Purification, crystallization and preliminary crystallographic analysis of the vacuole-type ATPase subunit E from Pyrococcus horikoshii OT3

Abstract

The vacuole-type ATPases in eukaryotic cells translocate protons across various biological membranes including the vacuolar membrane by consuming ATP molecules. The E subunit of the multisubunit complex V-ATPase from Pyrococcus horikoshii OT3, which has a molecular weight of 22.88 kDa, has been cloned, overexpressed in Escherichia coli, purified and crystallized by the microbatch method using PEG 4000 as a precipitant at 296 K. A data set to 1.85 Å resolution with 98.8% completeness and an Rmerge of 6.5% was collected from a single flash-cooled crystal using synchrotron radiation. The crystal belonged to the orthorhombic space group P212 121, with unit-cell parameters a = 52.196, b = 55.317, c = 77.481 Å, and is most likely to contain one molecule per asymmetric unit. © 2005 International Union of Crystallography All rights reserved.