Resonance assignments, automated

McFeeters, R. L., Swapna, G. V. T., Montelione, G. T., and Oswald, R. E. (2002). Semi-automated backbone resonance assignments of the extracellular ligand-binding domain of an ionotropic glutamate receptor. /. Biomol. NMR 22, 297-298. 

In addition to the in vitro studies of well-defined systems that have been discussed here, NMR spectroscopy can also be applied to living systems or complex substance mixtures (146). This broad applicability is an advantage of NMR spectroscopy over X-ray crystallography. Early in vivo NMR studies that mostly identify small metabolite molecules by P or H ID experiments have led to different applications. In-cell NMR uses isotopic labeling combined with two or higher dimensional NMR experiments for structural studies of the phosphorylation state of a given protein in the cellular environment or of intrinsically unstructured proteins. In-cell NMR applications in prokaryotic systems cover structural and functional studies (i.e., protein-protein interactions, protein dynamics, automated structure determination, and de novo resonance assignments (147-149)). 

G. T. Montelione, C. B. Rios, G. V. T. Swapna and D. E. Zimmerman, NMR Pulse Sequences and Computational Approaches for Automated Analysis of Sequence-Specific Backbone Resonance Assignments of Proteins , p. 81... 

With the dissemination of multinuclear multidimensional NMR experiments, the automated NMR resonance assignment became feasible and indispensable, simultaneously.23,35,41,99 Typically, 10-20 individual multidimensional NMR spectra are measured for a large protein. The NMR-derived information quality definitely relies on the resonance assignment. The precious assignment has been based on and assessed by the sequential NOE continuity on the peptide sequence and consistency with the amino acid spin... 

Keywords APSY Automated peak picking Automated resonance assignment GAPRO NMR Projection spectroscopy Protein backbone Protein side chains... 

Overall, the high quality of the GAPRO peak list of the 5D APSY-HC(CC-TOCSY)CONH experiment in terms of dimensionality, completeness, precision and very low number of artifacts provides an excellent basis for a reliable automated assignment of aliphatic side-chain atoms. Although the TOCSY mixing does not provide information on the direct covalent connectivities among the carbon nuclei, the 5D peaks can be used for reliable sequence-specific resonance assignment of aliphatic resonances, due to the availability of all five dimensions. 

In addition to providing automated peak picking and computation of the corresponding chemical shift lists, APSY supports automated sequential resonance assignment. Thus, APSY is a valid alternative to related NMR techniques. APSY can be the first step, after sample preparation, in a fully automated process of protein stmcture determination by NMR with successive automated algorithms for the NOESY spectrum analysis and structure calculation. 

Fiorito F, Hiller S, Wider G, Wiithrich K (2006) Automated resonance assignment of proteins 6D APSY-NMR. J Biomol NMR 35 27-37... 

Gossert AD, Hiller S, Fernandez C (2011) Automated NMR resonance assignment of large proteins for protein-ligand interaction studies. J Am Chem Soc 133 210-213... 

Hiller S, Joss R, Wider G (2008) Automated NMR assignment of protein side chain resonances using automated projection spectroscopy (APSY). J Am Chem Soc 130 12073-12079... 

NMR Pulse Sequences and Computational Approaches for Automated Analysis of Sequence-Specific Backbone Resonance Assignments of Proteins... 

Figure 6 Steps in automated assignment. (1) Select the lowest energy structures from iteration / — 1 that are used to interpret the spectra. (2) For each peak, list all possible assignments compatible with the resonances within a frequency mnge. (3) Extract a distance for each assignment possibility from the ensemble of structures. (4) Use the distances to assign ambiguous NOEs. (5) Calibrate the peak volumes to obtain distance restraints. (6) Calculate structures based on the new restraints.

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