Biology subjects

Ansell, T. B.

Publications and source records attributed to Ansell, T. B..

8 recordsLinked to original sources

bioRxiv2025

Cryogenic Electron Tomography by the Numbers: Charting Underexplored Lineages in Structural Cell Biology

Imaging of cells and their interactions across the whole biosphere, with molecular-scale resolution, is key for understanding structure-function relations. Cryogenic electron tomography (cryo-ET) is a powerful method for obtaining this critical information. However, cryo-ET studies are challenging and often limited to a small number of cell types per study. Here, we bring together cryo-ET data from hundreds of cells and tissues across the biosphere to (i) identify emerging methodological trends, (ii) reduce imaging time and costs, (iii) quantitively compare methods for cell freezing and sectioning and (iv) census cryo-ET species coverage across all domains of life. Comparison of the fraction of cellular material within lamellae across all domains of life reveals an order of magnitude difference between bacteria (9%) and archaea (15%) compared to eukaryotes (1%). We calculate the fraction of cellular material which can be imaged using distinct sectioning methods on multicellular communities and tissues - identifying serial lift-out as a powerful new approach for obtaining more complete cellular depictions. Finally, we show that the biodiversity of current cryo-ET studies is 2-3 order of magnitude lower than in sequence libraries and 4-5 lower than the total predicted on Earth. Our analyses reveal major evolutionary lineages which remain critically understudied and where future cryo-ET research would be most impactful. Significance StatementCryogenic electron tomography (cryo-ET) is a powerful method for imaging inside cells, but it is challenging and typically limited to a few cells per study. We consolidate and quantify data from hundreds of cellular cryo-ET studies across the biosphere. The methodological trends we identify will enhance cryo-ET throughput and heighten biological insight from underexplored lineages of life on Earth.

biophysics↗

bioRxiv2025

Structural basis of undecaprenyl phosphate glycosylation leading to polymyxin resistance in Gram-negative bacteria

In Gram-negative bacteria, the enzymatic modification of Lipid A with aminoarabinose (L-Ara4N) leads to resistance against polymyxin antibiotics and cationic antimicrobial peptides. ArnC, an integral membrane glycosyltransferase, attaches a formylated form of aminoarabinose to the lipid undecaprenyl phosphate, enabling its association with the bacterial inner membrane. Here, we present cryo-electron microscopy structures of ArnC from S. enterica in apo and nucleotide-bound conformations. These structures reveal a conformational transition that takes place upon binding of the partial donor substrate. Using coarse-grained and atomistic simulations, we provide insights into substrate coordination before and during catalysis, and we propose a catalytic mechanism that may operate on all similar metal-dependent polyprenyl phosphate glycosyltransferases. The reported structures provide a new target for drug design aiming to combat polymyxin resistance.

biochemistry↗

bioRxiv2023

Mapping Structural and Dynamic Divergence Across the MBOAT family

Membrane Bound O-acyltransferases (MBOATs) are membrane embedded enzymes which catalyse acyl chain transfer to a diverse group of substrates including lipids, small-molecules and proteins. Recent MBOAT structures reveal a conserved structural core, despite wide-ranging functional specificity across both prokaryotes and eukaryotes. The structural basis of catalytic specificity, regulation and interactions with the surrounding environment remain uncertain, hindering effective therapeutic targeting. Here, we combine comparative molecular dynamics (MD) simulations with bioinformatics to assess molecular and interactional divergence across the family. In simulations, MBOATs differentially distort the bilayer depending on their substrate type. Additionally, we identify specific lipid binding sites surrounding MBOAT reactant gates into the surrounding membrane. We use bioinformatics to reveal a conserved role for re-entrant loop-2 in stabilisation of the MBOAT fold and identify a key hydrogen bond involved in DGAT1 dimerisation. Finally, we predict differences in MBOAT core solvation and water gating properties across the family. These data are pertinent to the design of MBOAT specific inhibitors that encompass dynamic information within cellular mimetic environments.

biochemistry↗

bioRxiv2022

LipIDens: Simulation assisted interpretation of lipid densities in cryo-EM structures of membrane proteins.

Cryo-electron microscopy (cryo-EM) enables the determination of membrane protein structures in native-like environments. Characterising how membrane proteins interact with the surrounding membrane lipid environment is assisted by resolution of lipid-like densities visible in cryo-EM maps. Nevertheless, establishing the molecular identity of putative lipid and/or detergent densities remains challenging. Here we present LipIDens, a pipeline for molecular dynamics (MD) simulation-assisted interpretation of lipid and lipid-like densities in cryo-EM structures. The pipeline integrates the implementation and analysis of multi-scale MD simulations for identification, ranking and refinement of lipid binding poses which superpose onto cryo-EM map densities. Thus, LipIDens enables direct integration of experimental and computational structural approaches to facilitate the interpretation of lipid-like cryo-EM densities and to reveal the molecular identities of protein-lipid interactions within a bilayer environment. The LipIDens code is open-source and embedded within a notebook format to assist automation and usability.

biophysics↗

bioRxiv2021

Hepatocyte cholesterol content modulates glucagon receptor signalling

Glucagon decreases liver fat, and non-alcoholic fatty liver disease (NAFLD) is associated with hepatic glucagon resistance. Increasingly it is recognised that the function of G protein-coupled receptors can be regulated by their local plasma membrane lipid environment. The aim of this study was to evaluate the effects of experimentally modulating hepatocyte cholesterol content on the function of the glucagon receptor (GCGR). We found that glucagon-mediated cAMP production is inversely proportional to cholesterol content of human hepatoma and primary mouse hepatocytes after treatment with cholesterol-depleting and loading agents, with ligand internalisation showing the opposite trend. Mice fed a high cholesterol diet had increased hepatic cholesterol and a blunted hyperglycaemic response to glucagon, both of which were partially reversed by simvastatin. Molecular dynamics simulations identified potential membrane-exposed cholesterol binding sites on the GCGR. Overall, our data suggest that increased hepatocyte membrane cholesterol could directly contribute to glucagon resistance in NAFLD.

pharmacology and toxicology↗

bioRxiv2021

Structure and Mechanism of Hedgehog Acyl Transferase

The iconic Sonic Hedgehog (SHH) morphogen pathway is a fundamental orchestrator of embryonic development and stem cell maintenance, and is implicated in cancers in various organs. A key step in signalling is transfer of a palmitate group to the N-terminal cysteine residue of SHH, catalysed by the multi-pass transmembrane enzyme Hedgehog acyltransferase (HHAT) resident in the endoplasmic reticulum (ER). Here, we present the high-resolution cryo-EM structure of HHAT bound to substrate analogue palmityl-coenzyme A and a SHH mimetic megabody. Surprisingly, we identified a heme group bound to an HHAT cysteine residue and show that this modification is essential for HHAT structure and function. A structure of HHAT bound to potent small molecule inhibitor IMP-1575 revealed conformational changes in the active site which occlude substrate binding. Our multidisciplinary analysis provides a detailed view of the novel mechanism by which HHAT adapts the membrane environment to transfer a long chain fatty acid across the ER membrane from cytosolic acyl-CoA to a luminal protein substrate. This structure of a member of the protein-substrate membrane-bound O-acyltransferase (MBOAT) superfamily provides a blueprint for other protein substrate MBOATs, such as WNT morphogen acyltransferase Porcupine and ghrelin O-acyltransferase GOAT, and a template for future drug discovery.

biophysics↗

bioRxiv2021

Relative Affinities of Protein-Cholesterol Interactions from Equilibrium Molecular Dynamics Simulations

Specific interactions of lipids with membrane proteins contribute to protein stability and function. Multiple lipid interactions surrounding a membrane protein are often identified in molecular dynamics (MD) simulations and are, increasingly, resolved in cryo-EM densities. Determining the relative importance of specific interaction sites is aided by determination of lipid binding affinities by experimental or simulation methods. Here, we develop a method for determining protein-lipid binding affinities from equilibrium coarse-grained MD simulations using binding saturation curves, designed to mimic experimental protocols. We apply this method to directly obtain affinities for cholesterol binding to multiple sites on a range of membrane proteins and compare our results with free energies obtained from density-based equilibrium methods and with potential of mean force calculations, getting good agreement with respect to the ranking of affinities for different sites. Thus, our binding saturation method provides a robust, high-throughput alternative for determining the relative consequence of individual sites seen in e.g. cryo-EM derived membrane protein structures surrounded by a plethora of ancillary lipid densities.

biophysics↗

bioRxiv2021

Identification and assessment of cardiolipin interactions with E. coli inner membrane proteins

Integral membrane proteins are localised and/or regulated by lipids present in the surrounding bilayer. Whilst bacteria such as E. coli have relatively simple membranes when compared to eukaryotic cells, there is ample evidence that many bacterial proteins bind to specific lipids, especially the anionic lipid cardiolipin. Here, we apply molecular dynamics simulations to assess lipid binding to 42 different E. coli inner membrane proteins. Our data reveals a strong asymmetry between the membrane leaflets, with a marked increase of anionic lipid binding to the inner leaflet regions of membrane proteins, particularly for cardiolipin. From our simulations we identify over 700 independent cardiolipin binding sites, allowing us to identify the molecular basis of a prototypical cardiolipin binding site, which we validate against structures of bacterial proteins bound to cardiolipin. This allows us to construct a set of metrics for defining a high affinity cardiolipin binding site on (bacterial) membrane proteins, paving the way for a heuristic approach to defining more complex protein-lipid interactions.

biochemistry↗