ABSTRACT, CORE 2 Deciphering the molecular mechanisms underlying key steps of the HIV-1 replication cycle is essential to fully describe the biology of the virus, its interplay with host components, and for developing novel antiretroviral therapies. Toward this goal, structural biology plays an important role, as an atomic-level understanding of macromolecular structure and dynamics, and how alterations in structure affect function, provides mechanistic insights into the workings of biological macromolecules. The structural biology core (Core 2) will contribute complementary expertise, instrumentation, and resources for studying key steps of the viral replication cycle, as described within individual projects, including viral entry/uncoating, integration, and maturation/assembly. Core 2 includes: hydrogen deuterium exchange coupled to mass spectrometry (HDX-MS) and cross-linking mass spectrometry (XL-MS), nuclear magnetic resonance (NMR) spectroscopy, single-particle cryo-electron microscopy (cryo-EM), and cryo-electron tomography (cryo-ET). HDX-MS and XL-MS methods have emerged as powerful tools to probe protein interactions with ligands, co-regulatory proteins, and nucleic acids in solution to yield insights into macromolecular behavior on a residue-by-residue level. NMR spectroscopy enables determining high-resolution structures of small-to-medium size proteins and nucleic acids, probing macromolecular interactions at atomic resolution and studying conformational dynamics. Single-particle cryo- EM yields an understanding of macromolecular structure and dynamics for large assemblies, often with less material and more rapidly than with traditional methods. Cryo-ET and sub-tomogram averaging (STA) is ideally suited to studying irregular objects such as non-icosahedral protein lattices, where having 3D volumetric information for each unique assembly, prior to ensemble averaging, is necessary to gain insight into local and long-range structural perturbations. Core 2 will apply existing tools to address specific questions described within individual Projects, and each laboratory will also develop novel technologies to meet the needs of proposed research goals. The Core has established collaborations with most current B-HIVE investigators, has developed workflows for new projects that may emerge during the studies, and will provide ongoing consultation and training to B-HIVE members as projects develop. The established tools provide complementary and synergistic expertise toward the broad research goals of the center, while the training and dissemination module aligns with the broader impacts of the NIH.