Schnyder T, Cyrklaff M, Fuchs K, Wallimann T
Institute of Cell Biology, Swiss Federal Institute of Technology, Zürich.
J Struct Biol. 1994 Mar-Apr;112(2):136-47. doi: 10.1006/jsbi.1994.1015.
Polymorphic forms of crystals of mitochondrial creatine kinase (Mi-CK) octamers were generated by the lipid-layer technique, using cardiolipin as interphase adhesion matrix. Depending on the protein and lipid concentration, different types of monolayers and 3-D stacks thereof assembled in a low ionic strength crystallization buffer. Sodium tungstate was found to promote and stabilize the crystal formation, though in-plane crystallization was also possible in the absence of tungstate. All crystal forms exhibited a p4 symmetry with lattice parameters (a = b) ranging from 10.6 to 24.0 nm and with one or four octamers per unit cell in end-on orientation. In ice-embedded crystals, which showed a molecular packing different from that of negatively stained preparations, structural features of the Mi-CK octamer were observed at a resolution of 1 nm. The crystallization process took advantage of the electrostatic interaction between negatively charged lipid head groups of cardiolipin and positive charges located at the top/bottom faces of the Mi-CK octamer. In the absence of a cardiolipin support, Mi-CK formed linear filaments from a solution of phosphotungstate by association of octamers via their top/bottom faces. When tungstate was used instead of phosphotungstate, the filaments aligned themselves into large crystalline assemblies.
利用心磷脂作为相间粘附基质，通过脂质层技术生成了线粒体肌酸激酶（Mi-CK）八聚体晶体的多晶型形式。根据蛋白质和脂质浓度，在低离子强度结晶缓冲液中组装形成了不同类型的单层及其三维堆叠结构。发现钨酸钠可促进和稳定晶体形成，不过在没有钨酸盐的情况下也可能发生平面内结晶。所有晶体形式均呈现p4对称性，晶格参数（a = b）范围为10.6至24.0 nm，每个晶胞中有一个或四个八聚体以端对端方向排列。在冰包埋晶体中，其分子堆积与负染制剂不同，在1 nm的分辨率下观察到了Mi-CK八聚体的结构特征。结晶过程利用了心磷脂带负电荷的脂质头部基团与位于Mi-CK八聚体顶面/底面的正电荷之间的静电相互作用。在没有心磷脂支持的情况下，Mi-CK通过八聚体经由其顶面/底面缔合，从磷钨酸盐溶液中形成线性细丝。当使用钨酸盐代替磷钨酸盐时，细丝会自行排列成大型晶体聚集体。
