Contents
• Data processing
• 2D and nD data
• Making things look nicer
• Experiment examples
• Triple resonance experiments
• Metabonomics
• Screening
• Other software
• AMIX
• AUREMOL
2D processing
Workflow:
• Transform – xfb
• Phase correct
• Baseline – abs1, abs2,
abs2.water
• Set contours – levcalc
• Peak pick – pp
• Integrate – int2d
Making it look nice
• Improved spectra = improved peak picking
• QFIL baseline correction in F1 to remove water stripe
• WATERGATE to improve water suppression – wg in pulse
sequence / parameter set name
• Strip transforms to transform only part of the spectrum –
particularly useful for 3D, and 15N HSQC
• T1away! Nonlinear processing, so bad for integration, but
may be useful for peakpicking
Water “suppression” - QFIL
• Parameter bc_mod controls
baseline correction
• NO= nothing
• QUAD=dc offset
correction
• QPOL/QFIL = filter out
zero frequency component
• Need to also set bcfw
• Use COROFFS if water not
in the centre of the spectrum
Cropping the spectrum – strip transform
•Usually, centre of detected dimension is water frequency
•Usually, detect amide protons – all have shift > 4.7
•Less points = faster data manipulation
•Useful when viewing cubes/planes of cubes
•Parameters:
•STSR = first output point number, zero = left of spectrum
•STSI = number of output points, SI/2 = left half
•Note that points relate to SI , so to show only right half of
dimension, set STSR to SI/2 and STSI to SI/2
More dimensions
• As 2D, but:
• Need to get phase parameters from 2D planes
• Only real part of processed data kept (so phase in advance)
• Strip transform more commonly used (reduced processed data
size)
• Note that processing takes time!
• ftnd command handles up to 6D data, at least
• Slices of nD data can be processed with xfb
• Peak picking/integration still possible
FTND
• General FT of nD data
• Options – ftnd a b c
• a = dimensions to process and order, e.g. 31 processes f1 and f3,
0 = process all directions in default order
• b = output procno
• c = dlp – use delayed linear prediction
• Example: ftnd 321 998 dlp
• Transform f3, f2, and f1, store result in procno 998, use dlp.
• DLP ensures no distortions arise from linear prediction
• In Topspin 1.3, use ft3d for 3D data
Extracting subsets
• For nD data, one may want to view a subset, e.g. 2D plane
from 3D cube
• projcbp, projcbn, sumcb = positive, negative and sum cube
projections
• projplp, projpln, sumpl = positive, negative and sum planes
• To extract single planes/cubes, use xfb/ft3d
• Promts for plane/cube orientation and number
Peak picking/integration
• pp brings up appropriate dialogue box – any number of
dimensions
• Set thresholds using contour levels – remember to save!
• Check and adjust manually!
• Correlated windows useful
• Can import peak list from another dataset for comparison
• Can annotate peaks
• int for integration
Experiments – naming/information
• Pulprog.info – describes Bruker naming system
• NMR guide – pulse sequences, description, theory
• 3D triple resonance manual (help->manuals)
• Examples:
• HNCAGP3D – both intra- and inter-residue HN->N->Ca correlation
• HNCOCAGP3D – only inter-residue HN->N->Ca correlation
• C_CANCO_3D – carbon-detected Ca->N->CO
Pulprog.info
•Lives in $topspinhome/exp/stan/nmr/lists/pp
•Can see from edpul in 2.1
•Describes two-letter codes used for naming Bruker sequences
3D/triple resonance experiment families
• Backbone assignment, e.g.:
•
•
•
•
HNCO – inter-residue connection
HNCA – both intra- (strong) and inter-residue (weaker)
HN(CO)CA – only inter-residue
HN(CA)CO – both
• Backbone-sidechain, e.g.:
• HN(CO)CACB – Ca / Cb have opposite phase
• TOCSY experiments for whole sidechain
• Coupling constant measurements – IPAP
• NOESY type experiments
Worked examples – HNCA/HN(CO)CA
• HNCA shows intra- and inter-residue correlations
• HN(CO)CA only shows inter-residue correlations
•1JNCa = 11 Hz
•2JNCa = 7 Hz
•1JNCO = 15 Hz
•2JNCO = <2 Hz
Example: Hymenistatin
• 8 residue cyclic peptide
• Sequence: -Pro-Pro-Tyr-Val-Pro-Leu-Ile-Ile• No terminal residues, but prolines (no NH group) provide
key to assignment
• Few peaks, so can work with 2D planes of 3D experiments
• H-Ca plane of HNCA and HNCOCA for backbone assignments
• H-N plane for amide N
Interpreting HNCA/HN(CO)CA pair
Green = intra
HN(n)->Ca(n)
Black = inter
HN(n)->Ca(n-1)
Interpreting HNCA/HN(CO)CA pair
Vertical correlation:
HN(n)Ca(n) <->
HN(n)Ca(n-1)
Horizontal correlation:
HN(n)Ca(n) <->
HN(n+1)Ca(n)
Interpreting HNCA/HN(CO)CA pair
• Follow connections – vertical, then horizontal, then vertical –
move to increasing residue number
• Ca peak only in HNCA:
•
next residue is proline
•
next residue is N-methylated
•
current residue is terminus
• (inter) Ca peak missing in HNCA = preceding residue is proline
• No peaks seen for a proline residue preceding another proline
-Pro-Pro-Tyr-Val-Pro-Leu-Ile-IleInterpreting HNCA/HN(CO)CA pair
Interpreting HNCA/HN(CO)CA pair
Other aspects of biological NMR
• Bio-NMR is not just 3D triple resonance!
• Screening experiments to observe ligand binding
• SAR by NMR (15N HSQC)
• Saturation transfer difference
• WATER-LOGSY
• Metabonomics
• Study of relationships between metabolites and diseases etc.
• Can be combined with LC/MS
Screening - STD
• Saturation transfer difference (STD) – identify ligands which
bind to your protein
• Does not require much protein (has been done on 1
nmolar!)
• Observe ligand signals – large excess of ligand useful
• No labelling required
• Can observe competition between ligands
• Some information about binding site on ligand
• Suitable for large proteins (ideally >20kDa)
STD – how it works
1.
Selective irradiation of a well
separated protein signal
2.
Spin diffusion quickly spreads the
saturation to all protein-protons
3.
Intermolecular NOE transfers
saturation to ligand-protons at the
binding site
4.
Exchange between bound
(saturated) and free ligands
allows further ligand saturation if
ligand in excess
1. selective
irradiation
H
H
H
H
H
2. spin
H
diffusion
protein
H
H
H
H
H
H
H
H
ligand
H
H
H
H
3. intermolecular
NOE
H
H
H
H
H
H H
H
H
H
Patent: Bernt Meyer, University of Hamburg, Germany
Ref:
Mayer & Meyer JACS 123 (2001) 6108-6117
Review:
Angew. Chem. Int. Ed., 42 (2003, ) 864-890
STD - results
• Screening: identification of the binding component(s) from
a mixture through positive NOE signals
• Epitope mapping: parts of the ligand in contact with the
protein give strongest response
• Competition: add a known binder, response from candidate
reduced if it binds at correct site
STD - implementation
• Parameter sets e.g. STDIFFESGP
• Frequency list: protein on-res (e.g. <0 ppm – away from
ligand) and off-res (e.g. –20ppm)
• Can include CPMG (t2) filter to remove broad protein
signals (e.g. stdiffesgp.3)
• Standard bruker sequences make pseudo 2D
• Need to take the difference afterwards
• Sequence available with automatic subtraction (i.e. output
spectrum is difference) on request
Metabonomics
• Statistical analysis of metabolite solutions, e.g. urine,
plasma
• Need good quality data!
• Parameter sets available in TS2 which take full advantage
of hardware/software improvements
• Take care of temperature regulation/shimming
• Water suppression – noesygppr1d
• Good suppression, integratable spectra
• Not a NOESY! Mixing time short, d8=10msec
Metabonomics parameter sets
• MET_NOEGPPR1D – noesy presat
• MET_DIFFUFILT – diffusion filtered (remove smaller
molecules)
• MET_CPMGPR1D – CPMG t2 fiter (remove protein signals)
• MET_COSYGPPR – COSY, with presat
• Parameter sets use improved digital filters
• Noesygppr1d spectra – use akp0.noe for phasing
Example – water suppression
AUREMOL
AMIX – mixture analysis/statistics