Protein crystallization by design: chymotrypsinogen without precipitants.
Protein crystallization by design: chymotrypsinogen without precipitants.
复制标题
DOI：
10.1006/jmbi.2000.3851
复制
发表时间：
2000-07
影响因子：
5.6
通讯作者：
P. Pjura;A. Lenhoff;S. A. Leonard;A. Gittis
中科院分区：
文献类型：
--
作者：
P. Pjura;A. Lenhoff;S. A. Leonard;A. Gittis
蛋白质晶体通常是通过基于广泛筛选的经验方法获得的，以确定合适的结晶条件。相反，我们使用了系统的预测程序来生产数据质量的牛胰凝乳蛋白酶原A的晶体，并使用它们来获得3A分辨率的精细X射线结构。通过测量凝乳酶原溶液的渗透第二维里系数来确定合适的溶剂条件，然后在不使用任何沉淀剂的情况下，通过超速离心结晶生长晶体约30小时。在含有10-30%乙醇的溶液中可以得到凝乳酶原的现有结构，而在这里使用的是简单的缓冲的氯化钠溶液。该蛋白质属于四方空间群P4(1)2(1)2，每个不对称单元含有一个分子。精制的MAP的质量从头到尾都很高，除了四个残基外，所有的主链原子都被清楚地定义了，几乎所有的侧链也都被定义了。虽然与以前报道的结构相比只有很小的差异，但它们表明由于这些研究中使用的结晶条件，结构可能会发生变化。我们的结果表明，蛋白质的更系统的结晶是可能的，该过程可以扩大晶体成功生长的条件范围，并可以获得新的晶型。
Protein crystals are usually obtained by an empirical approach based on extensive screening to identify suitable crystallization conditions. In contrast, we have used a systematic predictive procedure to produce data-quality crystals of bovine chymotrypsinogen A and used them to obtain a refined X-ray structure to 3 A resolution. Measurements of the osmotic second virial coefficient of chymotrypsinogen solutions were used to identify suitable solvent conditions, following which crystals were grown for approximately 30 hours by ultracentrifugal crystallization, without the use of any precipitants. Existing structures of chymotrypsinogen were obtained in solutions including 10-30 % ethanol, whereas simple buffered NaCl solutions were used here. The protein crystallized in the tetragonal space group P4(1)2(1)2, with one molecule per asymmetric unit. The quality of the refined map was very high throughout, with the main-chain atoms of all but four residues clearly defined and with nearly all side-chains also defined. Although only minor differences are seen compared to the structures previously reported, they indicate the possibility of structural changes due to the crystallization conditions used in those studies. Our results show that more systematic crystallization of proteins is possible, and that the procedure can expand the range of conditions under which crystals can be grown successfully and can make new crystal forms available.