GP0.4 from bacteriophage T7: in silico characterisation of its structure and interaction with E. coli FtsZ.

BackgroundProteins produced by bacteriophages can have potent antimicrobial activity. The study of phage-host interactions can therefore inform small molecule drug discovery by revealing and characterising new drug targets. Here we characterise in silico the predicted interaction of gene protein 0.4 (GP0.4) from the Escherichia coli (E. coli) phage T7 with E. coli filamenting temperature-sensitive mutant Z division protein (FtsZ). FtsZ is a tubulin homolog which plays a key role in bacterial cell division and that has been proposed as a drug target.ResultsUsing ab initio, fragment assembly structure modelling, we predicted the structure of GP0.4 with two programs. A structure similarity-based network was used to identify a U-shaped helix-turn-helix candidate fold as being favoured. ClusPro was used to dock this structure prediction to a homology model of E. coli FtsZ resulting in a favourable predicted interaction mode. Alternative docking methods supported the proposed mode which offered an immediate explanation for the anti-filamenting activity of GP0.4. Importantly, further strong support derived from a previously characterised insertion mutation, known to abolish GP0.4 activity, that is positioned in close proximity to the proposed GP0.4/FtsZ interface.ConclusionsThe mode of interaction predicted by bioinformatics techniques strongly suggests a mechanism through which GP0.4 inhibits FtsZ and further establishes the latter's druggable intrafilament interface as a potential drug target

Helical ensembles outperform ideal helices in molecular replacement

The conventional approach in molecular replacement (MR) is the use of a related structure as a search model. However, this is not always possible as the availability of such structures can be scarce for poorly characterised families of proteins. In these cases, alternative approaches can be explored, such as the use of small ideal fragments that share high albeit local structural similarity with the unknown protein. Earlier versions of AMPLE enabled the trialling of a library of ideal helices, which worked well for largely helical proteins at suitable resolution. Here we explore the performance of libraries of helical ensembles created by clustering helical segments. The impacts of different B-factor treatments and different degrees of structural heterogeneity are explored. We observed a 30% increase in the number of solutions obtained by AMPLE when using this new set of ensembles compared to performance with ideal helices. The boost of performance was notable across three different folds: transmembrane, globular and coiled-coil structures. Furthermore, the increased effectiveness of these ensembles was coupled to a reduction of the time required by AMPLE to reach a solution. AMPLE users can now take full advantage of this new library of search models by activating the “helical ensembles” mode

Structural Insights into Pink-eyed Dilution Protein (Oca2).

Recent innovations in computational structural biology have opened an opportunity to revise our current understanding of the structure and function of clinically important proteins.  This study centres on human Oca2 which is located on mature melanosomal membranes. Mutations of Oca2 can result in a form of oculocutanous albinism which is the most prevalent and visually identifiable form of albinism. Sequence analysis predicts Oca2 to be a member of the SLC13 transporter family but it has not been classified into any existing SLC families. The modelling of Oca2 with AlphaFold2 and other advanced methods show that, like SLC13 members, it consists of a scaffold and transport domain and displays a pseudo inverted repeat topology that includes re-entrant loops. This finding contradicts the prevailing consensus view of its topology. In addition to the scaffold and transport domains, the presence of a cryptic GOLD domain is revealed that is likely responsible for its trafficking from the endoplasmic reticulum to the Golgi prior to localisation at the melanosomes. The GOLD harbours some known glycosylation sites. Analysis of the putative ligand binding site of the model shows the presence of highly conserved key asparagine residues that suggest Oca2 may be a Na+/dicarboxylate symporter. Known critical pathogenic mutations map to structural features present in the repeat regions that form the transport domain. Exploiting the AlphaFold2 multimeric modelling protocol in combination with conventional homology modelling allowed the building of plausible homodimers in both inward- and outward-facing conformations, supporting an elevator-type transport mechanism

Assessing the utility of CASP14 models for molecular replacement

Funder: CCP4Funder: Max‐Planck‐Gesellschaft; Id: http://dx.doi.org/10.13039/501100004189Abstract: The assessment of CASP models for utility in molecular replacement is a measure of their use in a valuable real‐world application. In CASP7, the metric for molecular replacement assessment involved full likelihood‐based molecular replacement searches; however, this restricted the assessable targets to crystal structures with only one copy of the target in the asymmetric unit, and to those where the search found the correct pose. In CASP10, full molecular replacement searches were replaced by likelihood‐based rigid‐body refinement of models superimposed on the target using the LGA algorithm, with the metric being the refined log‐likelihood‐gain (LLG) score. This enabled multi‐copy targets and very poor models to be evaluated, but a significant further issue remained: the requirement of diffraction data for assessment. We introduce here the relative‐expected‐LLG (reLLG), which is independent of diffraction data. This reLLG is also independent of any crystal form, and can be calculated regardless of the source of the target, be it X‐ray, NMR or cryo‐EM. We calibrate the reLLG against the LLG for targets in CASP14, showing that it is a robust measure of both model and group ranking. Like the LLG, the reLLG shows that accurate coordinate error estimates add substantial value to predicted models. We find that refinement by CASP groups can often convert an inadequate initial model into a successful MR search model. Consistent with findings from others, we show that the AlphaFold2 models are sufficiently good, and reliably so, to surpass other current model generation strategies for attempting molecular replacement phasing

Pathogenomic analyses of Shigella isolates inform factors limiting shigellosis prevention and control across LMICs

Shigella spp. are the leading bacterial cause of severe childhood diarrhoea in low- and middle-income countries (LMICs), are increasingly antimicrobial resistant and have no widely available licenced vaccine. We performed genomic analyses of 1,246 systematically collected shigellae sampled from seven countries in sub-Saharan Africa and South Asia as part of the Global Enteric Multicenter Study (GEMS) between 2007 and 2011, to inform control and identify factors that could limit the effectiveness of current approaches. Through contemporaneous comparison among major subgroups, we found that S. sonnei contributes ≥6-fold more disease than other Shigella species relative to its genomic diversity, and highlight existing diversity and adaptative capacity among S. flexneri that may generate vaccine escape variants in <6 months. Furthermore, we show convergent evolution of resistance against ciprofloxacin, the current WHO-recommended antimicrobial for the treatment of shigellosis, among Shigella isolates. This demonstrates the urgent need to integrate existing genomic diversity into vaccine and treatment plans for Shigella, providing a framework for the focused application of comparative genomics to guide vaccine development, and the optimization of control and prevention strategies for other pathogens relevant to public health policy considerations

The CCP4 suite : integrative software for macromolecular crystallography

The Collaborative Computational Project No. 4 (CCP4) is a UK-led international collective with a mission to develop, test, distribute and promote software for macromolecular crystallography. The CCP4 suite is a multiplatform collection of programs brought together by familiar execution routines, a set of common libraries and graphical interfaces. The CCP4 suite has experienced several considerable changes since its last reference article, involving new infrastructure, original programs and graphical interfaces. This article, which is intended as a general literature citation for the use of the CCP4 software suite in structure determination, will guide the reader through such transformations, offering a general overview of the new features and outlining future developments. As such, it aims to highlight the individual programs that comprise the suite and to provide the latest references to them for perusal by crystallographers around the world

Redeployment of automated MrBUMP searchmodel identification for map fitting in cryo-EM

In crystallography, the phase problem can often be addressed by the careful preparation of molecular-replacement search models. This has led to the development of pipelines such as MrBUMP that can automatically identify homologous proteins from an input sequence and edit them to focus on the areas that are most conserved. Many of these approaches can be applied directly to cryo-EM to help discover, prepare and correctly place models (here called cryo-EM search models) into electrostatic potential maps. This can significantly reduce the amount of manual model building that is required for structure determination. Here, MrBUMP is repurposed to fit automatically obtained PDB-derived chains and domains into cryo-EM maps. MrBUMP was successfully able to identify and place cryo-EM search models across a range of resolutions. Methods such as map segmentation are also explored as potential routes to improved performance. Map segmentation was also found to improve the effectiveness of the pipeline for higher resolution (<8 Å) data sets

MrParse: finding homologues in the PDB and the EBI AlphaFold database tor molecular replacement and more

Crystallographers have an array of search-model options for structure solution by molecular replacement (MR). The well established options of homologous experimental structures and regular secondary-structure elements or motifs are increasingly supplemented by computational modelling. Such modelling may be carried out locally or may use pre-calculated predictions retrieved from databases such as the EBI AlphaFold database. MrParse is a new pipeline to help to streamline the decision process in MR by consolidating bioinformatic predictions in one place. When reflection data are provided, MrParse can rank any experimental homologues found using eLLG, which indicates the likelihood that a given search model will work in MR. Inbuilt displays of predicted secondary structure, coiled-coil and transmembrane regions further inform the choice of MR protocol. MrParse can also identify and rank homologues in the EBI AlphaFold database, a function that will also interest other structural biologists and bioinformaticians

Evaluation of model refinement in CASP14

We report here an assessment of the model refinement category of the 14th round of Critical Assessment of Structure Prediction (CASP14). As before, predictors submitted up to five ranked refinements, along with associated residue-level error estimates, for targets that had a wide range of starting quality. The ability of groups to accurately rank their submissions and to predict coordinate error varied widely. Overall only four groups out-performed a “naïve predictor” corresponding to resubmission of the starting model. Among the top groups there are interesting differences of approach and in the spread of improvements seen: some methods are more conservative, others more adventurous. Some targets were “double-barrelled” for which predictors were offered a high-quality AlphaFold 2 (AF2)-derived prediction alongside another of lower quality. The AF2-derived models were largely unimprovable, many of their apparent errors being found to reside at domain and, especially, crystal lattice contacts. Refinement is shown to have a mixed impact overall on structure-based function annotation methods to predict nucleic acid binding, spot catalytic sites and dock protein structures