course contents aim is to show you how to use a modern bruker spectrometer for typical biomolecular...
TRANSCRIPT
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!
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
• rga;zg 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?
Contents
• Sample setup
• Calibration and parameter setup
• The prosol system
• Optimising parameters
• Acquisition
• Selecting and setup of bio-molecular sequences
• Cryo-probe notes
• Summary
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 don’t want floaters, layers, bent or dirty tubes (clean the inside, wipe the outside before inserting)!
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
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 23sx ej eject current sample only
• For NMR-CASE the sample changer is driven by ej/ij
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)
Lock
• If required first read some standard shims with rsh
• Use lock and select from list (or enter solvent directly with for example lock H2O+D2O)
• 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.
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)
tunemat
ch
Position of dip in curvewhen correctly optimised
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.
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!
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!
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!
Contents
• Sample setup
• Calibration and parameter setup
• The prosol system
• Optimising parameters
• Acquisition
• Selecting and setup of bio-molecular sequences
• Cryo-probe notes
• Summary
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 getprosol
UNDO
• 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
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!)
A pulse calibration tip
• Create a pulse program using 490° 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:
Best presat
3.153.203.253.303.353.403.453.503.553.603.653.703.753.803.85 ppm
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!)
Getprosol with calibrated pulses
• Having calibrated the observe pulse for your sample you should repeat getprosol to reset all related pulses:
getprosol nucleus length power
e.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 –1.0o1 2992.45sw 12.0…
Contents
• Sample setup
• Calibration and parameter setup
• The prosol system
• Optimising parameters
• Acquisition
• Selecting and setup of bio-molecular sequences
• Cryo-probe notes
• Summary
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, …)
Setup of prosol table• Standard setup (TopSpin 2):
• File->Set 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!
• Don’t forget ‘hardware’ pulses in ‘global’ set:
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
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
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/instr/<curinst>/prosol/relations/ usually the default file is used
• A different relations file can be used if specified in the pulse program: <prosol relations>=“triple”
• Entries can be simple assignment, or involve calculation: P[1]=P90[F1]; # 90 deg pulse F1
P[2]=P90[F1]*2; # 180 deg pulse F1 SP[19]=PLSH3[F1] +0.87; # 90 deg, F1, wet
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!
Contents
• Sample setup
• Calibration and parameter setup
• The prosol system
• Optimising parameters
• Acquisition
• Selecting and setup of bio-molecular sequences
• Cryo-probe notes
• Summary
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
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)
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 >= 64!
Contents
• Sample setup
• Calibration and parameter setup
• The prosol system
• Optimising parameters
• Acquisition
• Selecting and setup of bio-molecular sequences
• Cryo-probe notes
• Summary
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)
haltstopzg
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 …
Command spooler• Can queue acquisition and general commands using the
command spooler: qu <command>
• 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
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!
Contents
• Sample setup
• Calibration and parameter setup
• the prosol system
• Optimising parameters
• Acquisition
• Selecting and setup of bio-molecular sequences
• Cryo-probe notes
• Summary
Selecting your experiment
• NMR guide (Help - Start NMR Guide) a valuable resource!
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!
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
Accessing Pulse Programs
• Use edpul to view/edit all sequences, remember wildcards can be used (e.g. edpul hsqc*gp*)
• In Topspin can turn on comment display to aid selection (Options-Comment on/off)
• Use edcpul (or PulseProg tab) for current sequence
showpp
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!)
Contents
• Sample setup
• Calibration and parameter setup
• The prosol system
• Optimising parameters
• Acquisition
• Selecting and setup of bio-molecular sequences
• Cryo-probe notes
• Summary
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.
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.
Contents
• Sample setup
• Calibration and parameter setup
• the prosol system
• Optimising parameters
• Acquisition
• Selecting and setup of bio-molecular sequences
• Cryo-probe notes
• Summary
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)