Institute of Integrative Biology, University of Liverpool, Liverpool L69 7ZB, England.
Research Complex at Harwell, STFC Rutherford Appleton Laboratory, Didcot OX11 0FA, England.
Acta Crystallogr D Struct Biol. 2020 Mar 1;76(Pt 3):272-284. doi: 10.1107/S2059798320000443. Epub 2020 Feb 25.
The phase problem remains a major barrier to overcome in protein structure solution by X-ray crystallography. In recent years, new molecular-replacement approaches using ab initio models and ideal secondary-structure components have greatly contributed to the solution of novel structures in the absence of clear homologues in the PDB or experimental phasing information. This has been particularly successful for highly α-helical structures, and especially coiled-coils, in which the relatively rigid α-helices provide very useful molecular-replacement fragments. This has been seen within the program AMPLE, which uses clustered and truncated ensembles of numerous ab initio models in structure solution, and is already accomplished for α-helical and coiled-coil structures. Here, an expansion in the scope of coiled-coil structure solution by AMPLE is reported, which has been achieved through general improvements in the pipeline, the removal of tNCS correction in molecular replacement and two improved methods for ab initio modelling. Of the latter improvements, enforcing the modelling of elongated helices overcame the bias towards globular folds and provided a rapid method (equivalent to the time requirements of the existing modelling procedures in AMPLE) for enhanced solution. Further, the modelling of two-, three- and four-helical oligomeric coiled-coils, and the use of full/partial oligomers in molecular replacement, provided additional success in difficult and lower resolution cases. Together, these approaches have enabled the solution of a number of parallel/antiparallel dimeric, trimeric and tetrameric coiled-coils at resolutions as low as 3.3 Å, and have thus overcome previous limitations in AMPLE and provided a new functionality in coiled-coil structure solution at lower resolutions. These new approaches have been incorporated into a new release of AMPLE in which automated elongated monomer and oligomer modelling may be activated by selecting `coiled-coil' mode.
在利用 X 射线晶体学解决蛋白质结构的问题上，相位问题仍然是一个亟待克服的主要障碍。近年来，新的分子置换方法利用从头计算模型和理想的二级结构成分，为在 PDB 中没有明显同源物或实验相信息的情况下解决新结构做出了巨大贡献。这对于高度α-螺旋结构，特别是卷曲螺旋结构，特别成功，因为相对刚性的α-螺旋提供了非常有用的分子置换片段。在 AMPLE 程序中已经看到了这一点，该程序在结构解决中使用了大量从头计算模型的聚类和截断集合，并且已经完成了α-螺旋和卷曲螺旋结构的构建。本文报告了 AMPLE 在卷曲螺旋结构解决方面的扩展，这是通过对管道的一般改进、在分子置换中去除 tNCS 校正以及两种改进的从头计算建模方法实现的。在这两个改进中，强制对伸长螺旋的建模克服了对球状折叠的偏见，并提供了一种快速方法（相当于 AMPLE 中现有建模过程的时间要求），用于增强解决方案。此外，对二、三、四螺旋寡聚卷曲螺旋的建模，以及在分子置换中使用全/部分寡聚体，在困难和低分辨率情况下提供了额外的成功。总之，这些方法使许多平行/反平行二聚体、三聚体和四聚体卷曲螺旋的解决成为可能，分辨率低至 3.3 Å，从而克服了 AMPLE 中的先前限制，并在较低分辨率下提供了卷曲螺旋结构解决的新功能。这些新方法已被纳入 AMPLE 的新版本中，通过选择“卷曲螺旋”模式，可以自动激活伸长单体和寡聚物建模。
