Garboczi D N, Madden D R, Wiley D C
Department of Biochemistry and Molecular Biology, Harvard University, Cambridge, MA 02138.
J Mol Biol. 1994 Jun 17;239(4):581-7. doi: 10.1006/jmbi.1994.1398.
We prepared and crystallized five complexes of the human histocompatibility molecule HLA-A2 with peptides derived from human immunodeficiency virus type 1, human T lymphotropic virus type 1, influenza A virus and hepatitis B virus proteins. Each HLA-A2 complex was refolded in vitro from insoluble proteins produced in bacteria; to crystallize, two of the complexes required seeding with microcrystals of another complex. Maintained at -160 degrees C, single co-crystals of each of the five peptide-HLA-A2 complexes yielded complete X-ray diffraction data sets to a resolution of approximately 2.5 A. After a sufficient number of diffraction peaks were acquired during data collection, the direct analysis of integrated intensities established the point group of the co-crystal, thus allowing an efficient data collection strategy to be designed. The subsequent examination of systematic absences revealed that the five peptide-HLA-A2 co-crystals formed in space groups P1, P2(1), or P2(1)2(1)2(1). Molecular replacement structure solutions yielded unambiguous protein electron density maps, thus confirming the space group determinations. The system of obtaining HLA-A2 co-crystal structures described here is applicable to other crystallographic problems where structures of several related molecules from uncharacterized single crystals are required.
我们制备并结晶了5种人类组织相容性分子HLA - A2与源自1型人类免疫缺陷病毒、1型人类嗜T淋巴细胞病毒、甲型流感病毒和乙型肝炎病毒蛋白的肽段形成的复合物。每种HLA - A2复合物均由细菌产生的不溶性蛋白在体外重折叠而成；为了结晶，其中两种复合物需要用另一种复合物的微晶作为晶种。将5种肽 - HLA - A2复合物的单晶保存在 - 160℃，得到了分辨率约为2.5 Å的完整X射线衍射数据集。在数据收集过程中获得足够数量的衍射峰后，对积分强度进行直接分析确定了共晶体的点群，从而得以设计高效的数据收集策略。随后对系统消光的检查表明，这5种肽 - HLA - A2共晶体属于P1、P2(1)或P2(1)2(1)2(1)空间群。分子置换结构解析得到了明确的蛋白质电子密度图，从而证实了空间群的确定。本文所述的获得HLA - A2共晶体结构的系统适用于其他晶体学问题，即需要从未经表征的单晶中解析几种相关分子的结构。
