Institut für Strukturbiologie und Biophysik (ISB-3), Forschungszentrum Jülich, 52425 Jülich, Germany.
Nature. 2010 Apr 22;464(7292):1218-22. doi: 10.1038/nature08892. Epub 2010 Apr 7.
X-ray diffraction plays a pivotal role in the understanding of biological systems by revealing atomic structures of proteins, nucleic acids and their complexes, with much recent interest in very large assemblies like the ribosome. As crystals of such large assemblies often diffract weakly (resolution worse than 4 A), we need methods that work at such low resolution. In macromolecular assemblies, some of the components may be known at high resolution, whereas others are unknown: current refinement methods fail as they require a high-resolution starting structure for the entire complex. Determining the structure of such complexes, which are often of key biological importance, should be possible in principle as the number of independent diffraction intensities at a resolution better than 5 A generally exceeds the number of degrees of freedom. Here we introduce a method that adds specific information from known homologous structures but allows global and local deformations of these homology models. Our approach uses the observation that local protein structure tends to be conserved as sequence and function evolve. Cross-validation with R(free) (the free R-factor) determines the optimum deformation and influence of the homology model. For test cases at 3.5-5 A resolution with known structures at high resolution, our method gives significant improvements over conventional refinement in the model as monitored by coordinate accuracy, the definition of secondary structure and the quality of electron density maps. For re-refinements of a representative set of 19 low-resolution crystal structures from the Protein Data Bank, we find similar improvements. Thus, a structure derived from low-resolution diffraction data can have quality similar to a high-resolution structure. Our method is applicable to the study of weakly diffracting crystals using X-ray micro-diffraction as well as data from new X-ray light sources. Use of homology information is not restricted to X-ray crystallography and cryo-electron microscopy: as optical imaging advances to subnanometre resolution, it can use similar tools.
X 射线衍射在理解生物系统方面发挥着关键作用，它揭示了蛋白质、核酸及其复合物的原子结构，最近人们对核糖体等非常大的复合物产生了浓厚的兴趣。由于这些大复合物的晶体通常衍射较弱（分辨率差于 4Å），我们需要在如此低的分辨率下工作的方法。在大分子复合物中，一些成分可能具有高分辨率，而其他成分则未知：目前的精修方法失败了，因为它们需要整个复合物的高分辨率起始结构。原则上，应该有可能确定这些复合物的结构，因为在分辨率优于 5Å 的情况下，独立衍射强度的数量通常超过自由度的数量。在这里，我们引入了一种方法，该方法从已知同源结构中添加了特定信息，但允许这些同源模型的全局和局部变形。我们的方法利用了这样一种观察结果，即随着序列和功能的进化，局部蛋白质结构趋于保守。使用交叉验证的自由 R 因子（free R-factor）确定最佳变形和同源模型的影响。对于分辨率为 3.5-5Å 且具有高分辨率已知结构的测试案例，与传统精修相比，我们的方法在坐标精度、二级结构定义和电子密度图质量方面对模型进行了显著改进。对于来自蛋白质数据库的 19 个低分辨率晶体结构的代表性集合的重新精修，我们发现了类似的改进。因此，从低分辨率衍射数据中得出的结构可以具有类似于高分辨率结构的质量。我们的方法适用于使用 X 射线微衍射以及来自新型 X 射线光源的数据研究弱衍射晶体。同源信息的使用不仅限于 X 射线晶体学和冷冻电子显微镜：随着光学成像技术发展到亚纳米分辨率，它可以使用类似的工具。
