Cloning, expression and crystallization of VMA13p, an essential subunit of the vacuolar H+-ATPase of Saccharomyces cerevisiae

• Published in: Acta crystallographica. Section D, Biological crystallography. Vol. 56; no. 4; pp. 475 - 477
• Main Authors: Sagermann, Martin, Matthews, Brian W.
• Format: Journal Article
• Language: English
• Published: 5 Abbey Square, Chester, Cheshire CH1 2HU, England Munksgaard International Publishers 01.04.2000
• Subjects: Cloning, Molecular; Crystallization; Crystallography, X-Ray; Escherichia coli; Macromolecular Substances; Proton-Translocating ATPases - chemistry; Proton-Translocating ATPases - isolation & purification; Recombinant Proteins - biosynthesis; Recombinant Proteins - chemistry; Recombinant Proteins - isolation & purification; Saccharomyces cerevisiae - enzymology; V-ATPase; Vacuolar Proton-Translocating ATPases
• ISSN: 1399-0047; 0907-4449; 1399-0047
• DOI: 10.1107/S0907444900000950

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 P3121 or its enantiomorph, with unit‐cell parameters a = b = 118.8, c = 119.3 Å. Using an in‐house X‐ray source, the crystals diffract to about 3.5 Å resolution under rapidly frozen conditions.