ACME - Amplitude-Constrained Multiplet Evaluation
Version 2001.006.11.26

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CITATION

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:
  1. All multiplets in the spectrum have the same amplitude, and this amplitude must be determined in order to measure accurate coupling values.
  2. ACME can establish this amplitude from the diagonal signals of the COSY spectrum.
So, based on these points, consider the following:

WEB SITES

http://spin.niddk.nih.gov/bax/NMRPipe/acme
http://spin.niddk.nih.gov/bax/NMRPipe/diag

INSTALLING ACME

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 delaglio@nih.gov

DESCRIPTION

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:

  1. Measure COSY spectrum with conditions that allow the spin systems to be fully relaxed at the start of the COSY sequence.
  2. A numerical diagonal processing scheme is used to prepare two versions of the COSY spectrum:
  3. 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 multiplicity.
  4. Using the amplitude value determined from the diagonal, the ACME program will be used to measured the couplings in the cross peak spectrum.

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:
fid.com  Usual NMRPipe conversion of the raw FID data. This script creates the file "test.fid".
ft2.com COSY processing with diagonal suppression. This script creates the spectrum "test.ft2".
diag.com  Processing to create a diagonal-only spectrum. This script creates the spectrum "diag.ft2".
ext.tcl This script extracts small regions of interest from the processed data, for convenience. This script creates the extracted spectral data "ext.dat" and "diag.dat".

STARTING ACME

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:

   ACME -help

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


The Spectrum 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 Spectrum Viewing Window:
LEFT 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.
MIDDLE drags a zoom box which is used to select a region of cross-peaks for fitting in the Fit Window as described below.
RIGHT drags a zoom box to create an expanded view of the selected region. By default, the expanded view will be drawn in a new Spectrum Viewing Window.

ACME FIT WINDOW

The 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 Fit Window:
Fit 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.
Simulate Simulates a spectral model using the current settings, and displays the results.
Read This feature is currently not implemented; in future versions, it will read previously recorded settings.
Accept 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:
 
J-Active Active Coupling, Hz
Hi Amplitude
X Loc X-Axis Position of Multiplet Center, Pts
X Width 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 Loc Y-Axis Position of Multiplet Center, Pts
Y Width 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
Ass 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

  1. 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 Window.
  2. Use the LEFT mouse button in the ACME Fit Window one signal at the approximate center of each multiplet to be fit.
  3. Carefully adjust and review the parameter setting for each signal in the 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.
  4. Click the "Fit" button in the Fit Window.
  5. Evaluate the results. Things to check for:
  6. 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.


PROBLEMS IN THIS VERSION OF ACME


 
boulders

 
 
furanku
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