Fully automated sequence-specific resonance assignments of heteronuclear protein spectra

Full automation of the analysis of spectra is a prerequisite for high-throughput NMR studies in structural or functional genomics. Sequence-specific assignments often form the major bottleneck. Here, we present a procedure that yields nearly complete backbone and side chain resonance assignments starting from a set of heteronuclear three-dimensional spectra. Neither manual intervention, e. g., to correct lists obtained from peak picking before feeding these to an assignment program, nor protein-specific information, e. g., structures of homologous proteins, were required. By combining two earlier published procedures, AUTOPSY [Koradi et al. ( 1998) J. Magn. Reson., 135, 288 - 297] and GARANT [Bartels et al. ( 1996) J. Biomol. NMR, 7, 207 - 213], with a new program, PICS, all necessary steps from spectra analyses to sequence-specific assignments were performed fully automatically. Characteristic features of the present approach are a flexible design allowing as input almost any combination of NMR spectra, applicability to side chains, robustness with respect to parameter choices ( such as noise levels) and reproducibility. In this study, automated resonance assignments were obtained for the 14 kD blue copper protein azurin from P. aeruginosa using five spectra: HNCACB, HNHA, HCCH-TOCSY, N-15-NOESY-HSQC and C-13-NOESY-HSQC. Peaks from these three-dimensional spectra were filtered and calibrated with the help of two two-dimensional spectra: N-15-HSQC and C-13-HSQC. The rate of incorrect assignments is less than 1.5% for backbone nuclei and about 3.5% when side chain protons are also considered.