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.