title: Inorganic pyrophosphatase crystals from Thermococcus thioreducens for X-ray and neutron diffraction.
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.
authors.affiliation: Department of Biological Sciences and Laboratory for Structural Biology, University of Alabama in Huntsville, Huntsville, AL 35899, USA.
authors.forename: Ronny C
authors.initials: RC
authors.lastname: Hughes
authors.forename: Leighton
authors.initials: L
authors.lastname: Coates
authors.forename: Matthew P
authors.initials: MP
authors.lastname: Blakeley
authors.forename: Steve J
authors.initials: SJ
authors.lastname: Tomanicek
authors.forename: Paul
authors.initials: P
authors.lastname: Langan
authors.forename: Andrey Y
authors.initials: AY
authors.lastname: Kovalevsky
authors.forename: Juan M
authors.initials: JM
authors.lastname: García-Ruiz
authors.forename: Joseph D
authors.initials: JD
authors.lastname: Ng
mesh_terms: D019843:Archaeal Proteins / Q000737:chemistry* / Q000302:isolation & purification; D003460:Crystallization; D018360:Crystallography, X-Ray; D043564:Inorganic Pyrophosphatase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D033363:Neutron Diffraction / Q000379:methods; D019712:Thermococcus / Q000201:enzymology*; D014961:X-Ray Diffraction / Q000379:methods
chemical_list: D019843:Archaeal Proteins; D043564:Inorganic Pyrophosphatase