X射线晶体学的奇迹是从衍射图推导出的原子结构的美丽和精确。由于这些模式只记录振幅,衍射波的相位也必须进行评估,以确定系统的结构。因此,我们有相位问题作为一个中心并发症,无论是在智力上还是在许多分析上都是如此。这里,我讨论我们-我自己,我的实验室和衍射界-面临着相位问题,考虑到结构生物学发展到今天的阶段评估方法的演变。在大分子晶体学的爆炸性增长过程中,衍射分析的实践从对同构置换的普遍依赖发展到对从头结构确定的异常衍射的最终统治。随着蛋白质数据库(PDB)的发展和蛋白质之间的家族关系变得清晰,分子置换取代了所有其他定相方法;然而,实验阶段对于没有明显先例的分子来说仍然是必不可少的,以多波长和单波长反常衍射(MAD和SAD)为主。虽然基于数学的直接方法被证明对典型的大分子是不够的,他们在硒甲硫酰蛋白的SAD分析中恢复了大量的硒亚结构。原生SAD,利用生物分子的固有S和P原子,已经成为例行公事。硒甲硫酰SAD和MAD是结构基因组学努力的支柱,旨在用新型蛋白质填充PDB。PDB训练的人工智能方法在蛋白质结构预测方面取得了成功,这是最近的红利。目前,用AlphaFold模型进行分子替换通常无需进行实验阶段评估。出于多种原因,我们现在对阶段问题并不感到困惑。Cryo-EM分析是当今结构生物学家面临的许多应用的晶体学有吸引力的替代方法。它只是对相位问题进行罚款;然而,衍射分析的原理和程序仍然相关,并在生物分子的单粒子低温EM研究中采用。
The marvel of X-ray crystallography is the beauty and precision of the atomic structures deduced from diffraction patterns. Since these patterns record only amplitudes, phases for the diffracted waves must also be evaluated for systematic structure determination. Thus, we have the phase problem as a central complication, both intellectually for the field and practically so for many analyses. Here, I discuss how we - myself, my laboratory and the diffraction community - have faced the phase problem, considering the evolution of methods for phase evaluation as structural biology developed to the present day. During the explosive growth of macromolecular crystallography, practice in diffraction analysis evolved from a universal reliance on isomorphous replacement to the eventual domination of anomalous diffraction for de novo structure determination. As the Protein Data Bank (PDB) grew and familial relationships among proteins became clear, molecular replacement overtook all other phasing methods; however, experimental phasing remained essential for molecules without obvious precedents, with multi- and single-wavelength anomalous diffraction (MAD and SAD) predominating. While the mathematics-based direct methods had proved to be inadequate for typical macromolecules, they returned to crack substantial selenium substructures in SAD analyses of selenomethionyl proteins. Native SAD, exploiting the intrinsic S and P atoms of biomolecules, has become routine. Selenomethionyl SAD and MAD were the mainstays of structural genomics efforts to populate the PDB with novel proteins. A recent dividend has been paid in the success of PDB-trained artificial intelligence approaches for protein structure prediction. Currently, molecular replacement with AlphaFold models often obviates the need for experimental phase evaluation. For multiple reasons, we are now unfazed by the phase problem. Cryo-EM analysis is an attractive alternative to crystallography for many applications faced by today\'s structural biologists. It simply finesses the phase problem; however, the principles and procedures of diffraction analysis remain pertinent and are adopted in single-particle cryo-EM studies of biomolecules.