Abstract of grant 5 R01 GM069909-17: Karyopherin- protein transport between the nucleus and the cytoplasm. Of the 20 Kaps in human cells, 11 are import-Kaps or Importins, each recognizing a distinct set of protein cargos. This proposal describes structural, biochemical and biophysical analyses of nuclear import systems for core histones H2A/H2B and H3/H4. Here, we propose to understand how histones H2A/H2B are recognized by their primary human importer Importin-9 (Imp9) and by the homologous yeast Kap114. We will determine the mechanisms of how H2A/H2B is recognized by the Importins, how the GTPaseRan interacts with the Importin-H2A/H2B complex and how histone chaperones affect these interactions. We will also study how histones H3/H4 are recognized by their primary human importer,Importin-4 (Imp4). We will explain how histone chaperone Asf1 contributes to nuclear import of H3/H4 and how RanGTP regulates these multi-protein complexes Specific aims: Aim 2 seeks to determine structures of Kap114•H2A-H2B, Kap114•RanGTP, Kap114•Nap1 and Kap114•H2A-H2B-Nap1 complexes, and analyze the thermodynamics of their interactions. We have performed much of the biochemical and biophysical analyses, but crystallographic efforts stalled due to complexes dissociating in crystals and low resolution crystals. We have had success with cryo-EM (described below), which require new computational infrastructure to complete the work. Aim 3 seeks to solve structures of the Imp4•H3-H4•Asf1 complex. Low resolution crystals have been problematic. Cryo-EM success in Aim 2 suggests that the method should be used with Imp4•H3-H4•Asf1. This Aim will benefit from cryo-EM computational infrastructure in the lab. Aims 1 and 4 have been completed.