Crystallization, preliminary X-ray crystallographic and cryo-electron microscopy analysis of a bifunctional enzyme fucokinase/l-fucose-1-P-guanylyltransferase from Bacteroides fragilis

In this study, both recombinant F-FKP and C-FKP were expressed and purified. Size-exclusion chromatography experiments and analytical ultracentrifugation results showed that both F-FKP and C-FKP are trimers. Native F-FKP protein was crystallized by the vapour-diffusion method and the crystals belonged to space group P212121 and diffracted synchrotron X-rays to 3.7 Å resolution. The crystal unit-cell parameters are a = 91.36, b = 172.03, c = 358.86 Å, α = β = γ = 90.00°. The three-dimensional features of the F-FKP molecule were observed by cryo-EM (cryo-electron microscopy). The preliminary cryo-EM experiments showed the F-FKP molecules as two parallel disc-shaped objects stacking together. Combining all results together, it is assumed that there are six FKP molecules in one asymmetric unit, which corresponds to a calculated Matthews coefficient of 2.19 Å3 Da−1 with 43.83% solvent content. These preliminary crystallographic and cryo-EM microscopy analyses provide basic structural information on FKP.

Source: Acta Crystallographica Section F - August 28, 2014 Category: Biochemistry Authors: Cheng, C.Gu, J.Su, J.Ding, W.Yin, J.Liang, W.Yu, X.Ma, J.Wang, P.G.Xiao, Z.Liu, Z.-J. Tags: FKP Bacteroides fragilis cryo-EM crystallization communications Source Type: research