Structure of tetragonal hen egg-white lysozyme at 0.94 Å from crystals grown by the counter-diffusion method
Structure of tetragonal hen egg-white lysozyme at 0.94 Å from crystals grown by the counter-diffusion method
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DOI：
10.1107/s0907444901008873
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发表时间：
2001-08-01
影响因子：
2.2
通讯作者：
García-Ruiz, JM
中科院分区：
文献类型：
--
作者：
Sauter, C;Otálora, F;García-Ruiz, JM
在航天飞机上的高级蛋白质结晶设备（APCF）中，采用改进的自由界面扩散（FID）反应器，生长出了非常高质量的四季鸡卵清溶菌酶晶体，该反应器专门设计为具有更长的扩散路径，这种设计允许进行真正的反扩散实验。在50年前定义的经典化学条件下，用NaCl作为结晶剂，乙酸盐pH 4.5作为缓冲液，获得晶体。逆扩散结晶允许生长条件的“物理”而不是化学优化：事实上，这种方法在单次试验中筛选最佳过饱和条件，并产生非常高质量的晶体。一个完整的衍射数据集收集在原子分辨率从这些晶体之一，使用同步辐射在DESY-EMBL光束线。在31-0.94埃分辨率范围内的强度上的总体R-合并为5.2%，并且数据完成98.9%。用CNS和SHELX 97程序进行精修，得到72390次反射的最终晶体学R因子为12.26%。平均标准的原子位置的不确定度为0.024埃，估计从块最小二乘矩阵的反演。22个侧链显示出交替的构象，并且环59-75在相同的晶体包装中采用了在先前的高分辨率研究中对于三斜或四斜溶菌酶观察到的两种构象。除了255个水分子外，结晶剂（一个六配位钠离子和五个氯阴离子）参与了有序的溶菌酶水合壳。
Very high quality crystals of tetragonal hen egg-white lysozyme were grown in the Advanced Protein Crystallization Facility (APCF) on board the Space Shuttle using a modified free-interface diffusion (FID) reactor designed ad hoc to have a longer diffusion path. This design allows the performance of true counter-diffusion experiments. Crystals were obtained under the classical chemical conditions defined 50 y ago with NaCl as a crystallizing agent and acetate pH 4.5 as a buffer. Counter-diffusion crystallization allows a 'physical' instead of chemical optimization of growth conditions: indeed, this method screens for the best supersaturation conditions in a single trial and yields crystals of very high quality. A complete diffraction data set was collected at atomic resolution from one of these crystals using synchrotron radiation at the DESY-EMBL beamlines. The overall R-merge on intensities in the resolution range 31-0.94 Angstrom was 5.2% and the data were 98.9% complete. Refinement was carried out with the programs CNS and SHELX97 to a final crystallographic R factor of 12.26% for 72 390 reflections. A mean standard uncertainty in the atomic positions of 0.024 Angstrom was estimated from inversion of blocked least-squares matrices. 22 side chains show alternate conformations and the loop 59-75 adopts in the same crystal packing two conformations that were observed for either triclinic or tetragonal lysozyme in previous high-resolution studies. In addition to 255 water molecules, the crystallizing agent (one hexacoordinated sodium ion and five chloride anions) participates in the ordered lysozyme hydration shell.