Biology subjects

Naismith, J. H.

Publications and source records attributed to Naismith, J. H..

9 recordsLinked to original sources

bioRxiv2026

Femtosecond pulsed laser ablation for in-tissue electron cryo-tomography

In situ electron cryo-tomography (cryo-ET) is a powerful tool for visualising biology inside cells at the molecular scale. The routine application of cryo-ET in tissue samples promises biological insight beyond single cells, but is currently limited by the technical challenge of preparing high quality, electron-transparent specimens from thick (>100 m) biopsies efficiently. Here, we demonstrate that cryogenic femtosecond pulsed laser ablation (cryo-fs-PLA) can accelerate the preparation of vitreous specimens of mammalian brain tissue for cryo-ET. Using cryogenic fluorescence microscopy, regions of interest within high-pressure frozen tissue can be specifically targeted, and cryo-fs-PLA can be employed as a preparative step for serial cryogenic lift-out experiments. We define cryo-fs-PLA parameters and determine ablation rates for two different laser wavelengths, and demonstrate that tilt series collected on lamellae fabricated from sites exposed to cryo-fs-PLA largely maintain their vitrification state. Incorporating cryo-fs-PLA into existing cryo-ET workflows has the potential to unlock the sample preparation of tissues at the throughput necessary for routine in situ cell biology of multicellular samples.

biochemistry↗

bioRxiv2025

Isolation of functional supramolecular attack particles (SMAPs)

High density cultures of the GMP compatible NK-92 natural killer cell line release heterogeneous extracellular particles (EP), notably extracellular vesicles (EV) and non-vesicular extracellular particles (NVEP). The NVEP include a small proportion of supramolecular attack particles (SMAPs), the direct cytotoxic potential of which suggests therapeutic promise, yet scalable enrichment and functional evaluation alongside other extracellular particles (EPs) have been lacking. Here, we develop a high-resolution serial size-exclusion liquid chromatography (SELC) workflow that resolves EPs into 4 fractions F1-F4, in which EV are enriched in F2 and SMAPs are enriched in F3. Multi-modal characterization using Nanoparticle Tracking Analysis (NTA), Nano Flow Cytometry (Nano FCM), Transmission Electron Microscopy (TEM), Total Internal Reflection Microscopy (TIRFM) and Proteomics) demonstrated that F2 NK-92 EVs are [~]130-150 nm particles enriched for complement proteins, whereas SMAPs (F3) are 100-110 nm particles enriched for cytotoxic proteins (PRF1, GZMB) and SMAP shell components (e.g., THBS1, THBS4). Functionally, Ca2+-stabilized SMAPs (F3) trigger caspase-3-dependent apoptosis in tumor cell lines with robust dose responses, while F2 lacks direct cytotoxicity in vitro. Ex vivo, F3 SMAPs kill patient-derived breast cancer and chronic lymphocytic leukaemia cells (CLL) in a concentration-dependent manner. In NSG mice, intra-tumoral treatment with SMAPs (F3) restrains the growth of aggressive B16F10 melanoma and PANC-1 pancreatic cancer, confirming in vivo functionality. These results establish a scalable method to isolate and compare NK-derived particle classes and provide a foundation for targeted SMAP engineering as a promising approach for treatment of solid tumours. Significance StatementCell therapies face barriers in solid tumours, including lack of entry and suppression. NK cells release extracellular particles (EPs), including supramolecular attack particles (SMAPs), that can traffic into tumour tissue. We define a practical, scalable chromatography process that resolves NK-derived SMAPs from other EPs and demonstrate that SMAPs induce caspase mediated apoptosis in vitro and engage in cytotoxic lymphocyte independent anti-tumour activity in vivo. These insights open a route to engineering and testing SMAP-based therapeutics to overcome limitations of whole-cell approaches in solid cancers.

immunology↗

bioRxiv2024

Reduction of SEM charging artefacts in native cryogenic biological samples.

Scanning electron microscopy (SEM) of frozen-hydrated biological samples allows imaging of subcellular structures at the mesoscale in their native state. Combined with focused ion beam milling (FIB), serial FIB/SEM can be used to build a 3-dimensional picture of cells and tissues. The correlation of specific regions of interest with cryo-electron microscopy (cryoEM) can additionally enable subsequent high-resolution analysis. However, the adoption of serial FIB/SEM imaging-based methods is limited due to artefacts arising from insulating areas of cryogenically preserved samples. Here, we demonstrate the use of interleaved scanning to reduce charging artefacts, allowing the observation of biological features that otherwise would be masked or perturbed. We apply our method to samples where inherent features are not visible. These examples include membrane contact sites within mammalian cells, visualisation of the degradation compartment in the algae E.gracilis and observation of a network of membranes within different types of axons in an adult mouse cortex. We further propose an alternative scanning method that could also be widely applicable to imaging any non-conductive.

cell biology↗

bioRxiv2024

Xenon plasma focused ion beam lamella fabrication on high-pressure frozen specimens for structural cell biology

Cryo focused ion beam lamella preparation is a potent tool for in situ structural biology, enabling the study of macromolecules in their native cellular environments. However, throughput is currently limited, especially for thicker, more biologically complex samples. We describe how xenon plasma focused ion beam milling can be used for routine bulk milling of thicker, high-pressure frozen samples during lamellae preparation with a high success rate and determine a 4.0 [A] structure of the Escherichia coli ribosome on these lamellae using sub volume averaging. We determine the effects of increased ion currents on sample integrity during bulk milling of thicker planar samples, also showing that beyond an initial region of damage, no measurable structural damage propagates beyond this. The use of xenon results in substantial structural damage to particles up to 30 nm in depth from the milled surfaces, with detectable damage observed to 45 nm. Ours results outlines how the use of high currents using xenon plasma focused ion beam milling may be integrated into FIB milling regimes for preparing thin lamellae for high-resolution in situ structural biology.

biochemistry↗

bioRxiv2022

Cryo-plasma FIB/SEM volume imaging of biological specimens

Serial focussed ion beam scanning electron microscopy (FIB/SEM) enables imaging and assessment of sub-cellular structures on the mesoscale (10 nm to 10 m). When applied to vitrified samples, serial FIB/SEM is also a means to target specific structures in cells and tissues while maintaining constituents hydration shells for in-situ structural biology downstream. However, the application of serial FIB/SEM imaging of non-stained cryogenic biological samples is limited due to low contrast, curtaining and charging artefacts. We address these challenges using a cryogenic plasma FIB/SEM (cryo-pFIB/SEM). We evaluated the choice of plasma ion source and imaging regimes to produce high quality SEM images of a range of different biological samples. Using an automated workflow we produced three dimensional volumes of bacteria, human cells, and tissue, and calculated estimates for their resolution, typically achieving 20 to 50 nm. Additionally, a tag-free tool is needed to drive the application of in situ structural biology towards tissue. The combination of serial FIB/SEM with plasmabased ion sources promises a framework for targeting specific features in bulk-frozen samples (>100 m) to produce lamella for cryogenic electron tomography.

cell biology↗

bioRxiv2022

Plasma FIB milling for the determination of structures in situ

Structural biology inside cells and tissues requires methods able to thin vitrified specimens to electron transparent thicknesses. Until now, focused ions beams based on gallium have been used. However, ion implantation, changes to surface chemistry and an inability to access high currents limit Gallium as an ion beam source. Here, we show that plasma-coupled ion sources can produce cryogenic lamella of vitrified human cells in a robust and automated manner, with quality sufficient for pseudo-atomic structure determination. In addition, these lamellae were produced in a prototype microscope equipped for long cryogenic run times (>1 week) and with multi-specimen support fully compatible with modern-day transmission electron microscopes. We demonstrate for the first time that plasma ion sources can be used for structural biology within cells, determining a structure in-situ to 4.9 [A] and describing a workflow upon which different plasmas can be examined to streamline lamella fabrication further.

biochemistry↗

bioRxiv2022

Molecular basis for the enzymatic macrocyclization of multiply backbone N-methylated peptides

The enzyme OphP is essential for the biosynthesis of the macrocyclic peptide omphalotin A, a dodecamer with 9 backbone N-methylations produced by the wood-degrading fungus Omphalotus olearius. Heterologous expression of OphP and the peptide-precursor protein OphMA in yeast, yields omphalotin A. Thus, Oph P was hypothesized to have a dual function; catalyzing both endoproteolytic release of a peptide intermediate from OphMA, and macrocyclization of the multiply -N-methylated core peptide with concomitant release of a C-terminal follower peptide. In our in vitro activity assays, OphP showed robust endoproteolytic and macrocyclase activity on -N-methylated peptides but was unable to cleave OphMA. The enzyme had a strong preference for hydrophobic, highly -N-methylated peptides and an -N-methylated glycine residue at the P1 site. OphP adopts a canonical prolyl oligopeptidase (POP) fold with a predominantly hydrophobic substrate binding cleft, and a small and hydrophobic P1 binding pocket. We demonstrate that OphP is a POP-type macrocyclase with a specificity and a substrate route to the active site different from other members of the family. These results could be exploited for the biotechnological production of macrocyclic peptides with multiple backbone N-methylations, which are interesting due to their favorable pharmacological properties.

biochemistry↗

bioRxiv2021

Cryptic SARS-CoV2-spike-with-sugar interactions revealed by 'universal' saturation transfer analysis

Many host pathogen interactions such as human viruses (including non-SARS-coronaviruses) rely on attachment to host cell-surface glycans. There are conflicting reports about whether the Spike protein of SARS-CoV-2 binds to sialic acid commonly found on host cell-surface N-linked glycans. In the absence of a biochemical assay, the ability to analyze the binding of glycans to heavily- modified proteins and resolve this issue is limited. Classical Saturation Transfer Difference (STD) NMR can be confounded by overlapping sugar resonances that compound with known experimental constraints. Here we present universal saturation transfer analysis (uSTA), an NMR method that builds on existing approaches to provide a general and automated workflow for studying protein-ligand interactions. uSTA reveals that B-origin-lineage-SARS-CoV-2 spike trimer binds sialoside sugars in an end on manner and modelling guided by uSTA localises binding to the spike N-terminal domain (NTD). The sialylated-polylactosamine motif is found on tetraantennary human N-linked-glycoproteins in deeper lung and may have played a role in zoonosis. Provocatively, sialic acid binding is abolished by mutations in some subsequent SARS- CoV-2 variants-of-concern. A very high resolution cryo-EM structure confirms the NTD location and end on mode; it rationalises the effect of NTD mutations and the structure-activity relationship of sialic acid analogues. uSTA is demonstrated to be a robust, rapid and quantitative tool for analysis of binding, even in the most demanding systems. Extended AbstractThe surface proteins found on both pathogens and host cells mediate entry (and exit) and influence disease progression and transmission. Both types can bear host-generated post- translational modifications such as glycosylation that are essential for function but can confound biophysical methods used for dissecting key interactions. Several human viruses (including non- SARS-coronaviruses) attach to host cell-surface N-linked glycans that include forms of sialic acid (sialosides). There remains, however, conflicting evidence as to if or how SARS-associated coronaviruses might use such a mechanism. Here, we demonstrate quantitative extension of saturation transfer protein NMR methods to a complete mathematical model of the magnetization transfer caused by interactions between protein and ligand. The method couples objective resonance-identification via a deconvolution algorithm with Bloch-McConnell analysis to enable a structural, kinetic and thermodynamic analysis of ligand binding beyond previously-perceived limits of exchange rates, concentration or system. Using an automated and openly available workflow this universal saturation transfer analysis (uSTA) can be readily-applied in a range of even heavily-modified systems in a general manner to now obtain quantitative binding interaction parameters (KD, kEx). uSTA proved critical in mapping direct interactions between natural sialoside sugar ligands and relevant virus-surface attachment glycoproteins - SARS-CoV-2-spike and influenza-H1N1-haemagglutinin variants - by quantitating ligand signal in spectral regions otherwise occluded by resonances from mobile protein glycans (that also include sialosides). In B- origin-lineage-SARS-CoV-2 spike trimer end on-binding to sialoside sugars was revealed contrasting with extended surface-binding for heparin sugar ligands; uSTA-derived constraints used in structural modelling suggested sialoside-glycan binding sites in a beta-sheet-rich region of spike N-terminal domain (NTD). Consistent with this, uSTA-glycan binding was minimally- perturbed by antibodies that neutralize the ACE2-binding domain (RBD) but strongly disrupted in spike from the B1.1.7/alpha and B1.351/beta variants-of-concern, which possess hotspot mutations in the NTD. Sialoside binding in B-origin-lineage-NTD was unequivocally pinpointed by cryo-EM to a site that is created from residues that are notably deleted in variants (e.g. H69,V70,Y145 in alpha). An analysis of beneficial genetic variances in cohorts of patients from early 2020 suggests a model in which this site in the NTD of B-origin-lineage-SARS-CoV-2 (but not in alpha/beta-variants) may have exploited a specific sialylated-polylactosamine motif found on tetraantennary human N-linked-glycoproteins in deeper lung. Together these confirm a novel binding mode mediated by the unusual NTD of SARS-CoV-2 and suggest how it may drive virulence and/or zoonosis via modulation of glycan attachment. Since cell-surface glycans are widely relevant to biology and pathology, uSTA can now provide ready, quantitative, widespread analysis of complex, host-derived and post-translationally modified proteins with putative ligands relevant to disease even in previously confounding complex systems.

biophysics↗

bioRxiv2020

Structural basis for the neutralization of SARS-CoV-2 by an antibody from a convalescent patient

The COVID-19 pandemic has had unprecedented health and economic impact, but currently there are no approved therapies. We have isolated an antibody, EY6A, from a late-stage COVID-19 patient and show it neutralises SARS-CoV-2 and cross-reacts with SARS-CoV-1. EY6A Fab binds tightly (KD of 2 nM) the receptor binding domain (RBD) of the viral Spike glycoprotein and a 2.6[A] crystal structure of an RBD/EY6A Fab complex identifies the highly conserved epitope, away from the ACE2 receptor binding site. Residues of this epitope are key to stabilising the pre-fusion Spike. Cryo-EM analyses of the pre-fusion Spike incubated with EY6A Fab reveal a complex of the intact trimer with three Fabs bound and two further multimeric forms comprising destabilized Spike attached to Fab. EY6A binds what is probably a major neutralising epitope, making it a candidate therapeutic for COVID-19.

microbiology↗