BUDDING YEAST SEPTIN FILAMENTS: SAXS STUDIES

这个子项目是许多利用 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。 隔膜蛋白组装成细胞骨架聚合物。胞质分裂是细胞分裂的最后一步，细胞骨架是胞质分裂所必需的。在芽殖酵母中，在芽颈处可见丝状的隔蛋白环，并将其压缩以分离母细胞和子细胞。我们专注于可以在大肠杆菌中共表达的四种基本septins：Cdc 3，Cdc 10，Cdc 12和Cdc 11。我们最近在我们的负染色cryoEM研究中表明，隔蛋白自组装是盐依赖性的，并在高盐浓度（高于200 mM单价盐）下形成400 kDa的八聚体棒，每个八聚体具有两个拷贝的每个隔蛋白，而隔蛋白八聚体在低盐条件下形成成对的细丝。我们还证明了八聚体杆是反平行的，并显示了以下顺序的亚基相互作用：Cdc 11-Cdc 12-Cdc 3-Cdc 10-Cdc 10-Cdc 3-Cdc 12-Cdc 11。因此，聚合的细丝是非极性的，与最近的4 A分辨率的三种人septins的子集的晶体结构一致。septins的卷曲螺旋结构域在晶体结构中是不可见的，我们相信这些结构域参与了低离子强度下的细丝配对。3D EM重建正在进行中，目前在26 A分辨率下显示出杆状，但缺失了约20%的总蛋白质质量。缺失的质量可以归因于柔性卷曲螺旋域，因为晶体学确定的球状域可以拟合在EM模型中。 我们已经开始在SSRL的解决方案X射线散射研究，并获得了高品质。在0.5M单价盐下，我们在0.75-3 mg/ml蛋白质浓度范围内观察到散射数据没有浓度或时间依赖性变化。隔膜组件的回转半径和最大尺寸分别被确定为100和350 A。我们使用SAXS数据的3D重建产生了沿着长轴沿着具有温和锯齿状调制的棒形。

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. Septin proteins assemble into a cytoskeletal polymer. Septin cytoskeleton is essential for cytokinesis, the last step in cell division. In budding yeast, filamentous rings of septins have been visualized at the bud neck and constrict it in order to separate the mother and daughter cells. We focus on the essential four species of septins that can be co-expressed in E.coli: Cdc3, Cdc10, Cdc12 and Cdc11. We have recently shown in our negative-stain cryoEM study that septin self-assembly is salt-dependent and forms a 400 kDa octameric rod with two copies of each septin per octamer at high salt concentrations (above 200 mM monovalent salt) while septin octamers polymerize into a paired filament in low salt conditions. We have also demonstrated that the octameric rod is anti-parallel and dispalys the following sequence of subunit interactions: Cdc11-Cdc12-Cdc3-Cdc10-Cdc10-Cdc3-Cdc12-Cdc11. The polymerized filament is therefore non polar, consistent with the recent 4 A resolution crystal structure of a subset of three human septins. The coiled-coil domains of septins are invisible in the crystal structure and we believe these domains are involved in pairing the filament at low ionic strengths. 3D EM reconstruction is under way, and currently shows a rod shape at 26 A resolution, which however is missing ~20% of the total protein mass. The missing mass can be attributed to the flexible coiled-coil domains since the crystallographically determined globular domains can be fit in the EM model. We have begun solution x-ray scattering studies at SSRL and obtained a high quality. At 0.5 M monovalent salt, we observe no concentration- or time- dependent change in the scattering data in the protein concentration range 0.75-3 mg/ml. The radius of gyration and the maximum dimension of the septing assembly have been determined as 100 and 350 A, respectively. Our 3D reconstruction using the SAXS data resulted in a rod shape with a gentle saw-tooth like modulation along the long axis.