ACME - Amplitude-Constrained Multiplet Evaluation
Measurement of Homonuclear Proton Couplings from Regular 2D COSY Spectra. (HTML)
Frank Delaglio, Zhengrong Wu and Ad Bax (2001)
J. Magn. Reson., 149, 276-281. (PDF)
IMPORTANT NOTE ON EXPERIMENTAL REQUIREMENTS
As described in the paper cited above, typical use of ACME is
based on two assumptions:
So, based on these points, consider the following:
- All multiplets in the spectrum have the same amplitude,
and this amplitude must be determined in order to measure
accurate coupling values.
- ACME can establish this amplitude from the diagonal signals of
the COSY spectrum.
The spectrum must be fuly relaxed in order for all multiplets
to have the same amplitude, so a suitably long interscan delay
should be used.
Since the diagonal signals have to be measured, use ACME with an "ordinary"
phase-sensitive COSY, rather than a more typical DQF-COSY (which has
the diagonal suppressed).
Solvent suppression methods will attenuate signals differently,
so that multiplet intensities will no longer be uniform.
For this reason, common applications of ACME use samples in D2O.
There may be other, sample-specific issues such as chemical exchange
that will affect the analysis.
To use ACME, you will first have to get and install the latest version of
NMRPipe from the FTP site. The tools of ACME are included in the current
release. A directory of sample COSY data and scripts for ACME is provided
in the file "pub/acme.tar.Z" at the FTP site.
Once downloaded, the sample data directory, called "acme",
can be extracted with the command:
zcat acme.tar.Z | tar xvf -
For the latest NMRPipe FTP instructions and password information, send
email to email@example.com
ACME is an interactive interface for measuring coupling constants from
cross peaks in regular 2D COSY spectra. ACME is built from tools in the
NMRPipe System. The "acme" demonstration directory contains spectra and
scripts for examples of coupling constant extraction for the protein ubiquitin.
The main idea behind ACME is that active couplings can be extracted accurately
from individual COSY multiplets if the amplitude of the multiplet
is known and held fixed during a fitting procedure. If the spin systems
in the sample are fully relaxed, the amplitude will be uniform for all
cross peaks, and it can be measured readily from one or more diagonal peaks.
So, in practice, use of ACME will involve the following steps:
Measure COSY spectrum with conditions that allow the spin systems to
be fully relaxed at the start of the COSY sequence.
A numerical diagonal processing scheme is used to prepare two versions
of the COSY spectrum:
One version will contain the diagonal signals
only, phased in absorptive mode. This version of the spectrum will be
used to measure the amplitude.
The other version will contain the cross peaks only, with the
diagonal numerically subtracted. This version will be used to measure
active couplings in the cross peaks.
The ACME program will first be used in diagonal mode to fit one
or more peaks in the diagonal-only spectrum. Fitting more than
one diagonal peak will help establish average amplitude as well
as its uncertainty. Note well that the amplitudes for diagonal
peaks may have to be scaled by 1/2, 1/3, etc. to account for
Using the amplitude value determined from the diagonal, the ACME
program will be used to measured the couplings in the cross peak
PROCESSING COSY SPECTRA
In order to prepare for using ACME, two versions of the COSY spectrum are
created, one with the diagonal numerically subtracted, and another with
the diagonal alone, phased so that the diagonal signals are absorptive.
An example of the steps involved is given in the following scripts:
||Usual NMRPipe conversion of the raw FID data. This script creates the
||COSY processing with diagonal suppression. This script creates the
||Processing to create a diagonal-only spectrum. This script creates
the spectrum "diag.ft2".
||This script extracts small regions of interest from the processed data,
for convenience. This script creates the extracted spectral data "ext.dat"
The ACME program is usually invoked with several command-line arguments
describing the data to analyze. As with all other programs in the NMRPipe
system, command line arguments can be listed by using the "-help" flag:
Since there are several important command-line arguments required by ACME,
it is convenient to create a short UNIX script to invoke ACME with suitable
arguments. The demo directory includes two such scripts, "diagView" (for
analyzing the diagonal-only data) and "cosyView" (for analyzing the the
COSY cross peak data). A typical usage for diagonal peak analysis is:
ACME -diag -parent test.ft2 -in diag.dat -noise 3.0e+4 -hi 1.3e+7
The average amplitude determined by diagonal fitting will then be used as
the "-amp" argument for cross peak coupling analysis. A typical
usage for cross peak analysis is:
ACME -amp 1340 -parent test.ft2 -in ext.dat -noise 3.0e+4 -hi 8.5e+5
When the ACME program is started, the File Window
will appear. This window allows selection of the input spectrum and color
choices. In order to display the selected spectrum and begin using ACME,
click the Start button here. When the Start button is clicked, a Spectrum Viewing Windowwill appear, and the selected spectrum will be drawn as a contourplot. If the spectrum is large or complicated, it may takea while before the spectral graphic appears.
SPECTRUM VIEWING WINDOW
Viewing Window is used to zoom and select regions of the spectrum for
analysis. It also has controls for adjusting the contour heights and redrawing
the spectral display. Note that more than one Spectrum
Viewing Window can be used at a given time, so that multiple regions
of the spectrum can be manipulated. The mouse buttons perform the following
actions when used on the contour display of a
||drags a set of cursor lines through the spectrum. If more than one
Spectrum Viewing Window is available, the cursors
will track in all windows.
||drags a zoom box which is used to select a region of cross-peaks for
fitting in the Fit Window as described below.
||drags a zoom box to create an expanded view of the selected region.
By default, the expanded view will be drawn in a new
ACME FIT WINDOW
Fit Window displays three corresponding spectral
displays, from top to bottom: the spectral region selected for ACME analysis,
the current ACME model of the multiplets in the region, and the difference
between the spectrum and model (the residual).
Clicking the LEFT mouse button on a multiplet
position in the
Fit Window will insert a signal
at that location.
The following actions are available as buttons in the
||Starts the iterative nonlinear least-squares ACME fitting procedure
with the current settings (see below), updates the settings according to
the fit parameters, and displays the results. During the fit, a terminal
window will appear to display the results at each iteration. During
the fit, you can type Control-C to stop the iterations, although the program
will first complete one cycle of iterations before stopping. The results
displayed in the terminal window give the active couplings for each signal,
and the current RMS of the residual.
||Simulates a spectral model using the current settings, and displays
||This feature is currently not implemented; in future versions, it will
read previously recorded settings.
||Records the current settings in a table. Each accepted fit region will
be recorded in a separate table, in the "acmeDir" subdirectory.
ACME PARAMETER WINDOW
The ACME Parameter Window shows the current parameters
for each manually selected multiplet signal in the Fit
Window. The parameter listed for each signal are:
||Active Coupling, Hz
||X-Axis Position of Multiplet Center, Pts
||X-Axis Linewidth, Hz
|X Passive A
||X-Axis First Passive Coupling, Hz
|X Passive B
||X-Axis Second Passive Coupling, Hz
|X Passive C
||X-Axis Third Passive Coupling, Hz
||Y-Axis Position of Multiplet Center, Pts
||Y-Axis Linewidth, Hz
|Y Passive A
||Y-Axis First Passive Coupling, Hz
|Y Passive B
||Y-Axis Second Passive Coupling, Hz
|Y Passive C
||Y-Axis Third Passive Coupling, Hz
||Used to record assignment information, if any
Clicking with the mouse on a Parameter Checkbox will toggle the state
of that parameter for fitting purposes. If the checkbox is "on" (green)
the parameter will be varied during the fit. If it is "off" (gray), the
parameter will be held fixed during the fit.
Clicking with the mouse on a Parameter Value will reset the value for
that parameter. Clicking multiple times will also toggle the Parameter
Checkbox, so that you can more conveniently turn particular passive couplings
on and off.
Clicking with the mouse on a Parameter Label will change the Multiplicity
Factor (if any) for that parameter. Only the amplitude and passive couplings
have multiplicity factors.
STEPS FOR ANALYZING A MULTIPLET REGION
Use the MIDDLE mouse button to select a multiplet region from a Spectrum
View Window. Best results will be obtained if the selected region is
as simple as possible for the given coupling measurement, and includes
some baseline. The selected region will then be drawn in the Fit
Use the LEFT mouse button in the
ACME Fit Window
one signal at the approximate center of each multiplet to be fit.
Carefully adjust and review the parameter setting for each signal
ACME Parameter Window. If you are not
sure about how many passive couplings to include, try using none at first,
then retry the fit with additional passive couplings included.
Click the "Fit" button in the Fit Window.
Evaluate the results. Things to check for:
If needed, adjust the parameter settings, and retry the Fit. Otherwise,
click the "Accept" button in the Fit Window, which will record the current
parameters in a table.
Is the reported RMS of the residual too high?
Does the appearance of the model match that of the spectrum?
Is there a substantial residual visible in the regions of the fitted multiplets?
Do any of the parameters have unreasonable values, for example, negative
widths or couplings?
PROBLEMS IN THIS VERSION OF ACME
The fitting module does not always identify convergence, especially when
the parameters are mostly degenerate. So, the fit must often be stopped
manually, by typing Control-C in the terminal window where iteration results
The parameter window does not report the peak position in PPM.
The position of peak labels in the Fit Window is not updated to reflect
changes in position parameters during the fit.
The method for saving and organizing the fit results could be better. There
is currently no summary of all results, no way to read previous results,
and no way to accommodate saving results for multiple sessions.
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