Abstract for Technology Development Program 2 - MAS NMR It has become clear that dynamics within protein molecules comprising HIV-1 assemblies play critical roles in regulating viral infectivity, including uncoating and maturation. Current progress in the field is hampered by the paucity of structural-biological methods that yield atomic-level information into structure and dynamics simultaneously, particularly when large-amplitude conformational rearrangements take place. Magic angle spinning (MAS) NMR is uniquely positioned to yield such information and, when integrated with molecular dynamics (MD) simulations, provides dynamic information inaccessible by any other means. While MAS NMR is a very powerful technique, it currently suffers from three major drawbacks: i) inherent low sensitivity, resulting in long measurement times; ii) high spectral congestion for large assemblies due to numerous overlapping signals; and iii) extensive and time-consuming data analysis for large systems (resonance assignments and structure calculation), hampering the widespread use of the method. We propose to develop a new methodological framework for atomic-level structural, dynamic, and mechanistic characterization of HIV-1 protein assemblies by MAS NMR, which will overcome the main roadblocks, limited sensitivity and resolution, as well as long data analysis time. To accomplish this goal, we will integrate high magnetic fields (17.6–28.2 T) with ultrafast MAS frequencies, proton detection, streamlined data acquisition, processing, and analysis. We will also employ dynamic nuclear polarization (DNP)-based experiments for analysis of low-concentration species as well as for investigations of the conformational space accessible to the dynamically disordered states. We will develop new experiments suitable for studies of large assemblies of HIV-1 proteins and their complexes with host proteins and small-molecule interactors, in a fraction of time and with a small fraction of material that is required for conventional experiments. The dramatic sensitivity and/or resolution enhancements foreseen to become attainable through the combination of proposed MAS NMR and DNP methods will greatly expand the range of systems amenable to in-depth characterization. The streamlined data analysis through the integration of experiment and computation will dramatically improve the throughput of MAS NMR and make the technique accessible to a very wide cohort of researchers. The technologies developed for the analysis of HIV-1 assemblies through the proposed studies will be broadly applicable to other biological assemblies. Finally, we envision that the proposed methodological framework will pave the way for the atomic-resolution structural and dynamics characterization of viral proteins in the context of intact viruses.