Loris Remy, Garcia-Pino Abel, Buts Lieven, Bouckaert Julie, Beeckmans Sonia, De Greve Henri, Wyns Lode
Vrije Universiteit Brussel and Vlaams Instituut voor Biotechnologie, Laboratorium voor Ultrastructuur (ULTR), Pleinlaan 2, B-1050 Brussel, Belgium.
Acta Crystallogr D Biol Crystallogr. 2005 Jun;61(Pt 6):685-9. doi: 10.1107/S0907444904028707. Epub 2005 May 26.
The Man/Glc-specific legume lectin from the seeds of the African bloodwood tree (Pterocarpus angolensis) was crystallized in the presence of the disaccharide ligand Man(alpha1-3)ManMe. Small crystals initially appeared from a preliminary screen, but proved difficult to reproduce. The initial crystals were used to prepare microseeds, leading to a reproducible crystallization protocol. All attempts to obtain crystals directly of the ligand-free protein or of other carbohydrate complexes failed. However, the Man(alpha1-3)ManMe co-crystals withstand soaking with ten other carbohydrates known to bind to the lectin. Soaking for 15 min in 100 mM carbohydrate typically resulted in complete replacement of Man(alpha1-3)ManMe by the desired carbohydrate despite the involvement of lattice contacts at the binding site. Transferring the crystals for two weeks in carbohydrate-free artificial mother liquor resulted in the complete removal of the sugar from one of the two monomers in the asymmetric unit. Additional treatment of these crystals with 100 mM EDTA for two weeks resulted in removal of the structural calcium and manganese ions, which is accompanied by significant structural rearrangements of the loops that constitute the carbohydrate-binding site.
来自非洲红木树（安哥拉紫檀）种子的人/葡萄糖特异性豆科凝集素在二糖配体甘露糖（α1-3）甘露糖甲基酯存在下结晶。最初在初步筛选中出现了小晶体，但难以重现。最初的晶体用于制备微种子，从而得到了可重现的结晶方案。所有直接获得无配体蛋白或其他碳水化合物复合物晶体的尝试均告失败。然而，甘露糖（α1-3）甘露糖甲基酯共晶体能够经受住与已知与凝集素结合的其他十种碳水化合物的浸泡。在100 mM碳水化合物中浸泡15分钟通常会导致甘露糖（α1-3）甘露糖甲基酯被所需碳水化合物完全取代，尽管结合位点存在晶格接触。将晶体在无碳水化合物的人工母液中放置两周会导致不对称单元中两个单体之一的糖完全去除。用100 mM EDTA对这些晶体进行两周的额外处理会导致结构钙和锰离子的去除，同时构成碳水化合物结合位点的环会发生显著的结构重排。
