Journal of Biomolecular NMR 27: 313–321, 2003. KLUWER/ESCOM © 2003 Kluwer Academic Publishers. Printed in the Netherlands. 313 Smartnotebook: A semi-automated approach to protein sequential NMR resonance assignments Carolyn M. Slupsky, Robert F. Boyko, Valerie K. Booth & Brian D. Sykes * Protein Engineering Network Centers of Excellence, 713 Heritage Medical Research Center, University of Alberta, Edmonton, Alberta, T6G 2S2, Canada Received 19 March 2003; Accepted 10 June 2003 Key words: assignment, automated, chemical shifts, NMR, NMRView, semi-automated Abstract Complete and accurate NMR spectral assignment is a prerequisite for high-throughput automated structure determ- ination of biological macromolecules. However, completely automated assignment procedures generally encounter difficulties for all but the most ideal data sets. Sources of these problems include difficulty in resolving correlations in crowded spectral regions, as well as complications arising from dynamics, such as weak or missing peaks, or atoms exhibiting more than one peak due to exchange phenomena. Smartnotebook is a semi-automated assignment software package designed to combine the best features of the automated and manual approaches. The software finds and displays potential connections between residues, while the spectroscopist makes decisions on which connection is correct, allowing rapid and robust assignment. In addition, smartnotebook helps the user fit chains of connected residues to the primary sequence of the protein by comparing the experimentally determined chemical shifts with expected shifts derived from a chemical shift database, while providing bookkeeping throughout the assignment procedure. Introduction High throughput structure determination is a requisite for structural genomics and proteomics and, of course, is also desirable in more traditional protein structure determination applications. In determining protein structures using NMR, the critical step of obtaining complete and accurate chemical shift assignments is often the most tedious and time consuming. Thus, the automated assignment of NMR spectra of bio- macromolecules is a critical objective for automating structure determinations by NMR. There are many software packages that address the issue of chemical shift assignment. Automated and semi-automated procedures for the assignment of 2D homonuclear NOESY and COSY 1 H NMR spectra in- clude ANSIG (Kraulis, 1989), PROSPECT (van de Ven, 1990), EASY (Eccles et al., 1991), CLAIRE * To whom correspondence should be addressed. E-mail: brian.sykes@ualberta.ca. (Kleywegt et al., 1991), CPA (Xu and Sanctuary, 1993), CAMRA (Gronwald et al., 1998), and others (Cieslar et al., 1988; Weber et al., 1989; Eads and Kuntz, 1989; Xu et al., 1994). Most of these programs implement at least a portion of the basic Wüthrich se- quential assignment strategy (Wüthruch, 1986) where the spin systems are detected and classified, followed by inter-residue correlation of these spin systems and matching to the primary sequence. The advent of modern triple resonance three and four-dimensional experiments has resulted in a number of programs for automated assignment of multinuclear NMR data that include ALFA (Bern- stein et al., 1993), ANSRS (Kraulis, 1994), Pronto (Kjaer et al., 1994), AUTOASSIGN (Zimmerman et al., 1994, 1997; Szyperski et al., 2002), CON- TRAST (Olson and Markley, 1994), GARANT (Bar- tels et al., 1996), PASTA (Leutner et al., 1998), AURELIA (Görler et al., 1999), RESCUE (Pons and Delsuc, 1999), JIGSAW (Bailey-Kellogg et al., 2000), NOAH/DIAMOND (Oezguen et al., 2002), CANDID