Open AccessInorganic pyrophosphatase crystals from Thermococcus thioreducens for X‐ray and neutron diffraction
Author(s) -
Hughes Ronny C.,
Coates Leighton,
Blakeley Matthew P.,
Tomanicek Steve J.,
Langan Paul,
Kovalevsky Andrey Y.,
GarcíaRuiz Juan M.,
Ng Joseph D.
Publication year - 2012
Publication title -
acta crystallographica section f
Language(s) - English
Resource type - Journals
ISSN - 1744-3091
DOI - 10.1107/s1744309112032447
Subject(s) - crystallography , inorganic pyrophosphatase , neutron diffraction , resolution (logic) , monoclinic crystal system , crystallization , x ray crystallography , diffraction , materials science , crystal structure , chemistry , enzyme , optics , physics , biochemistry , organic chemistry , artificial intelligence , pyrophosphate , computer science
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 mm 3 . 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 C 2, 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.