Inorganic pyrophosphatase crystals from Thermococcus thioreducens for X-ray and neutron diffraction.

Ronny C Hughes, Leighton Coates, Matthew P Blakeley, Steve J Tomanicek, Paul Langan, Andrey Y Kovalevsky, Juan M García-Ruiz, Joseph D Ng

Index: Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 68(Pt 12) , 1482-7, (2012)

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Abstract

Inorganic pyrophosphatase (IPPase) from the archaeon Thermococcus thioreducens was cloned, overexpressed in Escherichia coli, purified and crystallized in restricted geometry, resulting in large crystal volumes exceeding 5 mm3. IPPase is thermally stable and is able to resist denaturation at temperatures above 348 K. Owing to the high temperature tolerance of the enzyme, the protein was amenable to room-temperature manipulation at the level of protein preparation, crystallization and X-ray and neutron diffraction analyses. A complete synchrotron X-ray diffraction data set to 1.85 Å resolution was collected at room temperature from a single crystal of IPPase (monoclinic space group C2, unit-cell parameters a=106.11, b=95.46, c=113.68 Å, α=γ=90.0, β=98.12°). As large-volume crystals of IPPase can be obtained, preliminary neutron diffraction tests were undertaken. Consequently, Laue diffraction images were obtained, with reflections observed to 2.1 Å resolution with I/σ(I) greater than 2.5. The preliminary crystallographic results reported here set in place future structure-function and mechanism studies of IPPase.