THE PERIPHERAL STALK OF YEAST VACUOLAR ATPASE

酵母液泡ATP酶的外周柄

• 批准号: 8363542
• 负责人: Stephan Wilkens
• 金额: $ 1.78万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-01 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8363542
• 关键词: 3-Dimensional; ATP Hydrolysis; ATP phosphohydrolase; Berry; Binding; Cells; Collaborations; Complex; Crystallization; Data Set; Enzymes; Eukaryotic Cell; F1F0-ATP synthase; Funding; Grant; Heavy Metals; L-Selenomethionine; Link; Maps; Membrane; Molecular; Morphology; Motor; National Center for Research Resources; Peripheral; Principal Investigator; Proteins; Proton Pump; Protons; Research; Research Infrastructure; Resolution; Resources; Source; Structure; System; Time; Torque; United States National Institutes of Health; Work; X-Ray Crystallography; Yeasts; beamline; cost; falls; protein function; reconstruction; vacuolar H+-ATPase

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. The vacuolar ATPase (V-ATPase) is a multi subunit rotary motor enzyme that functions as an ATP hydrolysis driven proton pump in the endomembrane system of eukaryotic cells. The V-ATPase consistes of two motor domains, a cytoplasmic ATPase (V1) and a membrane bound proton channel (V0). The two domains are linked by three peripheral stator proteins that function as a structural link to counteract the rotational torque that is generated during ATP hydrolysis. We have recently obtained a 3-D reconstruction of the intact V-ATPase from yeast (Zhang et al., JBC 283, 35983) and we are now using X-ray crystallography to determine the atomic resolution structures of V-ATPase subunits and subunit domains for fitting into the EM derived map. We have crystallized the peripheral stalk forming subunits of yeast V-ATPase (subunits E&G) in complex with a domain of subunit C. A preliminary diffraction analysis performed at the Chess beamline F1 (Fall 2009; in collaboration with Dr. Edward Berry) resulted in ~5.5 ¿ diffraction. The crystals belong to spacegroup P212121 with unit cell parameters of 95.2, 114.1, 133.9 ¿ , 90,90.5,90¿¿. Currently, there is no crystal structure available for the peripheral stalk(s) of the V-ATPase (or any of the related rotary ATPases including the F1F0-ATP synthase or the archaeal A-ATPase). SInce last fall, we have optimized crystallization conditions including a 96 condition additive screen, leading to numerous conditions with varying crystal morphologies. We will use the beam time at Chess, if approved, to screen crystals for high quality diffraction and to collect native data sets if time and crystal quality permits. Subsequent work, for which a full proposal is planned, would include heavy metal soaks and/or SeMet containing protein. Molecular replacement may be possible as a crystal structure for subunit C is available.

该子项目是利用资源的众多研究子项目之一 由 NIH/NCRR 资助的中心拨款提供。子项目的主要支持 并且子项目的主要研究者可能是由其他来源提供的， 包括其他 NIH 来源。 子项目可能列出的总成本 代表子项目使用的中心基础设施的估计数量， NCRR 赠款不直接向子项目或子项目工作人员提供资金。 液泡 ATP 酶 (V-ATP 酶) 是一种多亚基旋转马达酶，在真核细胞内膜系统中充当 ATP 水解驱动的质子泵。 V-ATP 酶由两个运动结构域组成，即细胞质 ATP 酶 (V1) 和膜结合质子通道 (V0)。这两个结构域由三个外围定子蛋白连接，这些定子蛋白充当结构链接以抵消 ATP 水解过程中产生的旋转扭矩。我们最近获得了来自酵母的完整 V-ATP 酶的 3-D 重建（Zhang 等人，JBC 283, 35983），我们现在使用 X 射线晶体学来确定 V-ATP 酶亚基和亚基结构域的原子分辨率结构，以便拟合到 EM 衍生图谱中。我们已经将酵母 V-ATP 酶的外周茎形成亚基（亚基 E&G）与亚基 C 的结构域形成复合物结晶。在国际象棋光束线 F1（2009 年秋季；与 Edward Berry 博士合作）进行的初步衍射分析产生了约 5.5 ¿ 的衍射。该晶体属于P212121空间群，晶胞参数为95.2、114.1、133.9 ¿、90、90.5、90 ¿。目前，V-ATP 酶（或任何相关的旋转 ATP 酶，包括 F1F0-ATP 合酶或古菌 A-ATP 酶）的外周柄尚无可用的晶体结构。 自去年秋天以来，我们优化了结晶条件，包括 96 种条件添加剂筛选，从而产生具有不同晶体形态的多种条件。如果获得批准，我们将使用 Chess 的光束时间来筛选晶体以进行高质量衍射，并在时间和晶体质量允许的情况下收集本机数据集。后续工作（已计划制定完整的提案）将包括重金属浸泡和/或含有 SeMet 的蛋白质。由于亚基 C 的晶体结构是可用的，分子替换是可能的。