Spectral Assignment - Applications & Techniques in NMR Research
Scientists at the NCI-FCRDC and Accelrys have used FELIX for spectral assignment of NMR data.

Rapid Assignment of Multidimensional NMR Spectra
R. Andrew Byrd and Kristine E. Miller, Macromolecular NMR Section, ABL-Basic Research Program, NCI-FCRDC, Frederick, MD.

Reducing Errors in NOE Crosspeak Assignment
Accelrys Inc.

Improved Structure Calculations Through Iterative Assignment Filtering Using NOESY Back-Calculation
Accelrys Inc.


-----------------------------------------------------------------------------------------------------------------------------------------

Rapid Assignment of Multidimensional NMR Spectra

23kb

Each strip plot is centered on the amide proton resonance in the plane of the amide nitrogen and contains the NOE crosspeaks between the aliphatic and amide protons used to determine sequential and long-range assignments among the first six residues of ubiquitin.


 






With the development of heteronuclear double and triple resonance 3D/4D NMR spectroscopy, the size limit of the protein, whose structure can be successfully tackled by NMR, has been continuously increased for the last five years. In the assignment process of these types of spectra, automated algorithms and good interactive tools are crucial. The capabilities within FELIX-Assign for working with multiple data sets (via separate or overlaid displays) facilitate the analysis procedure. Capabilities for shuffling between planes of 3D or 4D data (indicating whether a crosspeak is at maximum intensity in the currently displayed or adjacent plane) and drawing 1D vectors (parallel to any dimension) in real time vastly improve the efficiency of the analysis. These features enable the user to conduct the analysis directly on the workstation screen, without the time-consuming and cumbersome process of plotting on paper the many planes within a multidimensional spectrum.

The interactive ability to dea with both vector- and contour-mode data is extremely useful for analyzing less well-behaved data. The automatic tools for matching spin system patterns to amino acid types and for performing automatic neighbor finding among the identified spin systems speeds up the process for completing the assignments.

FELIX- Assign simplifies the interaction with the spectra and the resonance and peak databases, which permits the scientist to focus more on the analysis of the data (Figure 5 -25kb).

Contributed by Drs. R. Andrew Byrd and Kristine E. Miller, Macromolecular NMR Section, ABL-Basic Research Program, NCI-FCRDC, Frederick, MD.


-----------------------------------------------------------------------------------------------------------------------------------------

Reducing Errors in NOE Crosspeak Assignment

67kb

The image depicts a simple-to-use, menu-driven interface for suggesting and assigning individual spin frequencies (resonances) to each dimension of the NOE crosspeak (upper left). The full NOESY spectrum is depicted on the lower left with each NOE crosspeak colored accroding to its current assignment state. The yellow, purple and cyan lines in the right-hand spectrum represent the four possible frequencies (two in each dimension), color-coded based on the spin system to which they belong.

 

 





The accurate assignment of NOE crosspeaks is critical for the generation of accurate structures. A single, erroneous distance restraint can lead to an incorrect model. FELIX-Assign provides many tools for aiding the accurate assignment of the NOE crosspeaks (Figure 6 -67kb). For example, the LYS_6:HN assignment can be built by clicking the mouse on the various lists, preventing typographical errors and the introduction of erroneous restraints into the structure calculations. Additionally, the assignment state of each crosspeak in the database is stored for later use, such as in crosspeak coloring for visualizing the progress of the assignment procedure.


-----------------------------------------------------------------------------------------------------------------------------------------

Improved Structure Calculations Through Iterative Assignment Filtering Using NOESY Back-Calculation

63kb      107kb

The quality of NMR-generated structures can be dramatically improved by using starting models as guides to identify misassignments and to assign additional crosspeaks by imposing distance or NOE back-calculated intensity cutoffs to resolve assignment ambiguities. An iterative repetition of this cycle leads to more accurate and precise models. By combining the spectral visualization capabilities within FELIX-Model and the spectral simulation methods within NMR Refine, the NOESY spectrum can be generated from an ensemble of structures and used to verify and suggest additional assignments. The left-hand panel of Figure 7 (63kb) depicts the experimental 2D-NOESY spectra for Human Anaphylatoxin C3a with the crosspeaks colored according to their assignment state. The green colored peaks indicate those crosspeaks that were used as input constraints to DG-II calculations, and the red colored peaks represent those with ambiguous assignments. The right-hand panel depicts the back-calculated spectra simulated from an ensemble of 10 C3a structures using IRMA. The majority of individual proton resonances have been previously assigned(5), and all of the crosspeaks in the back-calculated spectra have assignments. It is evident that many more peaks are uniquely assigned in the back-calculated spectrum. These simulated crosspeak assignments can be used to verify the uniquely assigned experimental crosspeaks. They also suggest plausible assignments for the experimental crosspeaks that have ambiguous assignments due to resonance and crosspeak overlap(6).

Figure 8 (107kb) depicts the C3a DG-II-generated structures rendered as variable-width ribbons. The diameter of the ribbon is proportional to the average per-residue heavy-atom RMSD over the ensemble. The backbone atoms of the ensemble were superimposed prior to the RMS calculation. The two ribbons shown are before and after several iterations of assignment filtering using back-calculated 2D-NOESY spectra(7). This analysis led to an increase in the total number of constraints (~10%). The largest improvements in the quality of the structure arose from the removal or recategorization of erroneous crosspeak assignments. In addition, frequently a crosspeak in the experimental spectrum that was assigned as arising from a single proton pair turned out to contain more than one proton pair within the simulated spectrum, and thus was removed from the restraint list.


-----------------------------------------------------------------------------------------------------------------------------------------

Back to top
More Case Studies

   
     Product Overview

     Felix 2D/ND
    
Modules
        Felix 2D/ND
        Felix Assign
        Felix Autoscreen
        Felix Model
     Case Studies
        Spectral Assignment
        Spectral Processing & Analysis
        Structure Calculation & Analysis

     System Requirements
     How to Try/Buy Felix 
         
         
        www.FelixNMR.com 2007