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)