Untitled

Abstract

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, 15N-NOESY-HSQC and 13C-NOESY-HSQC. Peaks from these three-dimensional spectra were filtered and calibrated with the help of two two-dimensional spectra: 15N-HSQC and 13C-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.

Robust and versatile interpretation of spectra with coupled evolution periods using multi-way decomposition

“…26 The resulting shapes (not shown) exhibit unique peaks as indicated by the fact that every true peak exceeds the largest noise intensity in the same shape by at least a factor of 5.5. Thus, peak picking of these shapes provides unique chemical shifts, and indeed the resulting list of 5D peaks coincides closely with published chemical shifts 32,33 (Table 3). Residual discrepancies can be explained by measurements at different temperatures (24°C in the present study vs 30°C in Ref.…”

“…Residual discrepancies can be explained by measurements at different temperatures (24°C in the present study vs 30°C in Ref. 33), and the limited Figure 3. Illustration of the simultaneous use of all input projections in PRODECOMP to detect signals with low signal-to-noise.…”

Fully automated structure determinations of the Fes SH2 domain using different sets of NMR spectra

“…An ensemble of initial chemical shift assignments was obtained by multiple runs of a modified version of the GARANT algorithm 12,13 with different seed values for the random number generator. 14 The peak position tolerance was set to 0.03 ppm for the 1 H dimensions and to 0.4 ppm for the 13 C and 15 N dimensions. These initial chemical shift assignments were consolidated by CYANA into a single consensus chemical shift list.…”

Modern High Resolution NMR for the Study of Structure, Dynamics and Interactions of Biological Macromolecules

“…The highly systematic nature of heteronuclear J-correlated 3D experiments together with the limited number and specific NMR signatures of naturally occurring amino acid types triggered development of software for semiautomatic and fully automated resonance assignment of medium sized proteins (ca. 50 to 150 residues) [57][58][59][60]. Current efforts are directed towards extending automatic backbone assignment to significantly larger proteins [61].…”