Purification and crystallization of the entire recombinant subunit E of the energy producer A(1)A(o) ATP synthase

Abstract

A(1)A(o) ATP synthases are the major energy producers in archaea. Subunit E of the stator domain of the ATP synthase from Pyrococcus horikoshii OT3 was cloned, expressed and purified to homogeneity. The monodispersed protein was crystallized by vapour diffusion. A complete diffraction data set was collected to 3.3 A resolution with 99.4% completeness using a synchrotron-radiation source. The crystals belonged to space group I4, with unit-cell parameters a = 112.51, b = 112.51, c = 96.25 A, and contained three molecules in the asymmetric unit.

Figure 1

(a) SDS gel (17% total acrylamide and 0.4% cross-linked acrylamide) of the purified recombinant subunit E of the A-ATP synthase from P. horikoshii OT3 (lane 2). Lane 1, molecular-weight markers (kDa). (b) Far-UV CD spectrum of subunit E (2 mg ml⁻¹).

Figure 2

Crystals of subunit E of P. horikoshii OT3 A-ATP synthase. The crystals are approximately 0.2 × 0.3 × 0.3 mm in size.

Figure 3

Diffraction image of a subunit E crystal.