Cloning, expression, purification, crystallization and preliminary crystallographic analysis of selenomethionine-labelled KaiC-like protein PH0186 from Pyrococcus horikoshii OT3

Abstract

KaiC is the central protein in the circadian-clock system of cyanobacteria. A selenomethionine-labelled KaiC-homologous protein from Pyrococcus horikoshii OT3 (PH0186; 28 kDa) was crystallized by the sitting-drop vapour-diffusion method using ethanol as a precipitant. The crystals diffracted X-rays to beyond 2.0 A resolution using a synchrotron-radiation source. The space group of the crystals was determined to be C2, with unit-cell parameters a = 173.7, b = 51.8, c = 97.5 A, beta = 122.8 degrees. The crystal contains three molecules in the asymmetric unit (V(M) = 2.2 A(3) Da(-1)) and has a solvent content of 43.5%. Sixfold noncrystallographic symmetry was identified from self-rotation calculations, assuming the presence of a hexamer in the crystal.

Figure 1

Sequence alignment of PH0186 from P. horikoshii and KaiCI from Synechococcus sp. strain PCC 7942. The blue, red and black bars indicate the Walker A motif, Walker B motif and catalytic Glu residues, respectively. The sequences were aligned with ClustalW (Thompson et al., 1994 ▶) and the figure was produced using ESPript (Gouet et al., 1999 ▶).

Figure 2

A crystal of PH0186 (SeMet derivative) from P. horikoshii obtained by the sitting-drop vapour-diffusion method.

Figure 3

Diffraction pattern of a PH0186 crystal (SeMet derivative). The edge of the image corresponds to a resolution of 1.94 Å. The data were collected on BL38B1 at SPring-8.

Figure 4

Self-rotation function plot at κ = 60°. The rotation function was calculated with a radius of integration of 20 Å in the resolution range 20–3 Å. ω (the angle from the pole) is 0 or 180° in the centre and 90° at the edge; ϕ (the angle around the equator) is as marked on the periphery.