INTRODUCTION TO REVISED APPLICATION The A0 grant was submitted in May 2019, before Prof. Alexander Barnes moved to ETH-Zurich and before Prof. Chad Rienstra was recruited to UW-Madison. Now Rienstra is officially a Full Professor at UW-Madison, after having negotiated major investments in the solid-state NMR (SSNMR) program at NMRFAM (new and/or moving from Illinois) including three shielded 600 MHz magnets, one 750 MHz wide bore magnet, four spectrometers, several custom-designed magic-angle spinning (MAS) probes at 600-750 MHz, and upgrades to the 900 MHz spectrometer, which immediately have had an impact on data collection for DBP6 in late 2019 and for other experiments in progress during early 2020. Furthermore, the National Science Foundation Mid-Scale Research Infrastructure-2 proposal on "Network for Advanced NMR", which was submitted by UW-Madison PIs (Rienstra and Henzler-Wildman) in collaboration with Jeff Hoch at UConn Health and Art Edison at U. Georgia, is in late stages of negotiation and review with NSF. If funded, this grant would bring a 1.1 GHz dedicated SSNMR spectrometer to NMRFAM in ~2022-23. These developments have motivated several changes to this A1 application which more explicitly emphasizes the SSNMR program at NMRFAM: (1) Rienstra is now contact PI and Henzler-Wildman co-PI. (2) TR&D1 now includes sub-aims targeting development of micro-rotor packing and sample manipulation tools to leverage recent breakthroughs in ultra-fast MAS (>100 kHz) at <1 mm rotor diameters; this broadens the scope and impact of TR&D 1. Baselines and benchmarks for NMR under gradients are also more clearly described and proof-of-principle experiments are in place. (3) TR&D2 now addresses critical bottlenecks in SSNMR data collection, emphasizing: (a) automation for parameter optimization and spectrometer configuration; (b) new narrow bore magic-angle spinning probe designs at 600-900 MHz (that will be applicable at 1.1 GHz and higher in the future); and (c) real-time feedback interaction with data processing (TR&D3). (4) TR&D3 now leverages NMRFAM software products and continuing technology development for solid- state NMR, including assignment, structure determination, refinement and validation tools, and it is more clearly integrated with the rest of the proposal. (5) DBPs 5, 6 and 7 have been changed to include well-developed and impactful collaborations between Rienstra and Paul Kotzbauer (Wash. U. Medicine, Lewy bodies and synucleiopathies), Marty Burke (U. Illinois, antifungal drugs), and James Morrissey (U. Michigan, blood coagulation). Overall the proposal is now organized in (we think) a more logical/chronological manner, with TR&D1 emphasizing samples, TR&D2 the spectrometer and probes, and TR&D3 the software and analysis procedures. The proposal body further explains how these developments greatly augment the cost-benefit ratio for the project and integrate with the current user program and the long-term vision of NMRFAM. The revisions also address the overarching concerns of reviewers of the A0 application including "narrow scope/modest innovation in TR&D1"; "weak integration of TR&D3"; cost-benefit ratio; preliminary data; solution vs. solids emphasis of TR&D3; innovation; context (addressing competing ideas and precedents); and specificity of outcomes. Finally, we have clarified the premise and approach with respect to asymmetry in membrane proteins.

PROJECT NARRATIVE The scientific impact of an NMR experiment depends on the sample quality and biological relevance of the sample conditions, the quality of the NMR data, and the ability to interpret the NMR data. This proposal develops biological NMR technology across all three areas with the overarching goal of advancing NMR studies of biomedically important problems. By reducing the time, cost, effort and learning curve to acquire NMR data and improving the quality and reproducibility of data analysis, we will enable wider application and increase the scientific value of biomolecular NMR.