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Keegan, R. M.

Publications and source records attributed to Keegan, R. M..

9 recordsLinked to original sources

Predicting Protein Crystal Solvent Content from Patterson Maps Using Machine Learning

Estimating the solvent content of protein crystals is fundamental to identifying the correct symmetry and phasing of the unit cell. Typically, the number of molecules in the asymmetric unit is not known and probabilistic methods are used based on statistics derived from the Protein Data Bank (PDB). These methods tend to predict the number of molecules incorrectly in around 20% of cases, which can significantly impede the structure solution pipeline. Here multiple machine learning approaches are investigated to predict solvent content using Patterson Maps. Several architectures are shown to give a significant improvement over current approaches, with prediction errors being reduced by over 50%. In addition, the potential of embedded representations of Patterson Maps for clustering is demonstrated, which could lead to new approaches for identifying similar structures when processing novel data.

molecular biology↗

Slice'N'Dice: Maximising the value of predicted models for structural biologists

With the advent of next generation modelling methods, such as AlphaFold2, structural biologists are increasingly using predicted structures as search models for Molecular Replacement (MR) when experimental structures of homologues are unavailable. Inaccuracy in domain-domain orientations is often a key limitation when using predicted models for MR. SliceNDice is a software package designed to address this issue by first slicing models into distinct structural units and then automatically placing the slices using Phaser. The slicing step can use AlphaFold2s predicted aligned error (PAE), or can operate via a variety of C atom clustering algorithms, extending applicability to structures of any origin. The number of splits can be selected by the user. SliceNDice is available in CCP4 8.0 and is currently being adapted for cryo-EM use cases.

bioinformatics↗

MrParse: Finding homologues in the PDB and the EBI AlphaFold database for Molecular Replacement and more

Crystallographers have an array of search model options for structure solution by Molecular Replacement (MR). 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 use pre-calculated predictions retrieved from databases such as the EBI AlphaFold database. MrParse is a new pipeline to help streamline the decision process in MR by consolidating bioinformatic predictions in one place. When reflection data are provided, MrParse can rank any homologues found using eLLG which indicates the likelihood that a given search model will work in MR. In-built 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.

bioinformatics↗

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↗

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↗

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↗

Intercellular viral spread and intracellular transposition of Drosophila gypsy

It has become increasingly clear that retrotransposons (RTEs) are more widely expressed in somatic tissues than previously appreciated. RTE expression has been implicated in a myriad of biological processes ranging from normal development and aging, to age related diseases such as cancer and neurodegeneration. Long Terminal Repeat (LTR)-retrotransposons are evolutionary ancestors to, and share many features with, exogenous retroviruses. In fact, many organisms contain endogenous retroviruses (ERVs) that derive from an exogenous retrovirus that have integrated into the germ line. These ERVs are inherited in Mendelian fashion like RTEs, and some retain the ability to transmit between cells like viruses, while others develop the ability to act as RTEs. The process of evolutionary transition between LTR-RTE and retroviruses is thought to involve multiple steps by which the element loses or gains the ability to transmit copies between cells versus the ability to replicate intracellularly. But, typically, these two modes of transmission are incompatible because they require assembly in different sub-cellular compartments. Like murine IAP/IAP-E elements, the gypsy family of retroelements in arthropods appear to sit along this evolutionary transition. The fact that gypsy elements have been found to actively mobilize in neurons and glial cells during normal aging and in models of neurodegeneration raises the question of whether their replication in somatic cells occurs via intracellular retrotransposition, intercellular viral spread, or some combination of the two. These modes of replication in somatic tissues would have quite different biological implications. Here, we demonstrate that Drosophila gypsy is capable of both cell-associated and cell-free viral transmission between cultured S2 cells of somatic origin. Further, we demonstrate that the ability of gypsy to move between cells is dependent upon a functional copy of its viral envelope protein. This argues that the gypsy element has transitioned from an RTE into a functional endogenous retrovirus with the acquisition of its envelope gene. On the other hand, we also find that intracellular retrotransposition of the same genomic copy of gypsy can occur in the absence of the Env protein. Thus, gypsy exhibits both intracellular retrotransposition and intercellular viral transmission as modes of replicating its genome.

genetics↗

Cellular labeling of endogenous virus replication (CLEVR) reveals de novo insertions of the gypsy endogenous retrovirus in cell culture and in both neurons and glial cells of aging fruit flies

Evidence is rapidly mounting that transposable element expression and replication may impact biology more widely than previously thought. This includes potential effects on normal physiology of somatic tissues and dysfunctional impacts in diseases associated with aging such as cancer and neurodegeneration. Investigation of the biological impact of mobile elements in somatic cells will be greatly facilitated by use of donor elements that are engineered to report de novo events in vivo. In multicellular organisms, successful reporters of LINE element mobilization have been in use for some time, but similar strategies have not been developed to report Long Terminal Repeat (LTR) retrotransposons and endogenous retroviruses. We describe Cellular Labeling of Endogenous Virus Replication (CLEVR), which reports replication of the gypsy element in Drosophila. The gypsy-CLEVR reporter reveals gypsy replication both in cell culture and in individual neurons and glial cells of the aging adult fly. We also demonstrate that the gypsy-CLEVR replication rate is increased when the short interfering RNA silencing system is genetically disrupted. This CLEVR strategy makes use of universally conserved features of retroviruses and should be widely applicable to other LTR-retrotransposons, endogenous retroviruses and exogenous retroviruses.

genetics↗

Retrotransposon Activation Contributes to Neurodegeneration in a DrosophilaTDP-43 Model of ALS.

Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) are two incurable neurodegenerative disorders that exist on a symptomological spectrum and share both genetic underpinnings and pathophysiological hallmarks. Functional abnormality of TAR DNA-binding protein 43 (TDP-43), an aggregation-prone RNA and DNA binding protein, is observed in the vast majority of both familial and sporadic ALS cases and in [~]40% of FTLD cases, but the cascade of events leading to cell death are not understood. We have expressed human TDP-43 (hTDP-43) in Drosophila neurons and glia, a model that recapitulates many of the characteristics of TDP-43-linked human disease including protein aggregation pathology, locomotor impairment, and premature death. We report that such expression of hTDP-43 impairs small interfering RNA (siRNA) silencing, which is the major post-transcriptional mechanism of retrotransposable element (RTE) control in somatic tissue. This is accompanied by de-repression of a panel of both LINE and LTR families of RTEs, with somewhat different elements being active in response to hTDP-43 expression in glia versus neurons. hTDP-43 expression in glia causes an early and severe loss of control of a specific RTE, the endogenous retrovirus (ERV) gypsy. We demonstrate that gypsy causes the degenerative phenotypes in these flies because we are able to rescue the toxicity of glial hTDP-43 either by genetically blocking expression of this RTE or by pharmacologically inhibiting RTE reverse transcriptase activity. Moreover, we provide evidence that activation of DNA damage-mediated programmed cell death underlies both neuronal and glial hTDP-43 toxicity, consistent with RTE-mediated effects in both cell types. Our findings suggest a novel mechanism in which RTE activity contributes to neurodegeneration in TDP-43-mediated diseases such as ALS and FTLD.\n\nAUTHOR SUMMARYFunctional abnormality of TAR DNA-binding protein 43 (TDP-43), an aggregation-prone RNA and DNA binding protein, is observed in the vast majority of both familial and sporadic ALS cases and in [~]40% of FTLD cases, and mutations in TDP-43 are causal in a subset of familial ALS cases. Although cytoplasmic inclusions of this mostly nuclear protein are a hallmark of the disease, the cascade of events leading to cell death are not understood. We demonstrate that expression of human TDP-43 (hTDP-43) in Drosophila neurons or glial cells, which results in toxic cytoplasmic accumulation of TDP-43, causes broad expression of retrotransposons. In the case of glial hTDP-43 expression, the endogenous retrovirus (ERV) gypsy causally contributes to degeneration because inhibiting gypsy genetically or pharmacologically is sufficient to rescue the phenotypic effects. Moreover, we demonstrate that activation of DNA damage-mediated programmed cell death underlies hTDP-43 and gypsy mediated toxicity. Finally, we find that hTDP-43 pathology impairs small interfering RNA silencing, which is an essential system that normally protects the genome from RTEs. These findings suggest a novel mechanism in which a storm of retrotransposon activation drives neurodegeneration in TDP-43 mediated diseases such as ALS and FTLD.

neuroscience↗