氢(H)原子是丰富的大分子,往往发挥关键作用,在酶催化,配体识别过程和蛋白质-蛋白质相互作用。然而,通过衍射技术直接可视化是具有挑战性的。大分子X射线晶体学仅在(亚)原子分辨率(约1.2µ或更高)下提供最有序的H原子的定位。然而,许多具有生化意义的H原子仍然无法通过这种方法检测到。相比之下,中子衍射方法使大多数H原子可视化,通常以氘(2H)原子的形式,在更常见的分辨率值(优于2.5µ)。因此,中子晶体学,尽管技术要求很高,当寻求有关质子化状态的直接信息时,通常是选择的方法。来自协作计算项目编号的REFMAC5图4(CCP4)是用于针对X射线晶体学和低温EM数据改进大分子模型的程序。此贡献描述了其扩展,包括对从中子晶体学数据获得的结构模型的改进。具有H原子与其母体原子核之间精确键距的立体化学约束现在是CCP4单体库的一部分,精炼中使用的先前化学信息的来源。REFMAC5中中子数据分析的一个新特征是精化proi/氘(1H/2H)部分。该参数描述了氢同位素对中子散射的相对1H/2H贡献。通过使用(i)仅中子数据或(ii)补充外部约束的中子数据,对PDB中可用的几个条目和一种新型结构(FutA)进行(重新)改进,对新开发的REFMAC5算法进行了测试X射线晶体学结构。用REFMAC5重新细化提供了模型,其特征在于R因子值与,在某些情况下,最初存放的值。在改进过程中使用外部参考结构限制被认为是一种有价值的策略,特别是对于中低分辨率的结构。
Hydrogen (H) atoms are abundant in macromolecules and often play critical roles in enzyme catalysis, ligand-recognition processes and protein-protein interactions. However, their direct visualization by diffraction techniques is challenging. Macromolecular X-ray crystallography affords the localization of only the most ordered H atoms at (sub-)atomic resolution (around 1.2 Å or higher). However, many H atoms of biochemical significance remain undetectable by this method. In contrast, neutron diffraction methods enable the visualization of most H atoms, typically in the form of deuterium (2H) atoms, at much more common resolution values (better than 2.5 Å). Thus, neutron crystallography, although technically demanding, is often the method of choice when direct information on protonation states is sought.
REFMAC5 from the Collaborative Computational Project No. 4 (CCP4) is a program for the refinement of macromolecular models against X-ray crystallographic and cryo-EM data. This contribution describes its extension to include the refinement of structural models obtained from neutron crystallographic data. Stereochemical restraints with accurate bond distances between H atoms and their parent atom nuclei are now part of the CCP4 Monomer Library, the source of prior chemical information used in the refinement. One new feature for neutron data analysis in
REFMAC5 is refinement of the protium/deuterium (1H/2H) fraction. This parameter describes the relative 1H/2H contribution to neutron scattering for hydrogen isotopes. The newly developed
REFMAC5 algorithms were tested by performing the (re-)refinement of several entries available in the PDB and of one novel structure (FutA) using either (i) neutron data only or (ii) neutron data supplemented by external restraints to a reference X-ray crystallographic structure. Re-refinement with
REFMAC5 afforded models characterized by R-factor values that are consistent with, and in some cases better than, the originally deposited values. The use of external reference structure restraints during refinement has been observed to be a valuable strategy, especially for structures at medium-low resolution.