THE PERIPHERAL STALK OF YEAST VACUOLAR ATPASE

这个子项目是许多研究子项目中的一个 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， 因此可以在其他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的研究机构。 液泡膜ATP酶（vacuolar ATPase，V-ATPase）是真核细胞内膜系统中一种多亚基旋转马达酶，在ATP水解过程中起质子泵的作用。V-ATP酶由两个运动域组成，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周定子蛋白连接，其作为结构连接起作用以抵消在ATP水解期间产生的旋转扭矩。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），并且我们现在使用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，用于拟合EM衍生图。我们已经将酵母V-ATP酶的外围柄形成亚基（亚基E和G）与亚基C的结构域复合结晶。在Chess光束线F1上进行的初步衍射分析（2009年秋季;与Edward Berry博士合作）导致约5.5 <$衍射。晶体属于空间群P212121，晶胞参数分别为95.2，114.1，133.9。目前，V-ATP酶（或任何相关的旋转ATP酶，包括F1 F0-ATP合酶或古细菌A-ATP酶）的外周柄（一个或多个）没有可用的晶体结构。 自去年秋天以来，我们优化了结晶条件，包括96个条件的添加剂筛选，从而产生了具有不同晶体形态的许多条件。如果获得批准，我们将使用Chess的光束时间来筛选高质量衍射的晶体，并在时间和晶体质量允许的情况下收集本地数据集。随后的工作，其中一个完整的建议是计划，将包括重金属浸泡和/或SeMet含有蛋白质。分子置换可能是可能的，因为亚基C的晶体结构是可用的。

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. 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.