A practical overview of molecular replacement: Clostridioides difficile PilA1, a difficult case study

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.

Novel Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV2) and Other Coronaviruses: A Genome-wide Comparative Annotation and Analysis

“…Scientists across the globe are trying to elucidate the genome characteristics using phylogeny, structural, and mutational analysis. Recently, few scientists are able to crystalize the protein of this virus for future computational modeling and drug-related research [ 9 , 10 ].…”

Modes and model building in SHELXE

“…Finally, PilA1 (Crawshaw et al, 2020) was originally solved with ARCIMBOLDO_LITE and contains three chains of 150 amino acids. For structures such as this one, with NCS, the FASTA format file read by SHELXE should explicitly contain a copy of the sequence for each of the chains present.…”

Clostridioides difficile infection: traversing host–pathogen interactions in the gut

“…Pili structures in C. difficile were first described by Borriello et al, over 3 decades ago as fimbriae, describing multipolar protrusions of approximately 4-9 nm diameter and 6 µm long, although no correlation between binding efficiencies of strains with/ without these fimbriae were demonstrated [130]. The C. difficile genome encodes the type IV pili (T4P) system, consisting of nine different pilin genes, assembly and scaffold proteins [131,132]. The most studied genes are pilA1 and pilB1, which encode for the major pilin and pilus assembly ATPase, respectively [131][132][133].…”

“…The C. difficile genome encodes the type IV pili (T4P) system, consisting of nine different pilin genes, assembly and scaffold proteins [131,132]. The most studied genes are pilA1 and pilB1, which encode for the major pilin and pilus assembly ATPase, respectively [131][132][133]. Bacterial virulence associated with T4P has been attributed to increased biofilm formation, direct adhesion and colonization to epithelial cells [133][134][135].…”

“…The C. difficile genome encodes the type IV pili (T4P) system, consisting of nine different pilin genes, assembly and scaffold proteins [131, 132]. The most studied genes are pilA1 and pilB1 , which encode for the major pilin and pilus assembly ATPase, respectively [131–133].…”