pl7:f1
18 (p18:sp0 ph0):f1 ; spnam0=squ.1000, 100ms, sp0=pl7 loop
2u
lo to 18 times 10 ; off resonance presaturation
3u
20u pl1:f1
(p1 ph1):f1 90 1H
d2 pl3:f3 d2
(p2 ph3):f1 180 1H
(p3 ph2):f3 ; 90 deg 90 15N
3u
10u pl4:f3 change power level to pl4 (15N channel)
(p4 ph4):f3 ; 90/180 deg 90 (signal) or 180 (no signal)
3u
d22 pl13:f3 set decoupling power level
go=2 ph31 cpd3:f3 decoupling on
d11 wr #0 do:f3 writing data and switch decoupling off
exit
ph0=0
ph1=0
ph2=0 0 2 2
ph3=0
ph4=0 2
ph31=0 2 2 0
12
Calibration of carbon pulses
5 EMinvp90f2
calibration of 13C hard pulse
1 1 13 13
[off resonance presat 90 H delay 1/(2JHC) 180 H 90 C 90/180 C delay 1/(2JHC)
detection]
Optimize p4 and pl4 for pulse calibration, same principle as for the 15N calibration
; EMinvp90f2
; for calibration of rectangular pulse on F2 (13C)
; p3 pl3: ~ 90deg
; p4 pl4: 1) 90deg: phase spectrum
; 2) 180/360deg: optimize for zero signal
; F1 1H 4.7ppm (H2O)
; F3 13C ~20ppm (select methyl group with unique 1H shift in 1D)
"p2=p1*2"
"d2=3.8m" ; 1/2J(C,H)
"d22=d2-p3-p4-16u"
"p18=100m" ; for off resonance presaturation set SPOFFS0
1 ze
2 d1 do:f2
d12 pl7:f1
18 (p18:sp0 ph0):f1 ; spnam0=squ.1000, 100ms, sp0=pl7
2u
3u
20u pl1:f1
(p1 ph1):f1
d2 pl3:f3
(p2 ph3):f1
(p3 ph2):f2 ; 90 deg
3u
10u pl4:f2
(p4 ph4):f2 ; 90/180 deg
3u
d22 pl12:f2
go=2 ph31 cpd2:f2
d11 wr #0 do:f2
exit
ph0=0
ph1=0
ph2=0 0 2 2
ph3=0
ph4=0 2
ph31=0 2 2 0
6 EMinvq5
Use the shape tool in XWINNMR to check the difference in excitation profile
between the 90 and 180 deg hard pulses and the shaped pulses (Q5 for 90 deg, Q3
for 180 deg).
Calculate the power level of the shaped pulse from the known power level of the
hard pulse. (What is the basic assumption for the calculation?)
13
Calibration of 90 (Q5) C shaped pulses:
13
; EMinvq5
; for calibrating 90 shaped pulse on f3 (i.e. Q5, G4)
; 1) acquire spectrum with program as it is, phase to absorption
; 2) delete the ";;", acquire another spectrum,
; and optimize sp1 & sp2 for zero signal
; F1 1H 4.7ppm (H2O)
"p18=100m"
"p2=p1*2" Check o2p
"d2=3.8m" ; 1/(2J(CH))
"d13=3u"
"d20=d2+p13"
1 ze
2 d1 do:f2
3u
20u pl7:f1
3u
18 (p18:sp0 ph0):f1 ; sp0=squ.1000, 100ms, sp0=pl7
2u
d13
20u pl1:f1
(p1 ph1):f1
d20 ; d20 or d2 Compensate for p13
3u pl3:f3
(p13:sp1 ph3):f2 ; 90 Q5 or G4 (z->xy) spnam1 spoffs1
3u
(p2 ph0):f1 spnam2 spoffs2
(p13:sp2 ph4):f2 ; 90 Q5tr or G4tr(xy->z)
3u
(p13:sp1 ph4):f2 ; 90 Q5 or G4
3u
d2 pl12:f2
go=2 ph31 cpd2:f2
d11 wr #0 do:f2
exit
ph0=0
ph1=0
ph3=0 2
ph4=0 0 2 2
ph31=0 2 2 0
1. [90 1H delay 1/(2JHC) 90 shaped pulse (q5) (p13, sp1) 180 1H 90 shaped pulse (q5 time
reversed) (p13, sp2) delay 1/(2JHC) detection]
Exercise: Get a spectrum, phase it and store it as a reference (wrp 2).
2. [90 1H delay 1/(2JHC) delay = p13 90 shaped pulse (q5) (p13, sp1) 180 1H 90 shaped
pulse (q5 time reverse) (p13, sp2) 90 shaped pulse (q5) (p13, sp1) delay 1/(2JHC) detection.
Why change a delay here, and not in the hard pulse calibrations?
Exercise: Optimize sp1 and sp2 for having no signal (zero crossing).
Compare the calibrated power level with the one you calculated earlier!
14
EMinvq3
13
Calibration of 180 C shaped pulses
Change the pulse program in this dataset to EMinvq3: type pulprog EMinvq3 in
TOPSPIN.
1 1
[off resonance preset 90 H delay 1/(2JHC) 90 shaped pulse 180 H 180 shaped pulse on
13
C (q3 = p14 sp3 spoffs3, or an other 180 shaped pulse) delay 1/(2JHC) detection].
; EMinvq3
; M.Sattler (EMBL Heidelberg), J.Schleucher (U Umea), C.Griesinger (U Frankfurt)
; for calibration of 180deg shaped pulse (i.e. Q3, G3)

; 1) acquire and phase spectrum with pulse program EMinvq3
; 2) then use this pulse program, optimize sp3 for zero signal
; F1 1H 4.7ppm (H2O)
"p18=100m"
"p2=p1*2"
"d2=3.8m" ; 1/(2J(CH))
"d13=3u"
"d22=d2-p14+p13-d13"
1 ze
2 d11 do:f2
3u
18 (p18:sp0 ph0):f1 ; sp0=squ.1000, 100ms, sp0=pl7
2u
3u
30u pl1:f1
(p1 ph1):f1 90 1H
d2 ; d2=1/2J(HC)
(p13:sp1 ph3):f2 ; 90 Q5 or G4 90 13C, Q5
(p2 ph0):f1 180 1H
(p14:sp3 ph4):f2 ; 180 Q3 or G3 180 13C, Q3
d13
d22 pl12:f2
go=2 ph31 cpd2:f2
d11 wr #0 do:f2
d11
exit
ph0=0
ph1= 0
ph2= 0
ph3= 0 2
ph4= 0
;ph4 = 1
ph31=0
Exercise: check the effect the shaped pulses have, compared to the normal hard
pulses, by modifying the pulse program EMinvq5 and using hard pulses on
carbon.
15
Calibration of 2H pulse
7 EMzg2h
2
Calibration of 90 H pulse
Use the D2O Signal of the lock substance to calibrate the deuterium pulse (e.g. for
decoupling deuterium in deuterated proteins.)
2
[ 90 H detection].
IMPORTANT: Change the lock power to the lowest value!
;zg2h
;avance-version (07/04/03)
;1D sequence
;using 2H lockswitch unit or BSMS 2H-TX board
;
#include<Avance.incl>
#include<Sysconf.incl>
"d11=30m"
"acqt0=-p1*2/3.1416"
1 ze
d11 LOCKDEC_ON ; allow for 2H decoupling (lockswitch)
d11 H2_PULSE ; lock off, 2H transmitter on
2 30m
30m H2_LOCK ; lock on, 2H transmitter off
d1
d11 H2_PULSE ; lock off, 2H transmitter on
p1:D ph1
go=2 ph31
30m mc #0 to 2 F0(zd)
d11 H2_LOCK ; lock on, 2H transmitter off
d11 LOCKDEC_OFF ; disable for 2H decoupling (lockswitch)
exit
;pl1 : f1 channel - power level for pulse (default)
ph1=0 2 2 0 1 3 3 1 ;p1 : f1 channel - 90 degree high power pulse
ph2=0 2 2 0 1 3 3 1 ;d1 : relaxation delay; 1-5 * T1
ph3=2 0 0 2 3 1 1 3 ;d11: delay for disk I/O [30 msec]
ph31=0 2 2 0 1 3 3 1 ;NS: 1 * n, total number of scans: NS * TD0
;locnuc: off
Calibration of water-flip-back pulse

8 Calibration of water flip back pulse
Define a WFB pulse in the pulse program in cal/2 and calibrate it.
(use a 1ms sinc pulse, 90deg; (p12:sp11 ph11:r):f1 )
Calibrate both the power level and the phase correction:
1. Run the experiment with the 90deg hardpulse (no preset), NS=1 DS=0. Phase
the spectrum.
2. Calculate the power level of the WFB using the shape tool.
3. Run the experiment using the WFB pulse only. Determine the phase difference
by correcting the different phase in the spectrum.
4. optimize water suppression in gs by modification of shape power (sp11) and
phase correction (phcor11)
16
;EM1dfb
1 ze
2 d1
; (p27:sp11 ph27:r):f1 ; H2O sel. Gauss_1.1000, 2.5m, 270deg
(p12:sp11 ph11:r):f1 ; H2O sel. sinc_1.1000 1m, 90deg 90 selective pulse
d12 pl1:f1
p1 ph1 90 hard pulse
go=2 ph31
wr #0
exit
ph1=0 1 2 3
ph27=0 1 2 3 ;270 deg
ph11=2 3 0 1 ;90 deg
ph9=2 3 0 1
ph31=1 2 3 0
Calibration of soft pulse in WATERGATE

9 EM1dwgs
Use the same pulse as in cal/8 and optimize the water suppression in a soft pulse
WATERGATE sequence.
Optimize water suppression by modification of sp12 and phcor12 in gs.
;zggpwg
#include <Grad.incl>
"d12=20u"
"p16=600u"
"d16=200u"
"p2=2*p1"
1 ze
2 30m
d1
10u pl1:f1
50u UNBLKGRAD
p16:gp1 gradient
d16 pl0:f1
(p12:sp12 ph12:r):f1 90 selective pulse
4u
d12 pl1:f1
(p2 ph3) 180 hard pulse
4u
d12 pl0:f1
(p12:sp12 ph12:r):f1 90 selective pulse
46u
p16:gp1 gradient
d16
4u BLKGRAD
go=2 ph31 acquisition
d1 wr #0
exit
ph1=0 2
ph12=0 0 1 1 2 2 3 3
ph3=2 2 3 3 0 0 1 1
ph31=0 2 2 0
Why is the power level different than in cal/8 ?
17
Echo/Antiecho gradient calibration

10 EMgradcal
Optimize signal in the first scan of a sensitivity enhanced HSQC.
1. Record 1D with gradient ration 90/9.1 and phase the spectrum.
2. Define plotregion f1p = 11ppm and f2p = 5 ppm (amide protons)
3. paropt/popt gpz3 (e.g. sarting value 8.5 / increment 0.1 / 10 increments)
4. change the refocusing gradient: shorten duration (p19) and increase amplitude
(gpz3) type gpz 3 with a blank between gpz and 3 in paropt (topspin3) and
gpz3 w/o blank (topspin2)
5. repeat the optimization
Why do we want to have shorter p19? Why does the ratio between the gradients change?
; gradcal ;---------------------- 15N RT evol. (t1) ---------------
; X-nucleus on f2 d0 ; td1/2
; water-flip-back: p27 90deg 1ms sinc1.1000 ~31.8dB (cryo) (p14:sp3 ph0):f3 ; dec. Ca (on res. Caliph. G3 268u)
; set phcor27 !! (p2 ph0):f1 ; dec. 1H
; (p14:sp4 ph0):f3 ; dec. CO (G3 268u offset: +16500Hz)
d0 ; td1/2
#include <Avance.incl> p16:gp2*EA
#include <Grad.incl> d16
(p6 ph0):f3
"p2=p1*2"
"p6=p5*2" 3u
d28
"d29=p19+d16+3u+100u" ;----------- --------------SE (HzNy -> Hx) ------------------
"d27=(p6*2+8u-p2)/2" (p5 ph4):f2
"d4=2.7m" d8
;"d28=p2+p16+d16+6u" ; no p14, i.e. N15-only sample (p1 ph0):f1
"d28=p2+p16+d16+p14*2+6u" ; with p14 d4
(p6 ph0):f2
;"d0=3u+in0/2" ; 90/-180 phase in F1 (p2 ph0):f1
"d0=3u" d4
(p5 ph6):f2
"d8=p1" d9
"d9=p5" (p1 ph8):f1
"d16=250u" d4
(p5 ph8 4u p6 ph0 4u p5 ph8):f2 (d27 p2 ph0):f1
d4
(p1 ph0):f1
1 ze d29
2 d1 do:f2 (p2 ph0):f1
3 15m 3u
4 6m p19:gp3
5 3m d16 pl12:f2
6 1m ;--------------------------------------------------------------------
30u pl1:f1 100u BLKGRAD
30u pl2:f2 go=2 ph31 cpd2:f2
;------------------------- INEPT (Hz -> HzNy) ------------------------ d1 do:f2 wr #0
(p1 ph0):f1 exit
d4
d4 ph0=0
(p1 ph1):f1 ph1=1
3u ph2=0
20u pl11:f1 ph3=0 2
(p12:sp11 ph11:r):f1 ; H2O sel. sinc1.1000, 1m, 90deg ph4=0
3u ph5=0
100u UNBLKGRAD ph6=1
p16:gp1 ph8=1
d16 pl1:f1 ph11=2 ; ph11=2 for 90 deg flip-back
(p5 ph3):f2 ph31=0 2
18
Calibration of adiabatic pulses

11 EMwurstcal
Calibration of an adiabatic pulse
1. Generate an adiabatic pulse for proton inversion (e.g. Chirp 20kHz 2ms 40%)
2. Calculate the power level required for this shape (Analyze, Integrate adiabatic
shape)
3. Calibrate the hardpulse with the corresponding power level
4. Record a spectrum with the calibrated power level, phase and define f1p f2p
5. Look at the inversion for a range of shape powers using paropt (type SPdB 17
(topspin3) or sp17 (topspin2))
;EMwurstcal
;avance-version
; off resonance presat
"d12=20u"
"p18=100m"
1 ze
2 d1
d12 pl7:f1
4u
18 (p18:sp0 ph0):f1 ; squ.1000, 100m +55 db Presaturation on water
4u
lo to 18 times 10
3u
10u pl0:f1
; p2 ph0 Inversion of the magnetization
(p17:sp17 ph0):f1
4u
90 hard pulse
10u pl1:f1
p1 ph1
detection
go=2 ph31
50m wr #0
exit
ph0=0
ph1=0 2 2 0 1 3 3 1
ph10=0 2 2 0 1 3 3 1
ph27=0 2 2 0 1 3 3 1
ph31=0 2 2 0 1 3 3 1
Why is the pulse calibration so different from a hard pulse calibration? What
happens before/after the theoretical power level is reached? What are the basic
assumptions for the calculation of the power level?
19
4. 1D & basic 2D pulse sequences (heteronuclear correlations)
During this session some real experiments will be set up and analyzed.
Type "re 1h 1 1 w embo" to access the data sets for this practical.
1
101 EMzgpr presat on resonance and 90 H. (same pp than cal/2)
102 EM1dwg 1D spectrum using WATERGATE 3-9-19 for water suppression
Try to understand the WATERGATE pulse, e.g. by using the assumption that the
excitation profile corresponds to the Fourier transform of the pulse.
What happens if you decrease/increase d19?
; EM1dwg
; 1D with Watergate
"d12=20u"
"p16=600u"
"d16=200u"
1 ze
2 d1
d12 pl1:f1
50u UNBLKGRAD
p16:gp1 gradient
d16
p1*0.231 ph2
d19*2
p1*0.692 ph2
d19*2
p1*1.4621 ph2
d19*2
p1*1.4621 ph3
d19*2
p1*0.692 ph3
d19*2
p1*0.231 ph3
46u
p16:gp1 gradient
d16
4u BLKGRAD acquisition
go=2 ph31
d1 wr #0
exit
ph1=0 0 2 2 1 1 3 3
ph2=1
ph3=3
ph31=0 0 2 2 3 3 1 1
20
103 EM1dwgs 1D spectrum using soft pulse WATERGATE (same pp than cal/9)
Record the spectrum with and without the water flip back pulse in the beginning.
Compare the spectra.
; E1dwgs
; bs1dwgsp
; 1D with Watergate using 90 deg H2O selective pulses
#include <EMBL.incl>
"d13=4u"
"d12=20u"
"p2=p1*2"
1 ze
2 d1
d12 pl12:f1
(p12:sp11 ph11:r):f1 ; wfb 90 selective pulse
d13
d12 pl1:f1
50u UNBLKGRAD
p16:gp1 gradient
d16 pl0:f1
4u
20u pl1:f1
4u
20u pl0:f1
45u
p16:gp1 gradient
d16
5u BLKGRAD
go=2 ph31 acquisition
d1 wr #0
exit
ph1=0 0 ;2 2 1 1 3 3
ph2=0
ph11=2
ph12=2
ph31=0 0 ;2 2 3 3 1 1
21
104 EMzggpw5 1D spectrum using double WATERGATE
;zggpw5 4u
;1D sequence p16:gp2
;water suppression using watergate W5 pulse sequence with gradients d16
;using double echo p27*0.087 ph5
;M. Liu, X. Mao, C. He, H. Huang, J.K. Nicholson & J.C. Lindon, d19*2
; J. Magn. Reson. 132, 125 - 129 (1998) p27*0.206 ph5
d19*2
p27*0.413 ph5
#include <Avance.incl> d19*2
#include <Grad.incl> p27*0.778 ph5
d19*2
"p16=600u" p27*1.491 ph5
"d16=200u" d19*2
p27*1.491 ph6
d19*2
p27*0.778 ph6
1 ze d19*2
2 30m p27*0.413 ph6
d1 d19*2
10u pl1:f1 p27*0.206 ph6
p1 ph1 d19*2
50u UNBLKGRAD p27*0.087 ph6
p16:gp2
p16:gp1 d16
d16 pl18:f1
p27*0.087 ph3 4u BLKGRAD
d19*2 go=2 ph31
p27*0.206 ph3 d1 wr #0
d19*2 exit
p27*0.413 ph3
d19*2
p27*0.778 ph3 ph1=0 2
d19*2 ph3=0 0 1 1 2 2 3 3
p27*1.491 ph3 ph4=2 2 3 3 0 0 1 1
d19*2 ph5=0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1
p27*1.491 ph4 2222222233333333
d19*2 ph6=2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3
p27*0.778 ph4 0000000011111111
d19*2 ph31=0 2 2 0 0 2 2 0 2 0 0 2 2 0 0 2
p27*0.413 ph4
d19*2
p27*0.206 ph4
d19*2
p27*0.087 ph4
50u
p16:gp1
d16
Compare the water suppression and signal to noise of the different 1D sequences!
22
Sensitivity-enhanced 1H,15N HSQC

1
105 EMhnhsqcse H,15N HSQC using sensitivity enhancement with gradients
Change the pulse program so that there is no decoupling during t1 and t2.How
does the spectrum change compared to before?
; Ehnhsqc_dec
;------------------ SE (HzNy -> Hx) --------------------------
(p5 ph4):f3
#include <Avance.incl> d8
#include <Grad.incl> (p1 ph0):f1
d4
p2=p1*2 (p6 ph0):f3
p6=p5*2 (p2 ph0):f1
d4
d29=p16+d16+3u+100u (p5 ph6):f3
d27=(p6*2+6u-p2)/2 d9
d4=2.7m (p1 ph8):f1
;d28=p2+p16+d16+6u ; no p14, i.e. N15-only sample d4
d28=p2+p16+d16+p14*2+6u ; with p14 (p5 ph8 3u p6 ph0 3u p5 ph8):f3 (d27 p2 ph0):f1
d4
d0=3u+in0/2 ; 90/-180 phase in F1 (p1 ph0):f1
;d0=3u d29
(p2 ph0):f1
d8=p1 3u
d9=p5 p16:gp3
d16 pl12:f2 pl13:f3
1 ze ;-----------------------------------------------------------
2 d1 do:f3 do:f2 100u BLKGRAD
3 15m go=2 ph31 cpd3:f3 ;cpds2:f2
4 6m d1 do:f3 do:f2 wr #0 if #0 zd
5 3m 5m ip6 igrad EA
6 1m 5m ip6
30u pl1 :f1 lo to 3 times 2 ; E/AE
30u pl3 :f3 10m id0
;----------------- INEPT (Hz -> HzNy) ----------------------- 5m ip3
(p1 ph0):f1 5m ip3 ; States-TPPI
d4 5m ip31
(p5 ph8 3u p6 ph0 3u p5 ph8):f3 (d27 p2 ph0):f1 5m ip31
d4 lo to 4 times l0
(p1 ph1):f1 exit
3u
20u pl11:f1
(p12:sp11 ph11:r):f1 ; H2O sel. Sinc1.1000, 1m, 90deg ph0=0
3u ph1=1
100u UNBLKGRAD ph2=0
p16:gp1 ph3=0 2
d16 pl1:f1 ph4=0
(p5 ph3):f3 ph5=0
;--------------------- 15N RT evol. (t1) --------------------- ph6=1
d0 ; td1/2 ph8=1
(p14:sp3 ph0):f2 ; dec. Ca (on res. Caliph. G3 268u) ph12=2 ; ph27=2 for 90 deg bs
(p2 ph0):f1 ; dec. 1H ph31=0 2
(p14:sp4 ph0):f2 ; dec. CO (G3 268u offset: +16500Hz)
d0 ; td1/2
p16:gp2*EA
d16
(p6 ph0):f3
d28
23
WATERGATE 1H,15N HSQC

1
106 EMhnhsqcwgs H,15N HSQC using WATERGATE.
What are the advantages/disadvantages of sensitivity enhanced vs WATERGATE?

Compare a 1D trace of the two spectra (e.g. rsr 100 10) or the first FID (rser 1)
; Ehnhsqcwgs_dec
; HSQC with water flip back using selective 90 gauss pulses (p14:sp3 ph0):f2
(p2 ph0):f1 ; dec. 1H
#include <Avance.incl> (p14:sp4 ph0):f2
#include <Grad.incl> d0
#include <Delay.incl> (p6 ph0):f3
d27
"p2=p1*2" (p5 ph4):f3
"p6=p5*2" 50u
p16:gp2
"d4=2.6m" d16
"d0=3u+(in0/2)" ;-90/180 20u pl11:f1
;"d0=3u+in0" ;-180/360 (p12:sp11 ph11:r):f1 ; wfb
3u
"d24=d4-p16-d16" 20u pl1:f1
"d14=d4-p19-d16-p12-40u" (p1 ph0):f1
"CEN_HN2=(p6*2+6u-p2)/2" 20u
"d17=d16+p6*2+6u" p19:gp3
"d27=6u+p2+p14*2" d16
d14
(p12:sp12 ph12:r):f1
1 ze 3u
2 d1 do:f3 20u pl1:f1
3 3m (p5 ph8 3u p6 ph0 3u p5 ph8):f3 (CEN_HN2 p2 ph0):f1
4 6m 23u
5 3m (p12:sp12 ph12:r):f1
6 1m d14
30u pl1:f1 p19:gp3
30u pl3:f3 d16 pl123f3
50u UNBLKGRAD 20u BLKGRAD
d12 pl1:f1 go=2 ph31 cpd3:f3
(p1 ph0):f1 d1 do:f3 wr #0 if #0 zd
3u 3m ip3
p16:gp4 3m ip18
d16 3m ip19
d24 lo to 3 times 2
(p5 ph18 3u p6 ph19 3u p5 ph18):f3 (CEN_HN2 p2 ph0):f1 3m id0
d24 lo to 4 times l0
p16:gp4 exit
d16
3u ph0=0
(p1 ph1):f1 ph1=1
3u ph2=0
20u pl11:f1 ph3=0 2
(p12:sp11 ph11:r):f1 ; wfb ph4=0
3u ph5=0
20u pl1:f1 ph8=1
20u pl2:f3 ph18=1
p16:gp1 ph19=0
d16 ph11=0 ; -> 0
(p5 ph3):f3 ph12=2 ; -> 2
d0 ph31=0 2
1H, 15N SOFAST HMQC:

107 sfhmqcf3gpph (Bruker standard release)
24
Compare the SOFAST HMQC to the HSQC spectra. (Note the differences in the style due to
extended software features: frequency determination in F1, spectral width of F1, frequency
jumps, ..)
;sfhmqcf3gpph
;avance-version (07/08/28) ;pl3 : f3 channel - power level for pulse (default)
;SOFAST HMQC ;pl26: f3 channel - power level for CPD/BB decoupling (low power)
;2D H-1/X correlation via heteronuclear zero and double quantum ;sp13: f2 channel - shaped pulse 180 degree (adiabatic)
; coherence ;sp23: f1 channel - shaped pulse 120 degree
;phase sensitive ; (Pc9_4_120.1000 or Q5.1000)
;with decoupling during acquisition ;sp24: f1 channel - shaped pulse 180 degree (Rsnob.1000)
; ;p8 : f2 channel - 180 degree shaped pulse for inversion (adiabatic)
;P.Schanda and B. Brutscher, J. Am. Chem. Soc. 127, 8014 (2005) ;p16: homospoil/gradient pulse [1 msec]
; ;p21: f3 channel - 90 degree high power pulse
prosol relations=<triple> ;p39: f1 channel - 120 degree shaped pulse for excitation
; Pc9_4_120.1000 (120o) (3.0ms at 600.13 MHz)
#include <Avance.incl> ; (or Q5.1000 (90o) (2.0ms at 600.13 MHz) )
#include <Grad.incl> ;p40: f1 channel - 180 degree shaped pulse for refocussing
#include <Delay.incl> ; Rsnob.1000 (1.0ms at 600.13 MHz)
;d0 : incremented delay (2D) = in0/2-p21*4/3.1415
"d11=30m" ;d1 : relaxation delay; 1-5 * T1
"d12=20u" ;d11: delay for disk I/O [30 msec]
"d21=1s/(cnst4*2)" ;d12: delay for power switching [20 usec]
;d16: delay for homospoil/gradient recovery
"in0=inf1/2" ;d21 : 1/(2J)NH
;cnst4: = J(NH)
"d0=in0/2-p21*4/3.1415" ;cnst19: H(N) chemical shift (offset, in ppm)
;cnst39: compensation of chemical shift evolution during p39
"DELTA1=d21-p16-d16-p39*cnst39" ; Pc9_4_120.1000: 0.529
"DELTA2=d21-p16-d16-de-4u" ; Q5.1000: -0.07
;inf1: 1/SW(N) = 2 * DW(N)
"spoff23=bf1*(cnst19/1000000)-o1" ;in0: 1/(2 * SW(N)) = DW(N)
"spoff24=bf1*(cnst19/1000000)-o1" ;nd0: 2
;NS: 2 * n
1 ze ;DS: 16
d11 pl26:f3 ;aq: <= 50 msec
2 d1 do:f3 ;td1: number of experiments
3 d12 pl3:f3 ;FnMODE: States-TPPI, TPPI, States or QSEC
50u UNBLKGRAD ;cpd3: decoupling according to sequence defined by cpdprg3: garp4.p62
p16:gp2 ;pcpd3: f3 channel - 90 degree pulse for decoupling sequence
d16 ; use pulse of >= 350 usec
(p39:sp23 ph1):f1 ;use gradient ratio: gp 1 : gp 2
p16:gp1 ; 11 : 7
d16
DELTA1 ;for z-only gradients:
# ifdef LABEL_CN ;gpz1: 11%
(center (p40:sp24 ph2):f1 (p8:sp13 ph1):f2 (p21 ph3 d0 p21 ph4):f3 ) ;gpz2: 7%
# else
(center (p40:sp24 ph2):f1 (p21 ph3 d0 p21 ph4):f3 ) ;use gradient files:
# endif /*LABEL_CN*/ ;gpnam1: SINE.100
DELTA2 ;gpnam2: SINE.100
p16:gp1
d16 pl26:f3 ;preprocessor-flags-start
4u BLKGRAD ;LABEL_CN: for C-13 and N-15 labeled samples start experiment with
go=2 ph31 cpd3:f3 ; option -DLABEL_CN (eda: ZGOPTNS)
d1 do:f3 mc #0 to 2 ;preprocessor-flags-end
F1PH(ip3, id0)
exit ;$Id: sfhmqcf3gpph,v 1.1.2.2 2007/09/14 16:17:35 ber Exp $
ph1=0
ph2=0
ph3=0 2
ph4=0 0 2 2
ph31=0 2 2 0
1H, 15N TROSY:

1
108 EMtrosy H,15N TROSY with E/AE gradient selection
25
; Etrosysewgs_dec DELTA
; with E/AE gradient coherence selection p19:gp5*EA
; with wfb (many 1m Sinc pulses !!) d19
(p1 ph6)
#include <Avance.incl> 3u
#include <Delay.incl> (p12:sp11 ph26:r):f1 ; water-flip-back (+z)
#include <Grad.incl> 3u
10u
"p2=p1*2" p16:gp6
"p6=p5*2" d16 pl1:f1
d14
"p16=1m" (CEN_HN2 p2 ph0) (p6 ph0):f3 ; water -z
"d16=300u" d14
"p19=500u" p16:gp6
"d17=200u" d16
"d19=d17" 3u
(p12:sp11 ph28:r):f1 ; water +y
"d4=2.7m-p16-d16-4u" 3u
10u pl1:f1
"d0=100u" ; 0/0 phase (p1 ph0):f1 ; water +z
;"d0=100u+in0/2" ; 90/-180 phase (p5 ph1):f3
4u
"p12=1m" ; 90 selective H20 p16:gp7
"d14=d4-6u-p12" d16
"d27=p19+d17" d14
(p12:sp12 ph12:r):f1 ; wg
; for 13C sample use the following line and use [p14 4u p14] below! 3u
"DELTA=220u+(p5*4/3.1416)+(p14*2)+4u" 10u pl1:f1
(CEN_HN2 p2 ph0):f1 (p6 ph0):f3
"CEN_HN2=(p6-p2)/2" 3u
(p12:sp12 ph12:r):f1 ; wg
aqseq 312 d14
10u
1 30m ze p16:gp7
30m pl13:f3 d16
2 d1 do:f2 do:f3 4u
15m (p5 ph7):f3
3 5m d27
4 20m (p12:sp12 ph12:r):f1 ; wg
5 15m 3u
6 10m 10u pl1:f1
7 50u UNBLKGRAD (p2 ph0):f1
20u pl1:f1 3u
20u pl3:f3 10u
(p1 ph0):f1 (p12:sp12 ph12:r):f1 ; wg
4u 3u
p16:gp1 p19:gp8
d16 d17 pl13:f3
d4 4u BLKGRAD
(CEN_HN2 p2 ph10) (p6 ph0):f3 go=2 ph31
4u d1 do:f2 do:f3 wr #0 if #0 zd
d4 5m ip6*2 igrad EA
p16:gp1 5m ip26*2
d16 5m ip7*2
(p1 ph1) lo to 4 times 2 ; E/AE 15N
3u 5m id0
(p12:sp11 ph29:r):f1 ; water-flip-back 5m ip3*2
3u 5m ip31*2
10u pl1:f1 lo to 5 times l0 ; TD1/2
p16:gp2 exit
d16
20u ph0=0
(p5 ph3):f3 ; 90 N ph25=0 ; == ph0 water flip-flip
d0 gron0 ph1=1
10u groff ph3=0 2
(p14:sp3 ph0):f2 ph7=0 ; ph7=0 trosy H
4u ph6=1 ; trosy N
(p14:sp4 ph0):f2 ph26=3 ; == -ph6 water flip-back
d0 gron0*-1 ph12=2 ;
10u groff ph28=0
p19:gp5*EA*-1 ph10=0 ;0 (add) 1 (subtract) 15N Zeeman magn
d19 ph29=0 ; 0 (add) 2 (subtract) wfb for Zeeman phase
(p6 ph0):f3 ph31=0 2
109 EMhalftrosy The TROSY principle is only used in one dimension

26
; EMhalftrosy (p6 ph0):f3

; with E/AE gradient coherence selection DELTA
; with wfb (many 1m Sinc pulses !!) p19:gp5*EA
d19
#include <Avance.incl> (p1 ph6)
#include <Delay.incl> 3u
#include <Grad.incl> (p12:sp11 ph26:r):f1 ; water-flip-back (+z)
3u
"p2=p1*2" 10u
"p6=p5*2" p16:gp6
d16 pl1:f1
"p16=1m" d14
"d16=200u" (CEN_HN2 p2 ph0) (p6 ph0):f3 ; water -z
"p19=500u" d14
"d17=200u" p16:gp6
"d19=d17" d16
3u
"d4=2.7m-p16-d16-4u" (p12:sp11 ph24:r):f1 ; water +y
3u
"d0=100u" ; 0/0 phase 10u pl1:f1
;"d0=100u+in0/2" ; 90/-180 phase (p1 ph4):f1 ; water +z
(p5 ph16):f3
"p12=1m" ; 90 selective H20 4u
"d14=d4-6u-p12" p19:gp7
"d24=2.7m-p19-d19-4u-p12" d19
"d25=d24-p19-d17" d24
(p12:sp12 ph12:r):f1 ; wg
; for 13C sample use the following line and use [p14 4u p14] below! 3u
"DELTA=220u+(p5*4/3.1416)+(p14*2)+4u" ; with p14 (13C/15N 10u pl1:f1
sample) (CEN_HN2 p2 ph0):f1 (p6 ph0):f3
3u
"CEN_HN2=(p6-p2)/2" (p12:sp12 ph12:r):f1 ; wg
d25
10u
aqseq 312 p19:gp7
d19
1 30m ze 4u
2 d1 do:f2 do:f3 p19:gp8
15m d17 pl13:f3
3 5m 4u BLKGRAD
4 20m go=2 ph31 cpd3:f3
5 15m d1 do:f2 do:f3 wr #0 if #0 zd
6 10m 5m ip4*2 igrad EA
7 50u UNBLKGRAD 5m ip24*2
20u pl1:f1 lo to 4 times 2 ; E/AE 15N
20u pl3:f3 5m id0
(p1 ph0):f1 5m ip3*2
4u 5m ip31*2
p16:gp1 lo to 5 times l0 ; TD1/2
d16 exit
d4
(CEN_HN2 p2 ph10) (p6 ph0):f3 ph0=0
4u ph1=1
d4 ph3=0 2
p16:gp1 ph4=1 ; trosy N
d16 ph6=0 ; trosy N
(p1 ph1) ph16=0 ; trosy N
3u ph26=2 ; 0 =: -ph6 water flip-back
(p12:sp11 ph29:r):f1 ; water-flip-back ph12=2 ;
3u ph24=1 ; =: ph4 wfb
10u pl1:f1 ph28=0
p16:gp2 ph10=1 ; 0 (add) 1 (subtract) 15N Zeeman magnetization
d16 ph29=2 ; 0 (add) 2 (subtract) wfb for Zeeman phase
20u ; opposite for anti TROSY line!
(p5 ph3):f3 ; 90 N ph31=0 2
d0 gron0
10u groff
(p14:sp3 ph0):f2
4u
(p14:sp4 ph0):f2
d0 gron0*-1
10u groff
p19:gp5*EA*-1
d19
27
1. Confirm that the 15N Zeeman magnetization is added and not subtracted from
your signal
2. Change phases/gradients in order to select the other (undesired) multiplet
lines.
3. Check signal to noise compared to HSQC and TROSY
110 trosyetf3gpsi2 1H,15N TROSY with E/AE gradient selection, variant with improved
relaxation properties and efficient anti-trosy line suppression (Lit: D. Nietlsipach, J.
Biomol. NMR 31, 161-166 (2005))
1H,13C correlations
1
111 EMhchmqc H,13C HMQC using presat for water suppression
28
;mshmqc
;avance-version
;2D H-1/X correlation via heteronuclear zero and double quantum coherence
;A. Bax, R.H. Griffey & B.L. Hawkins, J. Magn. Reson. 55, 301 (1983)
"p2=2*p1"
;"d2=3.8m" ; 1/2J(H,C)protein (!)

"d2=3.4m" ; 1/2J(H,C)sugar(!)
;"d0=(in0-p2-p3*4/3.14)/2" ; 90/180
"d0=in0-(p2+p3*4/3.14)/2" ; 180/360
"d11=30m"
1 ze
2 d11 do:f2
20m
3 5m
4 20u pl7:f1
(p29 ph2):f1 Presaturation
45u UNBLKGRAD
p16:gp1 Gradient
d16
5u BLKGRAD
20u pl1:f1
30u fq=cnst20(bf ppm):f3 ; 13C o2p 40 ppm
;---------------- HMQC (Hz -> HxCy) -----------------
(p1 ph1):f1 90 pulse proton
23u
d2 pl2:f2 1/2J
(p3 ph3):f2 ; HxCy 90 pulse carbon
;----------------------- 13C RT ev. -------------
d0
(p2 ph2):f1 ; 1H dec 180 pulse proton
d0
;----------------------HxCy -> Hy --------------------
(p3 ph4):f2 ; HxCz 90 pulse carbon
3u 1/2J
d2 fq=cnst22(bf ppm):f3 ; 13C o2p 70 ppm for decoupling
20u pl12:f2
;------------------------------------------------
go=2 ph31 cpds2:f2 Acquisition
d11 do:f3 wr #0 if #0 zd
20m ip3
lo to 3 times 2
5m id0
lo to 4 times l0 ; l0=td1/2
exit
ph0=0
ph1=0 0 2 2
ph2=0 0 ;1 1 2 2 3 3
ph3=0 2 ;
ph4=0 0 ; 0 0 2 2 2 2
ph31=0 2 2 0; 2 0 2 0 0 2
; hchsqc, no gradient selection, no wfb
"p2=p1*2"
"p4=p3*2"
"d4=1.7m"
"d7=(p4*2+6u-p2)/2"
"d17=6u+p2"
;"d0=3u+in0/2"
"d0=3u"
29
"d11=30m"
1 ze
2 d11 do:f2
3 15m
4 6m
5 3m
6 1m
100u fq=cnst20(bf ppm):f2 13C o2p 40 ppm
30u pl7:f1
(p29 ph0):f1
50u UNBLKGRAD presaturation
p16:gp1
d16
30u pl1:f1 gradient
30u pl2:f2
30u pl3:f3
20u
d4 1/2J4
(p3 ph8 3u p4 ph0 3u p3 ph8):f2 (d7 p2 ph0):f1 180 pulse carbon and proton
d4
20u
3u
p16:gp2 Gradient
d16
(p3 ph3):f2 90 pulse carbon
d0
d0
(p4 ph0):f2 180 pulse carbon
d17
(p3 ph4):f2
90 pulse carbon
3u
p16:gp3
gradient
d16
50u BLKGRAD
(p1 ph0):f1
90 pulse proton
20u
d4
1/2J
3u
3u 180 pulse carbon and proton
d4 fq=cnst22(bf ppm):f2
20u pl12:f2 13C o2p 70 ppm for decoupling
go=2 ph31 cpds2:f2
d11 do:f3 wr #0 if #0 zd acquisition
5m ip3
lo to 3 times 2
10m id0
lo to 4 times l0
exit
ph0=0
ph1=0 0 2 2
ph2=1
ph3=0 2
ph4=0
ph8=1
ph12=0
ph22=1
ph23=3
ph31=0 2 2 0
1
112 EMhchsqc H,13C HSQC using presat for water suppression
Compare the 13C signals of aromatic and methyl signals in the HMQC vs. HSQC
implementation. Make sure the decoupling power is sufficient.
Modify the pulse sequence (rename it!) in order to use an adiabatic 180 pulse
during the 1H,13C INEPT transfers and compare the effect for the 13C signals far
off-resonance
30
Isotope Filter
The spectrum should show the unlabelled components, for example in a complex of protein
(doubly labelled) and unlabelled peptide and purge magnetization of protons directly bound to
15
N and 13C. These filters are used as building blocks for 2D and 3D experiments, i.e. TOCSY
and NOESY.
Compare the spectra with and without filter.
113 EMfilter 1D filter, using normal pulses.
; EMfilter (p13:sp5 ph5):f2 ;arom.

; 1D, presat d15 ;0.5m
; 12C aliph. + ar. double filter (G4) (p13:sp1 ph3):f2 ;90 aliph.
; 14N filter (p2 ph2):f1
; with Gradients (p5 ph14 3u p6 ph4 3u p5 ph14):f3
(p14:sp3 ph0):f2 ;180 aliph.
#include <Avance.incl> d6 ;0.5m
#include <Grad.incl> (p5 ph3):f3 ;90 (15N)
d7 ;~ 3.1m
"d14=3.1m-p16-d16" p16:gp1
"d15=0.5m" ; 0.5m d16
"d6=1.1m" 0.9m -> 1.1m (p13:sp2 ph4):f2 ;90 aliph
"d7=d14+p13+d15-p5*4-6u-p14-d6-p5" 3u
"p10=0.5m" ; trim pulse 1H 20u pl13:f3 pl12:f2 BLKGRAD
(p10 ph8):f1
1 ze go=2 ph31 ;;cpd2:f2 ;;cpd3:f3
2 d11 do:f3 do:f2 d11 do:f2 do:f3 wr #0
20u pl2:f2 exit
20u pl3:f3
20u pl7:f1 ph0= 0
(p29 ph29):f1 ph1= 0
3u ph2= 0 1 2 3
100u UNBLKGRAD ph3= 0 0 0 0 2 2 2 2
20u pl1:f1 ph4= 1 1 1 1 1 1 1 1 3 3 3 3 3 3 3 3
(p1 ph1):f1 ph14=0 0 0 0 0 0 0 0 2 2 2 2 2 2 2 2
23u ph5 =0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
p16:gp1 2222222222222222
d16 ph8=3
d14 ;3.1m ph29=0
ph31=0 2
What types of filters are used here simultaneously, single/double? For which spins?
Estimate amide proton T2 time

Run a jump-return spin-echo for HN to estimate the transverse relaxation time. Record the pulse
program with d4=4us, repeat with longer delay(s). For water-suppression run with gs and
optimize p0 and phcor2/4.
114/115 E1djrT2 1D experiment for amide proton T2 estimation.
31
; EM1djrT2
; 1D with Watergate
"d12=20u"
"p16=600u"
"d16=200u"
1 ze
20u pl13:f3 15N decoupling
2 d1 do:f3
20u pl1:f1 90 hard pulse
p1 ph1
d19 d19 (4 )-1 for max excitation @ 9.5ppm (e.g. 100/85u 500/600/MHz)
p0 ph2:r
10u
d4 of relaxation delay
p1 ph3
d19*2
p0 ph4:r
10u
d4
go=2 ph31 cpd3:f3
10u do:f3
wr #0
exit
ph1=0 1 2 3
ph2=2 3 0 1
ph3=0 0 0 0 1 1 1 1 2 2 2 2 3 3 3 3
ph31=0 3 2 1 2 1 0 3
Superimpose the spectra recorded with d4=4us and d4=?ms. From the ratio of intensities,
estimate the T2 time:
T2 = 2( A B)/ln(IA/IB)
The amide proton T2 is important for quality check and to decide which labeling strategy and
NMR experiments should be used ( lecture Daniel Nietlispach).
Standard experiments from the Bruker pp repository

Look into the pp directory of the spectrometer. Check which HNCACB experiments are
available. What is the difference between these experiments? Which one would you use? How
do you setup the experiment?
32

P1&P2

Uploaded by

Copyright:

Available Formats

P1&P2

Uploaded by

Document Information

Original Description:

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

P1&P2

Uploaded by

Copyright:

Available Formats

EMBO Practical Course:

Structure, Dynamics and function of biomacromolecules by solution NMR

Practical: Heteronuclear NMR

Pulse and parameter calibration

Bernd Simon & Michael Sattler

2. Bruker software commands and our pulse program conventions

with R being the resistance, usually 50 in a probe.

Example: p1:f1 (means a 90 deg pulse on protons)

Phases are defined at the bottom of the pulse program.

Phases can be incremented during the pulse program with ip

spnam = the name of the shaped pulse

We use the following convention:

(NOTE: normally on resonance is on C and off resonance is on C)

Example: 200u gron0

Gradients can be inverted using the following trick:

Example: 1D pulse program

PULSE PROGRAM |EXPLANATION:

#include <Avance.incl> Import nomenclature and definitions from file Avance.incl

1 ze Start (zero buffer)

ph1=0 2 2 0 1 3 3 1 ph1 is cycled as: x,-x,-x,x,y,-y,-y,y1 (incremented every scan)

;set Some remarks

2D spectra are processed with the statements below:

3. Calibration of frequency offset, pulses, gradients etc.

Is the calibrated o1 the same as in cal/1?

;EMzgpr Name of the pulse program

"d12=20u" Set up d12 to 20us

1 ze Start zero buffer

[presat (p29, pl7) 1H pulse (p1, pl1) detection]

Advanced procedure for quantitative 1D spectroscopy:

Why do we use off-resonance presaturation? Which offset do we use?

"d12=20u" Set up delays and/or pulses.

In general you can optimize a parameter by running a series of experiments,

Calibrate the proton pulse using the automation pulsecal

Calibration of nitrogen pulse

Why use d22 (which is < d2)???

Calibration of carbon pulses

Exercise: Get a spectrum, phase it and store it as a reference (wrp 2).

; for calibration of 180deg shaped pulse (i.e. Q3, G3)

Calibration of water-flip-back pulse

Calibration of soft pulse in WATERGATE

Why is the power level different than in cal/8 ?

Echo/Antiecho gradient calibration

Calibration of adiabatic pulses

4. 1D & basic 2D pulse sequences (heteronuclear correlations)

102 EM1dwg 1D spectrum using WATERGATE 3-9-19 for water suppression

104 EMzggpw5 1D spectrum using double WATERGATE

Sensitivity-enhanced 1H,15N HSQC

WATERGATE 1H,15N HSQC

What are the advantages/disadvantages of sensitivity enhanced vs WATERGATE?

1H, 15N SOFAST HMQC:

1H, 15N TROSY:

109 EMhalftrosy The TROSY principle is only used in one dimension

; EMhalftrosy (p6 ph0):f3

;"d2=3.8m" ; 1/2J(H,C)protein (!)

; hchsqc, no gradient selection, no wfb

Compare the spectra with and without filter.

113 EMfilter 1D filter, using normal pulses.

; EMfilter (p13:sp5 ph5):f2 ;arom.

Estimate amide proton T2 time