Purification, crystallization and preliminary X-ray diffraction analysis of ThiM from Staphylococcus aureus

Abstract

ThiM [5-(hydroxyethyl)-4-methylthiazole kinase; EC 2.7.1.50] from Staphylococcus aureus is an essential enzyme of thiamine or vitamin B(1) metabolism and has been crystallized by the vapour-diffusion method. The crystals belonged to the primitive space group P1, with unit-cell parameters a = 62.06, b = 62.40, c = 107.82 Å, α = 92.25, β = 91.37, γ = 101.48° and six protomers in the unit cell, corresponding to a packing parameter V(M) of 2.3 Å(3) Da(-1). Diffraction data were collected to 2.1 Å resolution using synchrotron radiation. The phase problem was solved by molecular replacement.

Figure 1

SDS–PAGE analysis of SaThiM after elution from the Ni–NTA column. The fraction was analysed on a 12.5% SDS gel and stained with Coomassie Blue. A single band with an approximate molecular weight of 30 kDa was observed.

Figure 2

DLS measurement of a 20 mg ml⁻¹ SaThiM solution, showing a monodisperse protein solution and a hydrodynamic radius (R _h) of approximately 3.2 nm.

Figure 3

SaThiM crystals grew to maximum dimensions after 5 d. A single lamella (0.3 × 0.2 × 0.02 mm) was prepared by separating the bundle of crystals with microtools prior to data collection.