STRUCTURE OF IMPORTIN B BOUND TO THE ADAPTOR SNURPORTIN

与适配器 SNURPORTIN 结合的导入蛋白 B 的结构

• 批准号: 7955543
• 负责人: Gino Cingolani
• 金额: $ 11.3万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-01 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7955543
• 关键词: Affinity; Ammonium Sulfate; Binding; Biochemical; Cell Nucleus; Cell Size; Complex; Computer Retrieval of Information on Scientific Projects Database; Crystallization; Cytoplasmic Protein; Data; Data Collection; Data Set; Funding; Goals; Grant; Guanosine Triphosphate; Hand; Hour; Housing; Human; Hydrolysis; Hydrophobic Interactions; Institution; Ionic Strengths; Maps; Measures; Monomeric GTP-Binding Proteins; Nuclear Import; Physiological; Proteins; Reaction; Research; Research Personnel; Resolution; Resources; Roentgen Rays; Small Nuclear Ribonucleoproteins; Solutions; Source; Structure; Time; United States National Institutes of Health; electron density; receptor

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).

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 Importin B是最佳特征的核进口受体，将细胞质蛋白进口到细胞核中。大多数细胞质进口底物是由Importin B与适配器importin a的复杂导入的，在需要小型GTPase RAN的反应中，该反应需要GTP水解。 SNRNP代表了进口货物的专门子集，该子集与特定的衔接子nurportin相关的Importin B导入到核中。我们的生化映射了跨越蛋白质1-68区的Snurportin（SIBB）中的最小进口蛋白B结合结构域，并获得了与SIBB结合的人类importin B（876个残基）的晶体（RES 1-68）。这些晶体是在硫酸铵高和衍射X射线下获得的，至〜1.8A。在结构中，仅SIBB的25-68区域在电子密度中可见，这在疏水相互作用（可能由硫酸铵高稳定）中与IMPOTIN BINCITIND绑定在一起。相比之下，SIBB的1-24区域对高亲和力的结合也很重要，在我们的晶体中不可见，这可能是由于结晶溶液的高离子强度（硫酸铵）。 为了可视化整个SIBB域（Res。1-68），我们在生理离子强度（〜175mm naCl）中获得了Importin B：SIBB的晶体。房屋中的初步衍射分析表明这些晶体还衍射X射线至高分辨率。 此快速访问建议的目的是在A1（或F1）上获得Beam-Time，并从我们的新晶体形式中进行完整的数据收集。考虑到单位细胞的大小，空间组（P212121）和手中晶体的高衍射质量，通常可以在2-3小时内测量完整的数据集。如果我们有24小时的束缚时间，我们可以从5-6个晶体中收集完整的X射线数据。即使是12小时，原则上就足够了。因此，请求的光束时间最小（12-24小时）。