Taylor Garry L
Centre for Biomolecular Sciences, University of St Andrews, St Andrews, Fife KY16 9ST, Scotland.
Acta Crystallogr D Biol Crystallogr. 2010 Apr;66(Pt 4):325-38. doi: 10.1107/S0907444910006694. Epub 2010 Mar 24.
When collecting X-ray diffraction data from a crystal, we measure the intensities of the diffracted waves scattered from a series of planes that we can imagine slicing through the crystal in all directions. From these intensities we derive the amplitudes of the scattered waves, but in the experiment we lose the phase information; that is, how we offset these waves when we add them together to reconstruct an image of our molecule. This is generally known as the 'phase problem'. We can only derive the phases from some knowledge of the molecular structure. In small-molecule crystallography, some basic assumptions about atomicity give rise to relationships between the amplitudes from which phase information can be extracted. In protein crystallography, these ab initio methods can only be used in the rare cases in which there are data to at least 1.2 A resolution. For the majority of cases in protein crystallography phases are derived either by using the atomic coordinates of a structurally similar protein (molecular replacement) or by finding the positions of heavy atoms that are intrinsic to the protein or that have been added (methods such as MIR, MIRAS, SIR, SIRAS, MAD, SAD or combinations of these). The pioneering work of Perutz, Kendrew, Blow, Crick and others developed the methods of isomorphous replacement: adding electron-dense atoms to the protein without disturbing the protein structure. Nowadays, methods from small-molecule crystallography can be used to find the heavy-atom substructure and the phases for the whole protein can be bootstrapped from this prior knowledge. More recently, improved X-ray sources, detectors and software have led to the routine use of anomalous scattering to obtain phase information from either incorporated selenium or intrinsic sulfurs. In the best cases, only a single set of X-ray data (SAD) is required to provide the positions of the anomalous scatters, which together with density-modification procedures can reveal the structure of the complete protein.
从晶体收集X射线衍射数据时，我们测量从一系列平面散射的衍射波的强度，这些平面可以想象成从各个方向穿过晶体。从这些强度中我们可以得出散射波的振幅，但在实验中我们失去了相位信息；也就是说，当我们将这些波相加以重建分子图像时，如何使这些波相互抵消。这通常被称为“相位问题”。我们只能从分子结构的一些知识中推导出相位。在小分子晶体学中，关于原子性的一些基本假设会产生振幅之间的关系，从中可以提取相位信息。在蛋白质晶体学中，这些从头算方法仅在极少数情况下使用，即数据分辨率至少达到1.2埃。在蛋白质晶体学的大多数情况下，相位是通过使用结构相似蛋白质的原子坐标（分子置换）或通过找到蛋白质固有的或添加的重原子的位置（如MIR、MIRAS、SIR、SIRAS、MAD、SAD等方法或这些方法的组合）来推导的。佩鲁茨、肯德鲁、布洛、克里克等人的开创性工作开发了同晶置换方法：在不干扰蛋白质结构的情况下向蛋白质中添加电子密度高的原子。如今，小分子晶体学的方法可用于找到重原子亚结构，并且可以从这些先验知识中引导出整个蛋白质的相位。最近，改进的X射线源、探测器和软件导致常规使用反常散射从掺入的硒或固有硫中获取相位信息。在最佳情况下，仅需要一组X射线数据（SAD）来提供反常散射体的位置，这与密度修正程序一起可以揭示完整蛋白质的结构。
