Cloning, expression and crystallization of VMA13p, an essential subunit of the vacuolar H+-ATPase of Saccharomyces cerevisiae.
The expression and crystallization of the VMA13p subunit of the vacuolar proton-translocating ATPase (V-ATPase) of Saccharomyces cerevisiae is described. This 478 amino-acid subunit is essential for activity but not for the assembly of this multisubunit complex. The protein has been recombinantly overexpressed in Escherichia coli and purified. Diffraction-quality crystals have been obtained using the hanging-drop vapor-diffusion method with ammonium sulfate as precipitant. Several different crystal forms were obtained. The most suitable crystal form for crystallographic characterization belongs to space group P3(1)21 or its enantiomorph, with unit-cell parameters a = b = 118.8, c = 119.3 A. Using an in-house X-ray source, the crystals diffract to about 3.5 A resolution under rapidly frozen conditions.
Sagermann
M
M
Institute of Molecular Biology, Howard Hughes Medical Institute and Department of Physics, 1229 University of Oregon, Eugene, Oregon 97403-1229, USA.
Matthews
B W
BW
D003001:Cloning, Molecular; D003460:Crystallization; D018360:Crystallography, X-Ray; D004926:Escherichia coli; D046911:Macromolecular Substances; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D025262:Vacuolar Proton-Translocating ATPases
D046911:Macromolecular Substances; D011994:Recombinant Proteins; D025262:Vacuolar Proton-Translocating ATPases; D006180:Proton-Translocating ATPases