Zhu De-Yu, Zhu Yong-Qun, Xiang Ye, Wang Da-Cheng
Center for Structural and Molecular Biology, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Beijing 100101, People's Republic of China.
Acta Crystallogr D Biol Crystallogr. 2005 Jun;61(Pt 6):772-5. doi: 10.1107/S0907444904028768. Epub 2005 May 26.
A dynamic seeding method that is different from the conventional method of seeding drops that have been equilibrated is described. The method basically consists of two steps. Firstly, microseeding was used in association with adjustment of the seeding-drop components, including buffer, additive and concentrations of the precipitants and protein, in order to screen suitable seeding conditions under which microseeds are seeded into a new non-equilibrated drop as the dynamic macroseed drop for the following step. Secondly, after being equilibrated for various times against the reservoir solution, the macroseed drops were used to prepare a dilution series with which the qualified crystals could be harvested using macroseeding. Compared with a conventional seeding technique, this method is distinct with a dynamic situation of macroseed drops before macroseeding and a non-equilibrium serial seeding where all the seeds are seeded into new non-equilibrated drops and the micro/macroseeding are efficiently combined into a whole system. The method simplifies control of the number of microseeds because an excess of microseeds has little effect on the final result. The method also simplifies the manipulation of macroseeds by optimizing the equilibration time and the dilution multiple of the macroseed drops before macroseeding. This dynamic seeding technique has been used in the crystallization of novel protein CutCm, which has a fast crystal-growth rate, and proved that the method is useful for optimizing protein crystallization.
本文描述了一种与传统的平衡液滴接种方法不同的动态接种方法。该方法主要包括两个步骤。首先，微接种与接种液滴成分的调整相结合，包括缓冲液、添加剂、沉淀剂和蛋白质的浓度，以筛选合适的接种条件，在该条件下，微种子被接种到一个新的非平衡液滴中，作为下一步的动态大种子液滴。其次，大种子液滴在与储液平衡不同时间后，用于制备稀释系列，通过大接种法收获合格晶体。与传统接种技术相比，该方法的独特之处在于大接种前大种子液滴的动态情况以及非平衡连续接种，即所有种子都接种到新的非平衡液滴中，并且微/大接种有效地组合成一个完整的系统。该方法简化了微种子数量的控制，因为过量的微种子对最终结果影响很小。该方法还通过优化大接种前大种子液滴的平衡时间和稀释倍数，简化了大种子的操作。这种动态接种技术已用于新型蛋白质CutCm的结晶，该蛋白质具有快速的晶体生长速率，并证明该方法有助于优化蛋白质结晶。
