Biology subjects

Burton-Smith, R. N.

Publications and source records attributed to Burton-Smith, R. N..

10 recordsLinked to original sources

bioRxiv2026

MC-Bayes: A Python-based wrapper for MotionCor3 processing of EER files compatible with Bayesian polishing

Here, we present MC-Bayes, a Python-based script for processing cryo-electron microscopy EER movies on one or more GPUs using MotionCor3 in a user-friendly manner. Further, it generates the .star files necessary for RELION to perform Bayesian polishing (a.k.a.: reference-based motion correction) with EER movies. Until now, Bayesian polishing of EER data was only possible if the CPU-based "RELIONCor" implementation of MotionCor2 was used, which is sub-optimal on GPU-heavy cryo-EM processing systems. This wrapper was created for those facilities and/or users who may have (many) powerful GPUs, but for whatever reason have few CPU cores or less system RAM. Leveraging MotionCor3, MC-Bayes allows motion correction of EER data 2 or more times faster (depending on system) than the RELION CPU implementation, except in circumstances where dozens or hundreds of CPU cores with high quantities of system RAM can be utilised.

biophysics↗

bioRxiv2026

Near-atomic structures of human astrovirus capsids reveal a subunit-specific maturation mechanism

Human astrovirus (HAstV) is a major cause of acute gastroenteritis, yet the structural basis of capsid maturation and infectivity remains poorly understood. Here, we report near-atomic cryo-electron microscopy structures of HAstV serotype 4 in immature and mature states at 1.79 [A] and 1.73 [A] resolution, respectively. These structures reveal a subunit-specific maturation mechanism in which differential protease accessibility drives selective removal of spike (P2) domains. Structural comparisons show that only A-B dimers form a broad cavity beneath the P2 domains that is permissive for trypsin entry, whereas the corresponding region in C-C dimers is sterically restricted. Proteolytic maturation induces pronounced conformational rearrangements, particularly in subunit B, linking capsid geometry to asymmetric protease susceptibility. Despite these changes, the inner capsid architecture and presumptive RNA-associated densities remain conserved, indicating that genome release occurs at a later stage of infection. Together, our findings establish a mechanistic framework in which capsid geometry governs proteolysis-driven activation.

microbiology↗

bioRxiv2026

Structural basis for Ca2+-dependent, RyR2-selective stabilization by Ryanozole in CPVT

The cardiac ryanodine receptor (RyR2) is a Ca{superscript 2} release channel essential for excitation-contraction coupling. RyR2 mutations cause severe arrhythmogenic disorders, including catecholaminergic polymorphic ventricular tachycardia (CPVT), through gain-of-function (GOF) effects leading to aberrant Ca{superscript 2} release. We have recently developed Ryanozole, a potential therapeutic compound for CPVT, which selectively stabilizes RyR2 in a Ca2+-dependent manner. Here, we define the mechanism of action of Ryanozole by combining high-resolution cryo-electron microscopy, targeted mutagenesis and functional assays. Ryanozole binds to the interface between the Ca{superscript 2}-binding site and the pore-forming S6 helix, interfering with conformational changes required for Ca{superscript 2}-induced channel opening at low Ca2+. We identified key residues for the binding and isoform-specific modulation of Ryanozole. Notably, Ryanozole-bound RyR2 retains its ability to open at high Ca2+ via unique conformational changes. These findings provide a structural basis for CPVT-targeted therapy and redefine the paradigm of small molecule-based regulation of large ion channels.

biochemistry↗

bioRxiv2025

Integrative structural interactomics reveals protein organization and structure in a giant virus

Giant viruses are large DNA viruses that infect unicellular and multicellular eukaryotes and form exceptionally large extracellular particles. (Meta)genomics and (meta)transcriptomics have provided insight into their diverse coding repertoire, but many of the proteins remain to be characterized as they lack homology with known proteins. Here, we integrated cross-linking mass spectrometry, quantitative proteomics, computational tools and cryo-EM data to characterize the protein architecture of intact melbournevirus particles. Based on this, we allocated 88 viral proteins to different virion sub-compartments and proposed topologies of 25 inner membrane proteins. We assigned eight components of the capsid in cryo-EM data, including proteins that tether the capsid shell to the membrane, reflecting key points in virion maturation. The data provide a valuable resource and demonstrate the power of an integrative approach to gain system-level structural insights into a poorly characterized biological system.

biochemistry↗

bioRxiv2024

Inconsistencies in the published rabbit ribosomal rRNAs: a proposal for uniformity in sequence and site numbering

Examination of all publicly available Oryctolagus cuniculus (rabbit) ribosome cryo-EM structures reveals numerous confusing inconsistencies. First, there are a plethora of single nucleotide differences among the various rabbit 28S and 18S rRNA structures. Second, two nucleotides are absent from the NCBI Reference Sequence for the 18S rRNA gene. Moving forward, we propose using the Broad Institutes rabbit whole genome shotgun sequence and numbering to reduce modeling ambiguity and improve consistency between ribosome models.

biophysics↗

bioRxiv2023

Post-acquisition super resolution for cryo-electron microscopy

Super resolution detector acquisition for cryo-EM has been used to improve the clarity of cryo-EM reconstructions. Recent reports have demonstrated achieving resolutions beyond the physical Nyquist limit using super resolution acquisition. Here, we demonstrate exceeding the physical Nyquist limitation by pre-processing the raw micrograph movies from "counting mode" data which has already reached physical Nyquist reconstruction resolution. To demonstrate functionality, micrograph movies of five datasets were pre-processed and demonstrate that it is possible to exceed the physical Nyquist limit via pixel doubling before motion correction. We call this "post-acquisition super resolution", or PASR. While this was originally developed for processing of giant virus datasets, where acquiring at high magnification is not always possible or desirable, it is also shown to work for smaller objects such as adeno-associated virus (AAV) and apoferritin, both of which are still high symmetry, and jack bean urease, with lower symmetry. PASR could reduce the magnification required to achieve desired resolutions, which may increase collection efficiency. PASR can also be of use for in vivo tomography and facilities with high storage demands. However, this method should only be used for data which is able to achieve the Nyquist limit without PASR pre-processing. It will not improve attained resolutions of data which does not already reach the Nyquist limit.

biophysics↗

bioRxiv2022

Six states of Enterococcus hirae V-type ATPase reveals non-uniform rotor rotation during turnover

The vacuolar-type ATPase from Enterococcus hirae (EhV-ATPase) is a thus-far unique adaptation of V-ATPases, as it performs Na+ transport and demonstrates an off-axis rotor assembly. Recent single molecular studies of the isolated V1 domain have indicated that there are subpauses within the three major states of the pseudo three-fold symmetric rotary enzyme. However, there was no structural evidence for these. Herein we activated the EhV-ATPase complex with ATP and identified multiple structures consisting of a total of six states of this complex by using cryo-electron microscopy. The orientations of the rotor complex during turnover, especially in the intermediates, were not as perfectly uniform as expected. The densities in the nucleotide binding pockets in the V1 domain indicated the different catalytic conditions for the six conformations. The off-axis rotor and its interactions with the stator a-subunit during rotation suggests that this non-uniform rotor rotation is performed through the entire complex.

molecular biology↗

bioRxiv2021

The 4.4 A structure of the giant Melbournevirus virion belonging to the Marseilleviridae family

Members of Marseilleviridae, one family of icosahedral giant viruses classified in 2012 have been identified worldwide in all types of environments. The virion shows a characteristic internal membrane extrusion at the five-fold vertices of the capsid, but its structural details need to be elucidated. We now report the 4.4 [A] cryo-electron microscopy structure of the Melbournevirus capsid. An atomic model of the major capsid protein (MCP) shows a unique cup structure on the trimer that accommodates additional proteins. A polyalanine model of the penton base protein shows internally extended N- and C-terminals, which indirectly connect to the internal membrane extrusion. The Marseilleviruses share the same orientational organisation of the MCPs as PBCV-1 and CroV, but do not appear to possess a protein akin to the "tape measure" of these viruses. Minor capsid proteins named PC-{beta}, zipper, and scaffold are proposed to control the dimensions of the capsid during assembly.

microbiology↗

bioRxiv2021

A novel capsid protein network allows the characteristic inner membrane structure of Marseilleviridae giant viruses

Marseilleviridae is a family of the new order of giant viruses, which exhibit a characteristic inner membrane. Here, we investigated the entire structure of tokyovirus, a species of Marseillevirus at 7.7 [A] resolution using 1 MV high-voltage cryo-EM and single particle analysis. The minor capsid lattice formed by five proteins, shows a novel structure compared to other icosahedral giant viruses. Under the minor capsid proteins, scaffold proteins connect two five-fold vertices and interact with the inner membrane. Previously reported giant viruses utilise "tape measure" proteins, proposed to control its capsid size, which could not be identified in tokyovirus, but scaffold proteins appear to perform a similar role. A density on top of the major capsid protein was identified, which suggested to be a 14kDa glycoprotein. Our observations suggest that the icosahedral particle of Marseilleviridae is constructed with a novel capsid protein network, which allows the characteristic inner membrane structure.

microbiology↗

bioRxiv2020

Sub-3 A resolution structure of apoferritin using a multi-purpose TEM with a side-entry cryo-holder

The structural analysis of protein complexes by cryo-electron microscopy (cryo-EM) single particle analysis (SPA) has had great impact as a biophysical method in recent years. Many results of cryo-EM SPA are based on state-of-the-art cryo-electron microscopes customized for SPA. These are currently only available in limited locations around the world, where securing machine time is highly competitive. One potential solution for this time-competitive situation is to reuse existing multi-purpose equipment. Here, we used a multi-purpose TEM with a side entry cryo-holder at our facility to evaluate the potential of high-resolution SPA. We report a 3 [A] resolution map of apoferritin with local resolution extending to 2.6 [A]. The map clearly showed two positions of an aromatic side chain. We also verified the optimal imaging conditions depending on different electron microscope and camera combinations. This study demonstrates the possibilities of more widely available and established electron microscopes, and their applications for cryo-EM SPA.

molecular biology↗