Automated methods for NMR structure determination of proteins are continuously becoming more robust. However, current methods addressing larger, more complex targets rely on analyzing 6-10 complementary spectra, suggesting the need for alternative approaches. Here, we describe 4D-CHAINS/autoNOE-Rosetta, a complete pipeline for NOE-driven structure determination of medium- to larger-sized proteins. The 4D-CHAINS algorithm analyzes two 4D spectra recorded using a single, fully protonated protein sample in an iterative ansatz where common NOEs between different spin systems supplement conventional through-bond connectivities to establish assignments of sidechain and backbone resonances at high levels of completeness and with a minimum error rate. The 4D-CHAINS assignments are then used to guide automated assignment of long-range NOEs and structure refinement in autoNOE-Rosetta. Our results on four targets ranging in size from 15.5 to 27.3 kDa illustrate that the structures of proteins can be determined accurately and in an unsupervised manner in a matter of days.

CEITEC Central European Institute of Technology Masaryk University Kamenice 5 Brno 62500 Czech Republic

Department of Biochemistry and Biophysics 2011 Ag and Life Sciences Bldg Oregon State University Corvallis OR 97331 USA

Department of Chemistry and Biochemistry University of California Santa Cruz Santa Cruz CA 95064 USA

Department of Chemistry Iowa State University 2438 Pammel Drive Ames IA 50011 USA

Department of Computer Science University of California Santa Cruz Santa Cruz CA 95064 USA

Roy J Carver Department of Biochemistry Biophysics and Molecular Biology Iowa State University Ames IA 50011 USA

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