Newcastle University Biosciences Institute, Faculty of Medical Sciences, Newcastle University, Newcastle upon Tyne, England.
Acta Crystallogr D Struct Biol. 2020 Mar 1;76(Pt 3):261-271. doi: 10.1107/S2059798320000467. Epub 2020 Feb 26.
Many biologists are now routinely seeking to determine the three-dimensional structures of their proteins of choice, illustrating the importance of this knowledge, but also of the simplification and streamlining of structure-determination processes. Despite the fact that most software packages offer simple pipelines, for the non-expert navigating the outputs and understanding the key aspects can be daunting. Here, the structure determination of the type IV pili (TFP) protein PilA1 from Clostridioides difficile is used to illustrate the different steps involved, the key decision criteria and important considerations when using the most common pipelines and software. Molecular-replacement pipelines within CCP4i2 are presented to illustrate the more commonly used processes. Previous knowledge of the biology and structure of TFP pilins, particularly the presence of a long, N-terminal α-helix required for pilus formation, allowed informed decisions to be made during the structure-determination strategy. The PilA1 structure was finally successfully determined using ARCIMBOLDO and the ab initio MR strategy used is described.
许多生物学家现在通常都在试图确定他们所选蛋白质的三维结构，这说明了这方面知识的重要性，也说明了简化和精简结构确定过程的重要性。尽管大多数软件包都提供了简单的流水线，但对于非专业人士来说，导航输出并理解关键方面可能会令人望而生畏。在这里，使用最常见的流水线和软件时，艰难梭菌（Clostridioides difficile）的 IV 型菌毛（TFP）蛋白 PilA1 的结构确定用于说明所涉及的不同步骤、关键决策标准和重要考虑因素。CCP4i2 中的分子置换流水线用于说明更常用的过程。对 TFP 菌毛蛋白的生物学和结构的先前知识，特别是形成菌毛所需的长、N 端α-螺旋的存在，使得在结构确定策略中可以做出明智的决策。最终使用 ARCIMBOLDO 成功确定了 PilA1 结构，并描述了所使用的从头开始 MR 策略。
