This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Importin b is the best characterized nuclear import receptor, which import cytoplasmic protein into the cell nucleus. The majority of cytoplasmic import substrates are imported by importin b in complex with the adaptor importin a, in a reaction which requires GTP hydrolysis by the small GTPase Ran. snRNP represents a specialized subset of import cargos that are imported into the nucleus by importin b in complex with the specific adaptor snurportin. We have biochemical mapped the minimum Importin b-Binding domain in Snurportin (sIBB), which spans region 1-68 of the protein, and obtained crystals of human importin b (876 residues) bound to the sIBB(res 1-68). These crystals were obtained under high ammonium sulphate and diffract X-rays to ~1.8A. In the structure only region 25-68 of the sIBB is visible in the electron density, which is bound to importin b via hydrophobic interactions (likely stabilized by the high ammonium sulphate). In contrast region 1-24 of the sIBB, also important for high affinity binding, is not visible in our crystal, likely due to the high ionic strength of the crystallization solution (~1.5M Ammonium Sulphate). In the attempt to visualize the entire sIBB-domain (res. 1-68), we have obtained crystals of importin b:sIBB at physiological ionic strength (~175mM NaCl). Preliminary diffraction analysis in house indicates these crystals also diffract X-rays to high resolution. The goal of this quick access proposal is to obtain beam-time at A1 (or F1) and carry out a complete data collection from our new crystal form. Given the unit cell size, space group (P212121) and the high diffraction quality of the crystals in hand, complete data set can be measured usually within 2-3 hours. If we had 24 hours of beamtime we could collect complete x-ray data from 5-6 crystals. Even 12-hours would be - in principle- sufficient. So the beam-time requested is minimal (12-24 hours).
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