BUDDING YEAST SEPTIN FILAMENTS: SAXS STUDIES

出芽酵母败丝：SAXS 研究

• 批准号: 8169940
• 负责人: HIROTSUGU TSURUTA
• 金额: $ 0.24万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-01 至 2011-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8169940
• 关键词: Cell division; Coiled-Coil Domain; Computer Retrieval of Information on Scientific Projects Database; Cryoelectron Microscopy; Cytokinesis; Cytoskeleton; Data; Dimensions; Filament; Funding; Grant; Human; Institution; Ionic Strengths; Modeling; Mothers; Neck; Negative Staining; Polymers; Proteins; Radial; Research; Research Personnel; Resolution; Resources; Saccharomycetales; Shapes; Sodium Chloride; Solutions; Source; Structure; Time; Tooth structure; United States National Institutes of Health; daughter cell; flexibility; reconstruction; retinal rods; self assembly

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.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目及 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 Septin 蛋白组装成细胞骨架聚合物。 Septin 细胞骨架对于胞质分裂（细胞分裂的最后一步）至关重要。在芽殖酵母中，在芽颈处可以看到丝状的脓蛋白环，并将其收缩以分离母细胞和子细胞。我们重点关注可在大肠杆菌中共表达的四种重要脓毒蛋白：Cdc3、Cdc10、Cdc12 和 Cdc11。我们最近在负染色冷冻电镜研究中表明，septin 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 八聚体棒，每个八聚体每个 septin 有两个副本，同时 septin 八聚体聚合在低盐条件下形成成对的细丝。我们还证明了八聚杆是反平行的，并显示了以下亚基相互作用序列：Cdc11-Cdc12-Cdc3-Cdc10-Cdc10-Cdc3-Cdc12-Cdc11。因此，聚合丝是非极性的，与最近的三个人类脓毒症子集的 4 A 分辨率晶体结构一致。脓毒症的卷曲螺旋域在晶体结构中是不可见的，我们相信这些域参与低离子强度下的细丝配对。 3D EM 重建正在进行中，目前以 26 A 分辨率显示棒状形状，但缺失约 20% 的总蛋白质质量。缺失的质量可归因于柔性卷曲线圈域，因为晶体学确定的球状域可以适合电磁模型。 我们已经在 SSRL 开始了溶液 X 射线散射研究，并获得了高质量的结果。在 0.5 M 一价盐下，我们在 0.75-3 mg/ml 的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。隔离组件的回转半径和最大尺寸分别确定为 100 和 350 A。我们使用 SAXS 数据进行 3D 重建，得到了沿长轴具有柔和锯齿状调制的棒状形状。