HYBRID STRUCTURE DETERMINATION OF TRAFFICKING COMPLEXES BY SAXS, PROTEIN CRYSTAL

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 膜运输途径对于正常生理过程如信号转导、抗原呈递、细胞器生物发生等以及对于病理生理过程如HIV出芽是必不可少的。通过内体的膜运输由一系列多蛋白复合物进行，包括ESCRT复合物和逆转录复合物。我们已经获得了这些组件的核心的一系列晶体结构，并结合单个结构域的结构和同源建模，可以生成完整结构的模型。该模型已进行了评估，通过比较多个截断结构的流体动力学研究，提供完整的复合物的解决方案的结构上的限制。我们已经应用粗粒度的蒙特卡罗模拟，使用基于残差的潜力与刚性域和核心结构的组件建模。为了完成完整的复合物的结构分析，我们迫切希望获得SAXS数据，这将提供更强大的约束的解决方案结构模型比可能单独使用流体动力学数据。 两个结构性出版物目前正在为顶级期刊做准备，因此，如果实验成功，几乎可以立即在高知名度的场所进行。这些论文涉及竞争激烈的主题，出版物将在1-2个月内提交。如果访问延迟，我们可能无法将SAXS分析纳入这些研究。

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. Membrane trafficking pathways are essential for normal physiological processes such as signal transduction, antigen presentation, organelle biogenesis, and many others, and for pathophysiological processes such as HIV budding. Membrane trafficking via endosomes is carried out by a series of multi-protein complexes, including the ESCRT complexes and the retromer complex. We have obtained a series of crystal structures of the cores of these assemblies, and in combination with the structures of individual domains and homology modeling, models for the complete structures can be generated. The models have been assessed by comparison to hydrodynamic studies of multiple truncation constructs, providing constraints on the solution structures of the intact complexes. We have applied coarse-grained Monte Carlo simulations using residue-based potentials with rigid domains and core structures to model the assemblies. To complete the structural analysis of the intact complexes, we urgently wish to obtain SAXS data, which will provide more powerful constraints on the solution structural models than is possible using hydrodynamic data alone. Two structural publications are currently in preparative for top tier journals, thus the experiments, if successful, would be publishable almost immediately in high profile venues. These papers deal with highly competitive topics, with publications to be submitted in 1-2 months. If there is a delay in access, we will probably not be able to include the SAXS analysis in these studies.