An Alternative Strategy for Macromolecular Crystallization

描述（由申请人提供）：结构生物学对蛋白质、核酸、病毒和其他大分子复合物结晶的明显依赖已经发展起来，因为绝大多数分子模型现在都来自x射线晶体学。晶体生长在个体研究人员的实验室和大型结构基因组学中心的整个结构测定事业中起着至关重要的作用。对于膜蛋白和糖蛋白等较难处理的大分子，这种依赖性尤其明显。目前的大分子结晶方法虽然在大约40%的病例中取得了成功，但尚未证明自己能够成功地解决许多最重要的生物学和医学问题。我们建议开发一种大分子结晶的替代策略，它不像目前的方法那样依赖于传统变量（如pH和沉淀剂浓度）的优化，而是基于许多传统小分子可能在晶体中建立稳定的、分子间的、非共价交联的假设，从而促进晶格的形成。为了验证这一假设，我们进行了初步实验，包括18240次结晶试验，使用了81种不同的蛋白质和200种化合物。对结果的统计分析证明了该思想的有效性。此外，我们还对实验中生长的一些晶体进行了x射线衍射分析。这些结果清楚地显示了常规分子与蛋白质晶体晶格的结合，并进一步证实了潜在的假设。我们建议将研究扩展到更广泛和更多样化的蛋白质，包括膜蛋白和大分子复合物，扩大对传统和生物活性小分子的搜索，以及更广泛的沉淀剂。这里提出的策略基本上与当前的方法正交，其目标是将目前的成功率提高一倍。

DESCRIPTION (provided by applicant): A clear dependence of structural biology on the crystallization of proteins, nucleic acids, viruses, and other macromolecular complexes has developed, as the vast majority of molecular models are now derived from X-ray crystallography. Crystal growth has come to play a crucial role in the entire enterprise of structure determination in the laboratories of both individual investigators, and in large structural genomics centers. The dependence is particularly acute with regard to the more intractable macromolecules such as membrane proteins and glycoproteins. Current approaches to macromolecular crystallization, while successful in perhaps 40% of cases, have not proven themselves able to successfully address many of the most biologically and medically significant problems. We propose to develop an alternate strategy for the crystallization of macromolecules that does not, like current methods, depend on the optimization of traditional variables such as pH and precipitant concentration, but is based on the hypothesis that many conventional small molecules might establish stabilizing, intermolecular, non covalent crosslinks in crystals, and thereby promote lattice formation. To test the hypothesis, we carried out preliminary experiments encompassing 18,240 crystallization trials using 81 different proteins, and 200 chemical compounds. Statistical analysis of the results demonstrates the validity of the idea. In addition, we have also conducted X-ray diffraction analyses of some of the crystals grown in the experiments. These clearly show incorporation of the conventional molecules into the protein crystal lattices, and they further validate the underlying hypothesis. We propose to extend the investigations to include a broader and more diverse set of proteins, including membrane proteins and macromolecular complexes, an expanded search of conventional and biologically active small molecules, and a wider range of precipitants. The strategy proposed here is essentially orthogonal to current approaches and has an objective of doubling the success rate of today.