Purification, crystallization and preliminary crystallographic analysis of the vacuole-type ATPase subunit E from Pyrococcus horikoshii OT3.
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 A 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 P2(1)2(1)2(1), with unit-cell parameters a = 52.196, b = 55.317, c = 77.481 A, and is most likely to contain one molecule per asymmetric unit.
Lokanath
Neratur K
NK
Highthroughput Factory, RIKEN Harima Institute at SPring-8, 1-1-1 Kouto, Hyogo 679-5148, Japan.
Ukita
Yoko
Y
Sugahara
Mitsuaki
M
Kunishima
Naoki
N
D019843:Archaeal Proteins / Q000737:chemistry / Q000302:isolation & purification; D003460:Crystallization; D021122:Protein Subunits / Q000737:chemistry / Q000302:isolation & purification; D043902:Pyrococcus horikoshii / Q000201:enzymology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D025262:Vacuolar Proton-Translocating ATPases / Q000737:chemistry* / Q000302:isolation & purification; D014961:X-Ray Diffraction
D019843:Archaeal Proteins; D021122:Protein Subunits; D011994:Recombinant Proteins; D025262:Vacuolar Proton-Translocating ATPases