Title: Course Contents
1Course 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!
2Introduction
- 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?
3Contents
- Sample setup
- Calibration and parameter setup
- The prosol system
- Optimising parameters
- Acquisition
- Selecting and setup of bio-molecular sequences
- Cryo-probe notes
- Summary
4Sample 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)!
5Set 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
6Insert 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
7Set 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)
8Lock
- 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.
9Tune 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)
10Shim
- 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.
11Common 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!
12Optimise 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!
13Autoshim
- 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!
14Contents
- Sample setup
- Calibration and parameter setup
- The prosol system
- Optimising parameters
- Acquisition
- Selecting and setup of bio-molecular sequences
- Cryo-probe notes
- Summary
15General 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
16Pulse 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!)
17A 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
18Further 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!)
19Getprosol 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
20Contents
- Sample setup
- Calibration and parameter setup
- The prosol system
- Optimising parameters
- Acquisition
- Selecting and setup of bio-molecular sequences
- Cryo-probe notes
- Summary
21The 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, )
22Setup 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
23How 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
24How prosol works - Relations Files
- Relations files are the link between the values
stored in the prosol tables and the parameters in
pulse programs
getprosol
25Getprosol 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
26Creating 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!
27Contents
- Sample setup
- Calibration and parameter setup
- The prosol system
- Optimising parameters
- Acquisition
- Selecting and setup of bio-molecular sequences
- Cryo-probe notes
- Summary
28Optimising 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
29Solvent 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)
30Adjust 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!
31Contents
- Sample setup
- Calibration and parameter setup
- The prosol system
- Optimising parameters
- Acquisition
- Selecting and setup of bio-molecular sequences
- Cryo-probe notes
- Summary
32Start 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)
33Running 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
34Command 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
35Multi-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!
36Contents
- Sample setup
- Calibration and parameter setup
- the prosol system
- Optimising parameters
- Acquisition
- Selecting and setup of bio-molecular sequences
- Cryo-probe notes
- Summary
37Selecting your experiment
- NMR guide (Help - Start NMR Guide) a valuable
resource!
38Standard 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!
39Generating 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
40Accessing 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
41BioTools
- 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!)
42Contents
- Sample setup
- Calibration and parameter setup
- The prosol system
- Optimising parameters
- Acquisition
- Selecting and setup of bio-molecular sequences
- Cryo-probe notes
- Summary
43Cryo-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.
44Cryo-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.
45Contents
- Sample setup
- Calibration and parameter setup
- the prosol system
- Optimising parameters
- Acquisition
- Selecting and setup of bio-molecular sequences
- Cryo-probe notes
- Summary
46Summary
- 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)