NMR Technologies for Integrating Structure, Function and Disease

修订后的申请简介 A0奖学金是在2019年5月提交的，当时亚历山大·巴恩斯教授还没有搬到苏黎世理工学院，在他之前。 查德·里恩斯特拉被招募到威斯康星大学麦迪逊分校。现在Rienstra正式成为威斯康星大学麦迪逊分校的全职教授，在 在NMRFAM谈判了固态核磁共振(SS核磁共振)计划的主要投资(新的和/或正在移动的 包括三个屏蔽600 MHz磁铁，一个750 MHz宽孔磁铁，四个光谱仪， 几个定制设计的魔角旋转(MAS)探头，频率为600-750 MHz，并升级到900 MHz 光谱仪，这立即对2019年底DBP6和其他 2020年初，试验正在进行中。 此外，国家科学基金中等规模研究基础设施-2的提案 高级核磁共振“，由UW-Madison PI(Rienstra和Henzler-Wildman)合作提交 与杰夫·霍克在康涅狄格州大学健康与艺术爱迪生大学佐治亚州大学，正处于谈判和审查的后期阶段 国家科学基金会。如果获得资金，这笔赠款将在2022-23年为NMRFAM带来一台1.1 GHz的专用SS核磁共振光谱仪。 这些发展推动了对此A1应用程序的几项更改，这更明确地强调了 NMRFAM的SS核磁共振计划： (1)Rienstra现在正在联系Pi和Henzler-Wildman联合PI。 (2)tr&d1现在包括针对微型转子填充和样品处理的开发的子目标 利用最近在转子直径为1毫米的超高速MAS(&gt；100 kHz)方面的突破的工具；这 扩大了研发的范围和影响1.梯度下核磁共振的基线和基准也是 更清晰的描述和原则验证实验已经到位。 (3)tr&d2现在解决了SSNMR数据收集中的关键瓶颈问题，强调： (A)参数优化和光谱仪配置的自动化； (B)600-900兆赫的新窄口径魔角旋转探头设计(将适用于1.1兆赫 GHz及未来更高的频率)；以及 (C)与数据处理的实时反馈互动(tr&d3)。 (4)TR&D3现在利用NMRFAM软件产品和持续的技术开发，以实现固体- 状态核磁共振，包括赋值、结构确定、精化和验证工具，它更多 显然与提案的其余部分结合在一起。 (5)DBP 5、6和7已被更改，以包括两国之间发展良好和有影响力的合作 Rienstra和Paul Kotzbauer(华盛顿州医学、路易小体和联体核病)、马蒂·伯克(U。 伊利诺伊州，抗真菌药物)，詹姆斯·莫里西(密歇根大学，凝血)。 总体而言，提案现在以(我们认为)更合乎逻辑/更符合时间顺序的方式组织，包括tr&d1 强调样品，tr&d2光谱仪和探头，tr&d3软件和分析程序。 提案主体进一步解释了这些发展如何极大地提高了项目的成本效益比 并与当前的用户计划和NMRFAM的长期愿景相结合。 这些修订还解决了A0应用程序审查者的首要关切，包括 研发与研发的范围/适度创新“；研发与研发的弱整合”；成本效益比；初步数据；解决方案 Tr&d3；创新；上下文(处理相互竞争的想法和先例)；以及特殊性的强调 关于结果的。最后，我们阐明了膜中不对称性的前提和方法。 蛋白质。

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.