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APSY-NMR for protein backbone assignment in high-throughput structural biology
Authors:Samit Kumar Dutta  Pedro Serrano  Andrew Proudfoot  Michael Geralt  Bill Pedrini  " target="_blank">Kurt Wüthrich
Institution:1.Department of Integrative Structural and Computational Biology,The Scripps Research Institute,La Jolla,USA;2.Joint Center for Structural Genomics,La Jolla,USA;3.Skaggs Institute for Chemical Biology,The Scripps Research Institute,La Jolla,USA;4.Institute of Molecular Biology and Biophysics,ETH Zürich,Zurich,Switzerland;5.Institut des Sciences Analytiques, Centre de RMN à Très Hauts Champs, UMR 5280 CNRS, ENS Lyon, UCB Lyon 1,Université de Lyon,Villeurbanne,France;6.SwissFEL Project,Paul Scherrer Institute (PSI),Villigen,Switzerland
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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