Journal of Biomolecular NMR, 4 (1994) 385-410 385 ESCOM J-Bio NMR 184 Evaluation of an algorithm for the automated sequential assignment of protein backbone resonances: A demonstration of the connectivity tracing assignment tools (CONTRAST) software package John B. Olson Jr. and John L. Markley* Department of Biochemistry, Collegeof Agriculture and Life Sciences, Universityof Wisconsin-Madison, 420 Henry Mall, Madison, W153706, U.S.A. Received 25 May 1993 Accepted 22 November 1993 Keywords: Automated assignment; Proteins; Software SUMMARY The peptide sequential assignment algorithm presented here was implemented as a macro within the CONnectivity TRacing ASsignment Tools (CONTRAST) computer software package. The algorithm pro- vides a semi- or fully automated global means of sequentially assigning the NMR backbone resonances of proteins. The program's performance is demonstrated here by its analysis of realistic computer-generated data for III~lc, a 168-residue signal-transducing protein ofEscherichia coli [Pelton et al. (1991) Biochemistry, 30, 10043-10057]. Missing experimental data (19 resonances) were generated so that a complete assignment set could be tested. The algorithm produces sequential assignments from appropriate peak lists of nD NMR data. It quantifies the ambiguity of each assignment and provides ranked alternatives. A 'best first' approach, in which high-scoring local assignments are made before and in preference to lower scoring assignments, is shown to be superior (in terms of the current set of CONTRAST scoring routines) to approaches such as simulated annealing that seek to maximize the combined scores of the individual assignments. The robustness of the algorithm was tested by evaluating the effects of imposed frequency imprecision (scatter), added false signals (noise), missing peaks (incomplete data), and variation in user- defined tolerances on the performance of the algorithm. INTRODUCTION The development of homonuclear IH 2D NMR has allowed the proton NMR resonances of many small proteins (Mr < 10 kDa) to be unambiguously assigned through use of intraresidue through-bond J-coupling connectivities and interresidue through-space NOE connectivities (Wtithrich, 1986). The process of assigning the resonances of a protein is one of the most time- *To whom correspondence should be addressed. 0925-2738/$10.00 9 1994 ESCOM SciencePublishers B.V.