Abstract
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.
Highlights
Once complete diffraction data at an appropriate resolution have been recorded from a protein crystal sample, the challenge is to solve the phase problem (Taylor, 2003)
Other programs such as AMPLE and MrBUMP provide a more complete pipeline, requiring only scaled reflections, the target sequence and, in the latter case, a small number of fragments provided by another server, Rosetta (Bibby et al, 2012; Rigden et al, 2008). Another approach is to use BALBES, a fully automated pipeline that uses a reorganized protein structure database derived from the Protein Data Bank (PDB) in order to include multimeric and domain organization models (Long et al, 2008). These and other molecular replacement (MR) pipelines are available in different software packages such as CCP4i2 (Potterton et al, 2018) and Phenix (Liebschner et al, 2019), here we focus on CCP4i2
PilA1Á1–34 was expressed in Escherichia coli Rosetta cells by inoculating 1 l Luria–Bertani (LB) medium supplemented with 50 mg mlÀ1 kanamycin and 30 mg mlÀ1 chloramphenicol with 10 ml of an overnight culture (100 ml LB with the same antibiotics)
Summary
Once complete diffraction data at an appropriate resolution (usually at least around 3–3.5 A ) have been recorded from a protein crystal sample, the challenge is to solve the phase problem (Taylor, 2003). Other programs such as AMPLE and MrBUMP provide a more complete pipeline, requiring only scaled reflections, the target sequence and, in the latter case, a small number of fragments provided by another server, Rosetta (Bibby et al, 2012; Rigden et al, 2008) Another approach is to use BALBES, a fully automated pipeline that uses a reorganized protein structure database derived from the PDB in order to include multimeric and domain organization models (Long et al, 2008). Our attempts to determine the structure of a PilA1 construct lacking part of the N-terminal 1 helix to prevent self-polymerization are used here as an example of molecularreplacement approaches
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callMore From: Acta crystallographica. Section D, Structural biology
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
