Abstract
A standard set of three APSY-NMR experiments has been used in daily practice to obtain polypeptide backbone NMR assignments in globular proteins with sizes up to about 150 residues, which had been identified as targets for structure determination by the Joint Center for Structural Genomics (JCSG) under the auspices of the Protein Structure Initiative (PSI). In a representative sample of 30 proteins, initial fully automated data analysis with the software UNIO-MATCH-2014 yielded complete or partial assignments for over 90 % of the residues. For most proteins the APSY data acquisition was completed in less than 30 h. The results of the automated procedure provided a basis for efficient interactive validation and extension to near-completion of the assignments by reference to the same 3D heteronuclear-resolved [1H,1H]-NOESY spectra that were subsequently used for the collection of conformational constraints. High-quality structures were obtained for all 30 proteins, using the J-UNIO protocol, which includes extensive automation of NMR structure determination.
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Acknowledgments
This work was supported by the Joint Center for Structural Genomics through the NIH Protein Structure Initiative (PSI) grant U540-GM074898 from the National Institute of General Medical Sciences (www.nigms.nih.gov). Kurt Wüthrich is the Cecil H. and Ida M. Green Professor of Structural Biology at The Scripps Research Institute. We thank our TSRI graduate students Bryan Martin, Sergey Shnitkind, Lukas Susac and Arndt Wallmann, who each provided the APSY-based assignments for one of the proteins in Table 1. T.H. thanks his graduate student Viet Dung Duong for contributions to the software UNIO-MATCH-2014.
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Dutta, S.K., Serrano, P., Proudfoot, A. et al. APSY-NMR for protein backbone assignment in high-throughput structural biology. J Biomol NMR 61, 47–53 (2015). https://doi.org/10.1007/s10858-014-9881-8
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DOI: https://doi.org/10.1007/s10858-014-9881-8