BUDDING YEAST SEPTIN FILAMENTS: SAXS STUDIES

出芽酵母败丝：SAXS 研究

• 批准号: 8362059
• 负责人: HIROTSUGU TSURUTA
• 金额: $ 0.14万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-03-01 至 2012-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8362059
• 关键词: Cell division; Coiled-Coil Domain; Cryoelectron Microscopy; Cytokinesis; Cytoskeleton; Data; Dimensions; Escherichia coli; Filament; Funding; Grant; Human; Ionic Strengths; Modeling; Mothers; National Center for Research Resources; Neck; Negative Staining; Polymers; Principal Investigator; Proteins; Radial; Radiation; Research; Research Infrastructure; Resolution; Resources; Saccharomycetales; Shapes; Sodium Chloride; Solutions; Source; Structure; Time; Tooth structure; United States National Institutes of Health; cost; daughter cell; flexibility; reconstruction; retinal rods; self assembly; structural biology

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. 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.

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 Septin蛋白组装成一种细胞骨架聚合物。Septin细胞骨架是细胞分裂的最后一步，胞质分裂是必不可少的。在萌芽酵母中，芽颈上可见丝状的隔质环，并将其收缩，以分隔母细胞和子细胞。我们重点介绍了在大肠杆菌中可以共表达的四种主要的分隔素：CDC3、CDC10、CDC12和CDC11。我们最近在我们的负染色低温EM研究中表明，Septin的自组装是依赖于盐的，并且在高盐浓度(高于200 mM一价盐)下形成一个400 kDa的八聚体，每个八聚体有两个拷贝，而在低盐条件下，Septin八聚体聚合成一对细丝。我们还证明了八聚体杆是反平行的，并消除了以下亚基相互作用的序列：Cdc11-Cdc12-Cdc3-Cdc10-Cdc10-Cdc3-Cdc12-Cdc11.因此，聚合的细丝是非极性的，与最近三个人类隔膜的子集的4A分辨率晶体结构一致。在晶体结构中，间隔蛋白的卷曲线圈结构域是看不见的，我们相信这些结构域参与了低离子强度下的纤维配对。3DEM重建正在进行中，目前在26A分辨率下显示为杆状，但它丢失了总蛋白质质量的20%。丢失的质量可以归因于柔性的盘绕线圈结构域，因为结晶学确定的球状结构域可以用EM模型来拟合。我们已经在SSRL开始了溶液X射线散射研究，并获得了高质量的研究。在0.5M的单价盐溶液中，蛋白质浓度在0.75-3 mg/ml范围内的散射数据没有随浓度或时间的变化，最大组装尺寸和旋转半径分别为100和350A。我们使用SAXS数据进行的三维重建得到了一个杆状形状，沿长轴方向有一个温和的锯齿状调制。