Hunsicker-Wang Laura M, Pacoma Ronald L, Chen Ying, Fee James A, Stout C David
Department of Molecular Biology, The Scripps Research Institute, MB8, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
Acta Crystallogr D Biol Crystallogr. 2005 Mar;61(Pt 3):340-3. doi: 10.1107/S0907444904033906. Epub 2005 Feb 24.
Cytochrome ba(3) oxidase is an integral membrane protein identified in the thermophilic bacterium Thermus thermophilus. The enzyme has now been expressed recombinantly and purified with a histidine tag. As such, it crystallizes under similar conditions and in the same space group (P4(3)2(1)2) as the native protein. A novel cryoprotection scheme is described here to obtain high-resolution diffraction from these crystals, which involves soaking in a mixture of glycerol and ethylene glycol under a layer of oil. The unit-cell parameters for these crystals are larger than the native protein, apparently deriving from increased ordering of the N-terminus and an internal loop (residues 495-500) in subunit I. Hence, compared with native cytochrome ba(3) oxidase, the recombinant His-tagged protein is accommodated in an expanded but equally well ordered lattice via an alternate set of specific intermolecular contacts. The structure was refined against data to 2.3 angstroms resolution to an R factor of 21.7% and an R(free) of 23.7%.
细胞色素ba(3)氧化酶是在嗜热栖热菌中发现的一种整合膜蛋白。该酶现已通过重组表达并用组氨酸标签进行了纯化。因此，它在与天然蛋白相似的条件下、以相同的空间群（P4(3)2(1)2）结晶。本文描述了一种新的冷冻保护方案，以从这些晶体中获得高分辨率衍射，该方案包括在油层下浸泡在甘油和乙二醇的混合物中。这些晶体的晶胞参数比天然蛋白大，这显然源于亚基I中N端和一个内部环（残基495 - 500）的有序性增加。因此，与天然细胞色素ba(3)氧化酶相比，重组的带组氨酸标签的蛋白通过另一组特定的分子间接触被容纳在一个扩展但同样有序的晶格中。该结构针对分辨率为2.3埃的数据进行了精修，R因子为21.7%，R(free)为23.7%。
