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Course Contents

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Course Contents Aim is to show you how to use a modern Bruker spectrometer for typical Biomolecular NMR experiments Setup and acquisition coffee – PowerPoint PPT presentation

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Title: Course Contents


1
Course Contents
  • Aim is to show you how to use a modern Bruker
    spectrometer for typical Biomolecular NMR
    experiments
  • Setup and acquisition
  • coffee
  • Processing, applications
  • lunch
  • Workshops (on spectrometer)
  • We will not be trying to teach you protein NMR
    (neither of us are experts!), this course is
    about getting the best out of the instrument for
    this kind of work the science is up to you!
  • Please ask questions, we are here to help!

2
Introduction
  • Sample acquisition for Protein/biomolecular NMR
    is not really any different to any other
    technique!
  • You can run standard experiments (manually or in
    automation) using the concepts
  • rpar read standard parameters
  • getprosol insert standard pulse calibrations
  • rgazg or xaua run acquisition steps
  • But you do need to do things properly (less room
    for mistakes) and think a little more!
  • Solvent suppression optimisation?
  • Sweep optimisation?
  • Relaxation problems?
  • Experiment time vs instrument availability?

3
Contents
  • Sample setup
  • Calibration and parameter setup
  • The prosol system
  • Optimising parameters
  • Acquisition
  • Selecting and setup of bio-molecular sequences
  • Cryo-probe notes
  • Summary

4
Sample Preparation
  • Samples should ideally be
  • Filled to 40 mm in the tube
  • Much more can give convection problems
  • Much less can give shimming problems (even if
    centred)
  • With Shigemi tubes make sure to use Bruker
    length chamfered bottoms (especially on
    cryo-probe)
  • Smaller tubes (e.g. 3mm) can also be used (useful
    when not concentration limited, and/or for higher
    salt concentrations)
  • Obviously you dont want floaters, layers, bent
    or dirty tubes (clean the inside, wipe the
    outside before inserting)!

5
Set starting dataset
  • It is no bad thing before you do anything else to
    ensure that you are in a suitable dataset
    something with the correct routing setup for your
    later experiments, and in your data storage area
    with a name you can later find!
  • Either
  • Start from an existing dataset and use new to
    copy parameters to new filename (can also setup
    alias names to aid this)
  • Or use new, then read suitable parameter with
    rpar
  • Use edasp and check the routing diagram if
    necessary

6
Insert sample
  • To insert a sample
  • Turn on lift air with ej (eject) - wait for air
    before releasing sample!
  • Turn off lift air with ij (inject)
  • If you have a BSMS keypad you can also use the
    Lift ON-OFF button
  • Or you can start the software equivalent of the
    keypad using bsmsdisp
  • Or for a BACS/Sample Jet sample changer use sx
    and holder position sx 23 insert sample in
    holder 23 sx ej eject current sample only
  • For NMR-CASE the sample changer is driven by ej/ij

7
Set temperature
  • Start edte, check
  • Target temperature is correct
  • Heater is on
  • Airflow is correct (higher flow usually better to
    avoid temperature gradients, but ensure sample
    does not lift!)
  • Temperature (and heater output) are stable if
    not the vt unit should be tuned!
  • After the temperature appears correct it can take
    a further 5 minutes for the sample temperature to
    fully equilibrate!
  • If temperature is important you should calibrate
    the VT unit (use deuterated MeOH on high-field
    systems)

8
Lock
  • If required first read some standard shims with
    rsh
  • Use lock and select from list (or enter solvent
    directly with for example lock H2OD2O)
  • For manual lock use bsmsdisp (or keypad)
  • Make sure SWEEP is on
  • Set reasonable defaults for frequency, power and
    gain (lopo)
  • Adjust FIELD to put ringing pattern in centre of
    lockdisp
  • Click LOCK button.

9
Tune probe
  • First make sure the nucleus setup is as you want
    to use (e.g 1H/13C/15N)
  • With automatic tuning probe use atma or atmm.
  • With manual probe use wobb and the Tune and Match
    wands at the base of the probe make sure to
    turn the correct ones!
  • Note that if you only have two preamplifiers you
    might have to temporarily recable to tune the 3rd
    channel!
  • In principle you could do this after shimming
    (but there are a very few probes where shimming
    can change slightly with tuning)

10
Shim
  • To optimise the shimming you would use one or
    more of
  • Topshim gradient shim - easiest and best, but
    TopSpin 2 only
  • Gradshim good results (if setup correctly!)
  • Tune/simplex take longer that gradient methods
  • Manual shimming
  • For samples in H2O/D2O then 1H gradient shimming
    can be performed in 1D (quick, on-axis only) or
    3D modes (slower, but all shims)
  • For deuterated solvents 2H gradient shimming can
    be used to correct on-axis shims only, followed
    by a tune to correct major off-axis shims.
  • It can be helpful to periodically use a H2O/D2O
    sample and 3D gradshim to keep high-order
    off-axis shims updated in your default shim
    files.

11
Common TopShim options
  • topshim run default 1D topshim (optimises for
    current observe nucleus, with method according to
    solvent)
  • Topshim 3d full 3D shimming (5-30 minutes)
  • Topshim 3dfast 3D shimming (5-10 minutes)
  • Topshim tunea 1D gshim, then off-axis tune
    x,y,z, xz,yz
  • Topshim tuneaxyz as above, but only shims x, y,
    z
  • Topshim shigemi ignore weak signals from edges
  • Topshim report view results
  • Topshim gui open graphical user interface
  • Topshim help open manual for full details!

12
Optimise lock
  • For maximum stability, and minimum recovery time
    (e.g. following gradient pulses) lock quality is
    important, especially the phase.
  • Check lock power is appropriate (avoid
    saturation)
  • Run loopadj to perform
  • autophase
  • autogain
  • set lock filter parameters according to final
    gain (i.e. noise)

lock phase wrong!
13
Autoshim
  • For long-term experiments the shim needs to be
    maintained against the effects of field drift
    if uncorrected in extreme cases you might even
    lose lock!
  • Use bsmsdisp and the autoshim tab to set
  • INTERVAL to 2 or 3 s (no shorter)
  • Shim step of 1 for Z, Z2, Z3, X, Y, XZ, YZ (avoid
    large steps)
  • Turn AUTOSHIM on!
  • Note that autoshim can be used within gradient
    experiments lock fluctuations during gradient
    pulses are handled!

14
Contents
  • Sample setup
  • Calibration and parameter setup
  • The prosol system
  • Optimising parameters
  • Acquisition
  • Selecting and setup of bio-molecular sequences
  • Cryo-probe notes
  • Summary

15
General setup of parameters
  • Read a standard set of experiment parameters
    rpar
  • Set pulse and power levels getprosol
  • Check and alter specific parameters
  • eda view all parameters AcquPars
  • ased view significant parameters (from pulse
    program, use edcpul PulseProg to view)
  • Or enter parameter name (ns, d1, p1, etc.)
  • Use expt to determine the acquisition time

16
Pulse calibration
  • Most non-trivial experiments require accurate
    calibration of pulses to work at their best, or
    even to work at all.
  • For samples in water, especially if containing
    salts or buffers, the pulse length can vary
    significantly even if the probe is perfectly
    tuned and matched.
  • Use au program pulsecal to perform proton
    calibration
  • If sample is in H2O/D2O and O1 is correct use
    pulsecal fast
  • Warning can give wrong answer if there are no
    proton signals!
  • Or determine manually
  • Scan sequence of values with popt / paropt
  • Use trial and error with zg or gs
  • Remember to use a suitable pulse program! (not
    zg30!)

17
A pulse calibration tip
Best presat
  • Create a pulse program using 4?90 pulses (e.g.
    zg360)
  • Can then change p1 directly while looking for
    signal null.
  • Additionally, the sharp component of residual
    signal gives correct o1 position for water
    suppression

18
Further pulse calibration
  • Usually you can assume that existing pulse
    calibrations of low frequency nuclei (13C, 15N,
    2H) are correct but someone should check them
    occasionally!
  • To calibrate decouple pulses use pulprog of
    decp, e.g.
  • decp90 inverse pulse on f2 (13C)
  • decp90f3 inverse pulse on f3 (15N)
  • decp902hf4 inverse pulse on f4 (2H)
  • decp90sp inverse shape pulse on f2
  • decp180 inverse 180 on f2
  • Remember to set heteronuclear offset (o2/o3/o4)
    correctly (especially if calibrating
    long/selective pulses!)

19
Getprosol with calibrated pulses
  • Having calibrated the observe pulse for your
    sample you should repeat getprosol to reset all
    related pulses getprosol nucleus length
    powere.g. getprosol 1H 14.5 1.0
  • If you intend running a number of different
    experiments on this sample it is worth making a
    macro to put all calibration actions together
    edmac name

getprosol 1H 14.5 1.0 o1 2992.45 sw 12.0
20
Contents
  • Sample setup
  • Calibration and parameter setup
  • The prosol system
  • Optimising parameters
  • Acquisition
  • Selecting and setup of bio-molecular sequences
  • Cryo-probe notes
  • Summary

21
The prosol system a reminder
  • Standard calibrations stored in table for each
    probe, and each solvent (if required, or all)
    edprosol to view/edit
  • Contains values for all commonly used pulses
  • hard pulses (90, decoupling, tocsy, )
  • irradiation powers (presat, noe, )
  • selective pulses (Calpha, Cali, )
  • adiabatic pulses (180 inversion, refocussing,
    decoupling)
  • hardware pulses (gradient pulse, trim pulse, )

22
Setup of prosol table
  • Standard setup (TopSpin 2)
  • File-gtSet default pulse widths (standard lengths,
    shapes etc.)
  • Insert your calibrated length and power for 90
    pulse
  • Save say yes to recalculate all powers
  • For TopSpin 1 you just have to put the values in!
  • Dont forget hardware pulses in global set

23
How prosol works - Pulse naming conventions
  • All Bruker pulse programs follow standard
    conventions for naming pulses/power levels, e.g.
  • Channel F1 high power pl1, 90 p1, 180 p2
  • Channel F2 high power pl2, 90 p3, 180 p4,
  • Described in Param.info

24
How prosol works - Relations Files
  • Relations files are the link between the values
    stored in the prosol tables and the parameters in
    pulse programs

getprosol
25
Getprosol problems
  • If getprosol appears to set something incorrectly
    then you should check the table entries if not
    obvious where the correct value should be look at
    the relations file!
  • The relations files are in TOPSPINHOME/conf/in
    str/ltcurinstgt/prosol/relations/ usually the
    default file is used
  • A different relations file can be used if
    specified in the pulse program ltprosol
    relationsgttriple
  • Entries can be simple assignment, or involve
    calculation P1P90F1
    90 deg pulse F1 P2P90F12
    180 deg pulse F1 SP19PLSH3F1 0.87
    90 deg, F1, wet

26
Creating a shaped pulse
  • Common shapes are installed as part of expinstall
    but if you need different ones then create them!
  • Start the shape tool with stdisp
  • Select shape from menu, then set size and any
    other parameters.
  • Can then calibrate, modulate, simulate,
  • and put into edprosol!

27
Contents
  • Sample setup
  • Calibration and parameter setup
  • The prosol system
  • Optimising parameters
  • Acquisition
  • Selecting and setup of bio-molecular sequences
  • Cryo-probe notes
  • Summary

28
Optimising solvent suppression
  • Depending upon the suppression sequence you want
    to use you might need to fine-tune the
    suppression
  • frequency/power for presaturation methods
  • power for flip-back pulse or other shapes (e.g.
    WET)
  • As for pulse calibration can scan a range of
    values with popt/paropt
  • Or use trial and error probably gs mode is
    essential here as steady-state conditions are
    often important.
  • gs (go setup) repeats the first scan of the
    experiment while allowing interactive change of
    parameters

29
Solvent suppression tips
  • If radiation damping is a problem more power
    might be required for pulses where full signal
    exists
  • increase presat power to say 100 Hz
  • increase power of first WET pulse
  • Gradients and/or volume selection sequences can
    be effective
  • noesygppr1d presat, gradient purge and
    composite pulse
  • Gradient sequences can sometimes benefit from
    increased gradient power
  • Use smoothed-square shape (SMSQ10.100 instead of
    SINE.100)

30
Adjust receiver gain
  • For correct digitisation the signal must fit
    within the ADC
  • if it is too large clipping creates large
    distortions in the spectrum
  • if it is very small then dynamic range and noise
    can suffer
  • Automatic adjustment is with the command rga
  • To manually adjust simply change the parameter rg
  • First start gs mode (repeats first scan
    endlessly)
  • View acquisition window (acqu)
  • Adjust rg so the signal is well within the height
    of the screen (in later software version this is
    shown explicitly with red lines)
  • Note that on a modern high dynamic range
    digitiser (DRU) you will get full sensitivity if
    rg gt 64!

31
Contents
  • Sample setup
  • Calibration and parameter setup
  • The prosol system
  • Optimising parameters
  • Acquisition
  • Selecting and setup of bio-molecular sequences
  • Cryo-probe notes
  • Summary

32
Start acquisition
  • To start an acquisition from scratch type zg,
    this zeroes any existing data and then goes.
  • To repeat an acquisition and add to the existing
    data use go
  • During a long acquisition you can look at the
    data accumulated so far by typing tr (transfer)
    at the end of the next scan the data is available
    for processing
  • To stop a long acquisition early use halt
  • To abort an acquisition use stop (any
    un-transferred data will be lost)

33
Running further experiments
  • At this point we should have acquired a decent 1D
    spectrum always worth doing to check everything
    OK!
  • Can now setup further experiments of whatever
    type are required
  • iexpno or new create a new dataset
  • rpar read parameters for next experiment
  • run your macro - set calibrations and
    optimisations (getprosol, set o1, etc)
  • Check one more time that everything is correct
    ased, expt,
  • Can then start with rga and zg
  • or setup a sequence of experiments and use
    multizg
  • or in TopSpin 2 use

34
Command spooler
  • Can queue acquisition and general commands using
    the command spooler qu ltcommandgt
  • Acquisition commands can be set to queue
    automatically (setres option) rga, go, zg,
    atma, etc.
  • A nicer way to set-up multiple experiments than
    multizg!
  • Can also use at to run command at specific time.

Right-click spooler in task bar to view/edit queue
35
Multi-dimensions
  • Can be setup and run essentially the same as a
    1D!
  • eda will now has multiple columns for each
    frequency axis
  • Can run planes of 3D by simply setting TD to 1 in
    appropriate axis a useful check on parameter
    setup, and for sensitivity tests
  • Data in nD experiments is automatically
    transferred after each increment good idea to
    perform xfb or ftnd with limited amount of data
    to check that experiment is working!

36
Contents
  • Sample setup
  • Calibration and parameter setup
  • the prosol system
  • Optimising parameters
  • Acquisition
  • Selecting and setup of bio-molecular sequences
  • Cryo-probe notes
  • Summary

37
Selecting your experiment
  • NMR guide (Help - Start NMR Guide) a valuable
    resource!

38
Standard Bio-molecular parameters
  • In addition to the prosol settings and basic
    sweep-widths you might see other common parameter
    settings
  • ZGOPTNS for some sequences can set to
    -DLABEL_CN to indicate that the sample is
    double labelled (e.g for N15 dec.)
  • CNST21/22/23 offset of CO, Calpha, Caliphatic
    in ppm
  • D20-D29 delays based on coupling constants
  • and many others!
  • All will be described in the pulse program
    comments, and mostly appear in ased.
  • If you read a standard parameter set (rpar) then
    of course they should have reasonable values
    already but they might need adjusting for your
    sample!

39
Generating New Parameter Sets
  • There are many more pulse programs than parameter
    sets which cover the many variations of the
    standard experiments. To implement these
  • Start from the closest standard parameter set
    (rpar)
  • Change to the new pulprog and read the comments
  • eda, check nd0 - if changed then check sw(f1)
  • check FN_mode (if incorrect ased will give
    error!)
  • ased, check delays and gradient ratios
  • If changing nucleus remember to set nuc1
    correctly in both dimensions. Use f1ref to
    adjust processing frequencies

40
Accessing Pulse Programs
  • Use edpul to view/edit all sequences, remember
    wildcards can be used (e.g. edpul hsqcgp)
  • In Topspin can turn on comment display to aid
    selection (Options-Comment on/off)
  • Use edcpul (or PulseProg tab) for current
    sequence

showpp
41
BioTools
  • Another approach to experiment selection and
    setup is the BioTools software
  • Leads user through setup and calibrations, and
    cascades these onto later experiments
  • Multiple experiments are automatically queued and
    executed (BioTools is a front-end to IconNMR!)

42
Contents
  • Sample setup
  • Calibration and parameter setup
  • The prosol system
  • Optimising parameters
  • Acquisition
  • Selecting and setup of bio-molecular sequences
  • Cryo-probe notes
  • Summary

43
Cryo-probes
  • In short, use them just like any other probe!
  • Power requirements are less ensure you are
    using powercheck. This also means rf heating is
    less of an issue!
  • Gas flow is important (670 l/h)
  • Radiation damping will have more of an influence,
    but is treated in the same way as normal probes.
  • Remember high-sensitivity can allow some
    otherwise impossible experiments for example
    carbon observe experiments can give excellent
    dispersion.
  • Fast methods such as SoFAST and Projection
    reconstruction are more often appropriate.

44
Cryo-probe Tubes
  • If samples are very conductive then the s/n is
    largely set by the solvent!
  • Small tubes (e.g. 3 mm) can be very effective
  • easier solvent suppression
  • shorter pulses
  • will tune to any salt concentration
  • at high salt will get same s/n for same
    concentration of sample (less sample), and if
    concentration can be increased will gain!
  • Or if specified on your system can use shaped
    tubes.

45
Contents
  • Sample setup
  • Calibration and parameter setup
  • the prosol system
  • Optimising parameters
  • Acquisition
  • Selecting and setup of bio-molecular sequences
  • Cryo-probe notes
  • Summary

46
Summary
  • There is nothing special! Lock, shim, pulse
    calibration are more important but that just
    means doing things properly.
  • Then most things can be run pretty easily
  • Use standard parameter sets (rpar)
  • Fill in calibrated numbers (getprosol, or by
    macro)
  • Adjust rg, timings (ns, etc), if required
  • run with spooler or multizg
  • And if you like a really automated life use
    BioTools!
  • We can try some of this stuff in the lab this
    afternoon
  • Or you can read the manual! (Help, manuals, 3D
    triple-resonance experiments)
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