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.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 Septin蛋白组装成细胞骨架聚合物。 Septin细胞骨架对于细胞因子（细胞分裂的最后一步）至关重要。在萌芽的酵母中，已在芽颈上可视化了七月的丝状环，并将其收缩以将母细胞分开。我们专注于可以在e.coli：cdc3，cdc10，cdc12和cdc11中共表达的四种septins。我们最近在我们的负污染冷冻研究中表明，septin自组装是盐的依赖​​性的，并形成400 kDa的八聚棒棒，在高盐浓度（高于200 mm单价盐）下，每个八聚体每八聚体中的每个septin二拷贝，而septin八聚体在低盐条件下聚合成配对的细丝。我们还证明了八接杆是反平行的，并分配了以下亚基相互作用的序列：CDC11-CDC12-CDC3-CDC3-CDC10-CDC10-CDC10-CDC3-CDC3-CDC12-CDC11。因此，聚合丝是非极性的，与最近的4个分辨率晶体结构一致，该子集的三个人类septins的子集。 Septins的盘绕型结构域在晶体结构中是看不见的，我们认为这些结构域参与了低离子强度以低离子强度配对丝。 3D EM重建正在进行中，目前在26 A分辨率下显示杆状，然而，这却缺少约20％的总蛋白质质量。缺失的质量可以归因于柔性螺旋线域，因为晶体学确定的球状域可以适合EM模型。 我们已经开始在SSRL上进行X射线散射研究，并获得了高质量。在0.5 m的单价盐下，我们观察到蛋白质浓度范围0.75-3 mg/ml的散射数据的浓度或时间依赖性变化。回旋半径和分子组件的最大维度分别确定为100和350a。我们使用SAXS数据的3D重建导致杆状形状，沿着长轴具有温和的锯齿状调制。