THE PERIPHERAL STALK OF YEAST VACUOLAR ATPASE

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 液泡型ATPase(V-ATPase)是一种多亚基旋转马达酶，在真核细胞的膜系统中起着ATP水解泵的作用。V-ATPase由两个运动域组成，即细胞质ATPase(V1)和膜结合质子通道(V0)。这两个结构域由三个外围的定子蛋白连接在一起，它们作为结构纽带发挥作用，以抵消在ATP水解过程中产生的旋转扭矩。我们最近从酵母中获得了完整的V-ATPase的三维重建(Zhang等人，JBC283,35983)，现在我们正在使用X射线结晶学来确定V-ATPase亚基和亚单位结构域的原子分辨率结构，以便适应EM衍生的图谱。我们已经将酵母V-ATPase的外围茎形成亚基(E和G亚基)结晶成具有C亚基结构域的复合体。在Chess Beamline F1(2009年秋季；与Edward Berry博士合作)上进行的初步衍射分析导致了~5.5°的衍射。晶体属于P212121空间群，晶胞参数分别为95.2,114.1,133.9，90，90.5，90°。目前，V-ATPase(或任何相关的旋转ATPase，包括F1F0-ATP合成酶或古代A-ATPase)的外周茎(S)还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个条件的添加剂屏幕，导致了许多不同晶体形态的条件。如果批准，我们将使用国际象棋的光束时间来筛选晶体的高质量衍射，并在时间和晶体质量允许的情况下收集本地数据集。随后的工作将包括重金属浸泡和/或含有蛋白质的SeMet，计划对其进行完整的提议。当C亚单位的晶体结构可用时，分子置换是可能的。

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.