PSEUDO-ATOMIC STRUCTURE OF THE NUCLEAR PORE COMPLEX (NPC) USING SAXS

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。子项目及 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。 核孔复合物（NPC，约50 MDa）是大分子物质穿过核膜的唯一通道。孔在许多关键的细胞过程中起着关键作用，如转录，其许多组分与人类疾病如癌症有关。Sali小组最近的工作首次描述了酵母NPC的大分子结构。这种结构定义了它的456个组成核孔蛋白（nup）蛋白的相对位置和接近度，基于来自实验数据的空间限制。进一步阐明NPC的进化起源和运输机制将需要更高分辨率的信息。为了帮助提高NPC结构的分辨率和精度，我们已经开始在SSRL进行小角度X射线散射研究。我们在洛克菲勒大学和礼来公司的合作者定期表达、生产和纯化单个nup蛋白和复合物。我们选择了几个nup蛋白的晶体结构已经解决，以建立一个基准。其中Nup 145属于在整个真核生物中发现的高度保守的同源物组。Nup 145 N，蛋白质的N-末端自蛋白水解的一半，通过其与Nup 98的类似性，已经涉及在细胞核和NPC之间携带RNA。然而，Nup 145 N（443-605）和Nup 98 N的晶体结构在其各自的晶体学不对称单元内显示出不同的缔合模式。我们初步的溶液X射线散射结果表明，Nup 145 N（443-605）形成的头到尾的二聚体在溶液中观察到的两个不同的空间群的不对称单位。

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. The Nuclear Pore Complex (NPC, about 50 MDa) is the sole passageway for the transport of macromolecules across the nuclear envelope. The pore plays a key role in numerous critical cellular processes such as transcription, and many of its components are implicated in human diseases such as cancer. The recent work by the Sali group provided the first description of the macromolecular architecture of the yeast NPC. This structure defined the relative positions and proximities of its 456 constituent nucleoporin (nup) proteins, based on spatial restraints derived from experimental data. Further elucidation of the evolutionary origin and transport mechanism of the NPC will require higher resolution information. To help improve upon the resolution and accuracy of the NPC structure, we have begun small angle x-ray scattering studies at SSRL. Individial nup proteins and complexes are being expressed, produced and purified by our collaborators at Rockefeller University and Eli Lilly on a regular basis. We selected several nup proteins whose crystal structures have been solved to establish a benchmark. Among them is Nup145 which belongs to a highly conserved group of homologs found throughout the eukaryotes. Nup145N, the autoproteolised N-terminal half of the protein has been implicated in carrying RNA between the nucleus and the NPC by its analogy with Nup98. The crystal structures of Nup145N(443-605) and Nup98N, however, shows different modes of association within their respective crystallographic asymmetric units. Our preliminary solution x-ray scattering results demonstrate that Nup145N(443-605) forms the head-to-tail dimer in solution as observed in the asymmetric units of two different space groups.