Biology subjects

Rigden, D. J.

Publications and source records attributed to Rigden, D. J..

28 records · Page 2Linked to original sources

bioRxiv2021

Assessing the utility of CASP14 models for molecular replacement

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 likelihood (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.

bioinformatics↗

bioRxiv2021

Informing shigellosis prevention and control through pathogen genomics

Shigella spp. are the leading bacterial cause of severe childhood diarrhoea in low- and middle-income countries (LMIC), are increasingly antimicrobial resistant and have no licensed vaccine. We performed genomic analyses of 1246 systematically collected shigellae from seven LMIC to inform control and identify factors that could limit the effectiveness of current approaches. We found that S. sonnei contributes [≥]20-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 the current recommended antimicrobial among shigellae. This demonstrates the urgent need to integrate existing genomic diversity into vaccine and treatment plans for Shigella, and other pathogens.

microbiology↗

bioRxiv2021

Identification of unknown proteins in X-ray crystallography and cryo-EM

Although experimental protein structure determination usually targets known proteins, chains of unknown sequence are often encountered. They can be purified from natural sources, appear as an unexpected fragment of a well characterized protein or as a contaminant. Regardless of the source of the problem, the unknown protein always requires tedious characterization. Here we present an automated pipeline for the identification of protein sequences from cryo-EM reconstructions and crystallographic data. We present the methods application to characterize the crystal structure of an unknown protein purified from a snake venom. We also show that the approach can be successfully applied to the identification of protein sequences and validation of sequence assignments in cryo-EM protein structures.

molecular biology↗

bioRxiv2020

The Paracaedibacter-like endosymbiont of Bodo saltans (Kinetoplastida) uses multiple putative toxin-antitoxin systems to maintain its host association

Bacterial endosymbiosis has been instrumental in eukaryotic evolution, and includes both mutualistic, dependent and parasitic associations. Here we characterize an intracellular bacterium inhabiting the flagellated protist Bodo saltans (Kinetoplastida). We present a complete bacterial genome comprising a 1.39 Mb circular chromosome with 40.6% GC content. Fluorescent in situ hybridisation confirms that the endosymbiont is located adjacent to the nuclear membrane, and a detailed model of its intracellular niche is generated using serial block-face scanning electron microscopy. Phylogenomic analysis shows that the endosymbiont belongs to the Holosporales, most closely related to other -proteobacterial endosymbionts of ciliates and amoebae. Comparative genomics indicates that it has a limited metabolism and is nutritionally host-dependent. However, the endosymbiont genome does encode diverse symbiont-specific secretory proteins, including a type VI secretion system and three separate toxin-antitoxin systems. We show that these systems are actively transcribed and hypothesize they represent a mechanism by which B. saltans becomes addicted to its endosymbiont. Consistent with this idea, attempts to cure Bodo of endosymbionts led to rapid and uniform cell death. This study adds kinetoplastid flagellates to ciliates and amoebae as hosts of Paracaedibacter-like bacteria, suggesting that these antagonistic endosymbioses became established very early in Eukaryotic evolution.

ecology↗

bioRxiv2020

Prophage-encoded phage defence proteins with cognate self-immunity

Temperate phages are pervasive in bacterial genomes, existing as vertically-inherited islands called prophages. Prophages are vulnerable to the predation of their host bacterium by exogenous phages. Here we identify BstA, a novel family of prophage-encoded phage defense proteins found in diverse Gram-negative bacteria. BstA drives potent suppression of phage epidemics through abortive infection. During lytic replication, the bstA-encoding prophage is not itself inhibited by BstA due to a self-immunity mechanism conferred by the anti-BstA (aba) element, a short stretch of DNA within the bstA locus. Inhibition of phage replication by distinct BstA proteins from Salmonella, Klebsiella and Escherichia prophages is functionally interchangeable, but each possesses a cognate aba element. The specificity of the aba element ensures that immunity is exclusive to the replicating prophage, and cannot be exploited by heterologous BstA-encoding phages. BstA allows prophages to defend host cells against exogenous phage attack, without sacrificing their own lytic autonomy.

microbiology↗

bioRxiv2020

In silico prediction of structure and function for a large family of transmembrane proteins that includes human Tmem41b

Recent strides in computational structural biology have opened up an opportunity to understand previously mysterious uncharacterised proteins. The under-representation of transmembrane proteins in the Protein Data Bank highlights the need to apply new and advanced bioinformatics methods to shed light on their structure and function. This study focuses on such a family; transmembrane proteins containing the Pfam domain PF09335 ( SNARE_ASSOC/ VTT / Tvp38). One prominent member, Tmem41b, has been shown to be involved in early stages of autophagosome formation and is vital in mouse embryonic development. Here we use evolutionary covariance-derived information not only to construct and validate ab initio models but also to make domain boundary predictions and infer local structural features. The results from the structural bioinformatics analysis of Tmem41b and its homologues show that they contain a tandem repeat that is clearly visible in evolutionary covariance data but much less so by sequence analysis. Furthermore, cross-referencing of other prediction data with the covariance analysis shows that the internal repeat features 2-fold rotational symmetry. Ab initio modelling of Tmem41b reinforces these structural predictions. Local structural features predicted to be present in Tmem41b are also present in Cl-/H+ antiporters. These results together strongly point to Tmem41b and its homologues as being transporters for an as-yet uncharacterised substrate and possibly using H+ antiporter activity as its mechanism for transport.

bioinformatics↗

bioRxiv2020

Helical ensembles out-perform 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.

bioinformatics↗

bioRxiv2019

Informatics investigations into anti-thyroid drug induced agranulocytosis associated with multiple HLA-B alleles

Adverse drug reactions have been linked with HLA alleles in different studies. These HLA proteins play an essential role in the adaptive immune response for the presentation of self and non-self peptides. Anti-thyroid drugs methimazole and propylthiouracil have been associated with drug induced agranulocytosis (severe lower white blood cell count) in patients with B*27:05, B*38:02 and DRB1*08:03 alleles in different populations: Taiwanese, Vietnamese, Han Chinese and Caucasian.\n\nIn this study, informatics methods were used to investigate if any sequence or structural similarities exist between the two associated HLA-B alleles, compared with a set of \"control\" alleles assumed not be associated, which could help explain the molecular basis of the adverse drug reaction. We demonstrated using MHC Motif Viewer and MHCcluster that the two alleles do not have a propensity to bind similar peptides, and thus at a gross level the structure of the antigen presentation region of the two alleles are not similar. We also performed multiple sequence alignment to identify polymorphisms shared by the risk but not by the control alleles and molecular docking to compare the predicted binding positions of the drug-allele combinations.\n\nTwo residues, Cys67 and Thr80, were identified from the multiple sequence alignments to be unique to these risk alleles alone. The molecular docking showed the poses of the risk alleles to favour the F-pocket of the peptide binding groove, close to the Thr80 residue, with the control alleles generally favouring a different pocket. The data are thus suggestive that Thr80 may be a critical residue in HLA-mediated anti-thyroid drug induced agranulocytosis, and thus can guide future research and risk assessment.

bioinformatics↗

bioRxiv2018

The heparin-binding proteome in normal pancreas and murine experimental acute pancreatitis

Acute pancreatitis (AP) is acute inflammation of the pancreas, mainly caused by gallstones and alcohol, driven by changes in communication between cells. Heparin-binding proteins (HBPs) play a central role in health and diseases. Therefore, we used heparin affinity proteomics to identify extracellular HBPs in pancreas and plasma of normal mice and in a caerulein mouse model of AP. Many new extracellular HBPs (360) were discovered in the pancreas, taking the total number of HBPs known to 786. Extracellular pancreas HBPs form highly interconnected protein-protein interaction networks in both normal pancreas (NP) and AP. Thus, HBPs represent an important set of extracellular proteins with significant regulatory potential in the pancreas. HBPs in NP are associated with biological functions such as molecular transport and cellular movement that underlie pancreatic homeostasis. However, in AP HBPs are associated with additional inflammatory processes such as acute phase response signalling, complement activation and mitochondrial dysfunction, which has a central role in the development of AP. Plasma HBPs in AP included known AP biomarkers such as serum amyloid A, as well as emerging targets such as histone H2A. Other HBPs such as alpha 2-HS glycoprotein (AHSG) and histidine-rich glycoprotein (HRG) need further investigation for potential applications in the management of AP. Pancreas HBPs are extracellular and so easily accessible and are potential drug targets in AP, whereas plasma HBPs represent potential biomarkers for AP. Thus, their identification paves the way to determine which HBPs may have potential applications in the management of AP.

biochemistry↗

bioRxiv2018

Critical assessment of approaches for molecular docking to elucidate associations of HLA alleles with Adverse Drug Reactions

Adverse drug reactions have been linked with genetic polymorphisms in HLA genes in numerous different studies. HLA proteins have an essential role in the presentation of self and non-self peptides, as part of the adaptive immune response. Amongst the associated drugs-allele combinations, anti-HIV drug Abacavir has been shown to be associated with the HLA-B*57:01 allele, and anti-epilepsy drug Carbamazepine with B*15:02, in both cases likely following the altered peptide repertoire model of interaction. Under this model, the drug binds directly to the antigen presentation region, causing different self peptides to be presented, which trigger an unwanted immune response. There is growing interest in searching for evidence supporting this model for other ADRs using bioinformatics techniques. In this study, in silico docking was used to assess the utility and reliability of well-known docking programs when addressing these challenging HLA-drug situations. Four docking programs: SwissDock, ROSIE, AutoDock Vina and AutoDockFR, were used to investigate if each software could accurately dock the Abacavir back into the crystal structure for the protein arising from the known risk allele, and if they were able to distinguish between the HLA-associated and non-HLA-associated (control) alleles. The impact of using homology models on the docking performance and how using different parameters such as including receptor flexibility affected the docking performance, were also investigated to simulate the approach where a crystal structure for a given HLA allele may be unavailable. The programs that were best able to predict the binding position of Abacavir were then used to recreate the docking seen for Carbamazepine with B*15:02 and controls alleles. It was found that the programmes investigated were sometimes able to correctly predict the binding mode of Abacavir with B*57:01 but not always. Each of the software packages that were assessed could predict the binding of Abacavir and Carbamazepine within the correct sub-pocket and, with the exception of ROSIE, was able to correctly distinguish between risk and control alleles. We found that docking to homology models could produce poorer quality predictions, especially when sequence differences impact the architecture of predicted binding pockets. Caution must therefore be used as inaccurate structures may lead to erroneous docking predictions. Incorporating receptor flexibility was found to negatively affect the docking performance for the examples investigated. Taken together, our findings help characterise the potential but also the limitations of computational prediction of drug-HLA interactions. These docking techniques should therefore always be used with care and alongside other methods of investigation, in order to be able to draw strong conclusions from the given results.

bioinformatics↗