Biology subjects

Radecke, J.

Publications and source records attributed to Radecke, J..

14 recordsLinked to original sources

bioRxiv2026

Cryo-ET and Sub-Volume Analysis of Fibrillar Pathology in the Neurodegenerative Human Brain

Structural studies of amyloid fibrils extracted from brain tissue have identified disease-specific fibril polymorphs. However, the mechanisms driving distinct polymorphs remain unclear because no method currently links the cellular context, composition and ultrastructure of individual aggregates to their constituent fibril polymorphs. Here, we present a workflow for in situ cryo-electron tomography (cryo-ET) to study amyloids within individual aggregates in the human neurodegenerative postmortem brain. Using chemically fixed tissue, we studied in situ tau fibrils in the hippocampus of an Alzheimer's disease (AD) donor and -synuclein fibrils within a glial nuclear inclusion in the cingulate gyrus of a multiple systems atrophy (MSA) donor. Subtomogram averaging and helical reconstruction of fibril subvolumes yielded low-resolution (~30-35A) density maps, which were compared to existing ex vivo structures. Preserved ultrastructure enabled detailed observations of pathology, including an alpha-synuclein fibril penetrating the nuclear envelope, providing mechanistic insight into intranuclear aggregate formation. Combined with existing high-resolution fibril structures lacking spatial context, cryo-ET offers a powerful approach to understanding amyloid polymorphism in neurodegenerative diseases, advancing anti-amyloid therapies and diagnostic tools.

neuroscience↗

bioRxiv2025

Correlative Ultrastructural Mapping of Lewy Pathology Reveals Regional Diversity in Parkinsons and Dementia with Lewy bodies

Lewy body diseases, including Parkinsons disease (PD) and dementia with Lewy bodies (DLB), are defined by neuronal accumulation of misfolded -synuclein (-Syn), yet the ultrastructural diversity of these inclusions across brain regions and disease contexts remains unclear. Here, we applied large-scale correlative light and electron microscopy (CLEM) to map -Syn pathology across cortical regions (entorhinal cortex, ENT; anterior cingulate cortex, AC; hippocampal CA2 region) and substantia nigra (SN) in clinically and pathologically confirmed PD and DLB donors. We identified pronounced regional heterogeneity in Lewy pathology, with cortical inclusions showing diverse maturation stages at the ultrastructural level, ranging from low-density fibrils interspersed with organelles to highly compact fibrillar inclusions. In the SN of DLB donors, we observed the full range of classical nigral LB morphologies previously described in PD. We additionally characterized diverse neuritic -Syn pathologies in DLB and identified a distinct population of electron-dense, degenerating, -Syn-positive cortical neurons not previously reported. Importantly, we found no significant difference in LB ultrastructure between PD and DLB in either cortical or nigral pathology. In contrast, quantitative analysis of >10,000 mitochondria revealed disease- and region-specific signatures of altered mitochondrial homeostasis. PD showed increased mitochondrial density and enlargement in the SN, whereas DLB showed increased mitochondrial density only in the ENT. Mitochondrial enlargement was exclusive to PD. These findings indicate that LB ultrastructure alone does not distinguish PD from DLB; instead, region-specific mitochondrial phenotypes may better reflect disease identity and regional susceptibility. Overall, we provide a high-resolution framework for human Lewy pathology in PD and DLB, revealing that ultrastructural responses to -Syn pathology are driven primarily by neuronal identity and regional vulnerability. Our results highlight the need for disease- and region-specific models that capture human phenotypes to advance mechanistic understanding and therapeutic targeting of synucleinopathies.

pathology↗

bioRxiv2025

CryoWriter: A Robotic Solution for Improved Cryo-EM Grid Preparation

Cryo-electron microscopy (cryo-EM) structure determination relies on preparing thin, vitreous films of sample solution on EM grids. Cryo-EM is a mature technology, but preparing the grids remains a major bottleneck. Here, we evaluate the cryoWriter, a blotting-free, microfluidic grid-preparation robot that writes nanoliter volumes onto EM grids in a controlled environment. Using capillary-writing in spiral or line patterns, we prepared high-quality grids from minimal sample volumes and obtained near-atomic reconstructions for test specimens, including TMV, apoferritin, and the membrane protein TRPM4. We further demonstrate programmable deposition modes, such as writing the sample twice to boost particle density, or two-line writing for on-grid mixing to visualize time-resolved protein-ligand binding. In a challenging case (NrS-1 DNA polymerase), the cryoWriter grids exhibited reduced orientation bias relative to conventional blotting, enabling a more isotropic reconstruction. These results show that the cryoWriter provides a versatile platform for reproducible low volume cryo-EM grid preparation and for on-grid biochemical workflows.

biophysics↗

bioRxiv2025

Pulsed-electron illumination does not reduce beam damage for imaging biological macromolecules

Radiation damage remains a fundamental limitation in cryo-electron microscopy (cryo-EM), constraining the total electron dose that can be used and thus limiting high-resolution imaging of biological specimens. Recent studies have proposed that temporally structured or pulsed electron beams could reduce radiation damage by allowing time for energy dissipation between individual electron interactions. To evaluate this hypothesis, we conducted a systematic investigation using a radiofrequency (RF) driven 300 kV Titan Krios microscope equipped with cold field emission gun (c-FEG) to generate highly regular pulsed electron beams for specimens under cryogenic conditions. We compared radiation damage in three representative samples: paraffin 2D crystals, bacteriorhodopsin (purple membrane) 2D crystals, and plunge-frozen tobacco mosaic virus (TMV) in vitreous ice, under both pulsed and conventional (= random) illumination, while keeping all other imaging conditions constant. Radiation damage was quantified by tracking the decay of computed diffraction intensities to determine the critical dose (Ne). We observed no statistically significant difference in critical dose between pulsed and random illumination across all 3 samples. Our findings provide a critical reference point for future development and evaluation of temporally modulated electron sources in cryo-EM instrumentation.

molecular biology↗

bioRxiv2024

CryoVesNet: A Dedicated Framework for Synaptic Vesicle Segmentation in Cryo Electron Tomograms

Cryo-electron Tomography (Cryo-ET) has the potential to reveal cell structure down to atomic resolution. Nevertheless, cellular cryo-ET data is often highly complex, and visualization, as well as quantification, of subcellular structures require image segmentation. Due to a relatively high level of noise and anisotropic resolution in cryo-ET data, automatic segmentation based on classical computer vision approaches usually does not perform satisfactorily. For this reason, cryo-ET researchers have mostly performed manual segmentation. Communication between neurons relies on neurotransmitter-filled synaptic vesicle (SV) exocytosis. Recruitment of SVs to the plasma membrane is an important means of regulating exocytosis and is influenced by interactions between SVs. Cryo-ET study of the spatial organization of SVs and of their interconnections allows a better understanding of the mechanisms of exocytosis regulation. Extremely accurate SV segmentation is a prerequisite to obtaining a faithful representation of SVs state of connectivity. Hundreds to thousands of SVs are present in a typical synapse, and their time-consuming manual segmentation is a bottleneck in this analysis. Several attempts to automate vesicle segmentation by classical computer vision or machine learning algorithms have not yielded robust results. We addressed this problem by designing a workflow consisting of a U-Net convolutional segmentation network followed by post-processing steps. This combination yields highly accurate results. Furthermore, we provide an interactive tool for accurately segmenting spherical vesicles in a fraction of the time required by available manual segmentation methods. This tool can be used to segment vesicles that were missed by the fully automatic procedure or to quickly segment a handful of vesicles while bypassing the fully automatic procedure. Our pipeline can in principle be used to segment any spherical vesicle in any cell type as well as extracellular vesicles.

neuroscience↗

bioRxiv2023

Intrinsically disordered CsoS2 acts as a general molecular thread for α-carboxysome shell assembly

Carboxysomes are a paradigm of self-assembling proteinaceous organelles found in nature, offering compartmentalisation of enzymes and pathways to enhance carbon fixation. In -carboxysomes, the disordered linker protein CsoS2 plays an essential role in carboxysome assembly and Rubisco encapsulation. Its mechanism of action, however, is not fully understood. Here we synthetically engineered -carboxysome shells using minimal shell components and determined cryoEM structures of these to decipher the principle of shell assembly and encapsulation. The structures reveal that the intrinsically disordered CsoS2 C-terminus is well-structured and acts as a universal "molecular thread" stitching through multiple shell protein interfaces. We further uncovered in CsoS2 a remarkable highly conserved repetitive key interaction motif, [IV]TG, which is critical to the shell assembly and architecture. Our study provides a general mechanism for the CsoS2-govern carboxysome shell assembly and cargo encapsulation and further advances synthetic engineering of carboxysomes for diverse biotechnological applications.

microbiology↗

bioRxiv2023

ChAdOx1 COVID vaccines express RBD open prefusion SARS-CoV-2 spikes on the cell surface

Vaccines against SARS-CoV-2 have been proven to be an effective means of decreasing COVID-19 mortality, hospitalization rates, and transmission. One of the vaccines deployed worldwide is ChAdOx1 nCoV-19, which uses an adenovirus vector to drive the expression of the original SARS-CoV-2 spike on the surface of transduced cells. Using cryo-electron tomography and subtomogram averaging, we determined the native structures of the vaccine product expressed on cell surfaces in situ. We show that ChAdOx1-vectored vaccines expressing the Beta SARS-CoV-2 variant produce abundant native prefusion spikes predominantly in one-RBD-up conformation. Furthermore, the ChAdOx1 vectored HexaPro stabilized spike yields higher cell surface expression, enhanced RBD exposure, and reduced shedding of S1 compared to the wild-type. We demonstrate in situ structure determination as a powerful means for studying antigen design options in future vaccine development against emerging novel SARS-CoV-2 variants and broadly against other infectious viruses.

microbiology↗

bioRxiv2023

Cryo-electron tomography sheds light on the elastic nature of the Trypanosoma brucei tripartite attachment complex

Trypanosomes only contain a single mitochondrion per cell. Within that singular mitochondrion, the protist carries a single mitochondrial genome that consists of a complex DNA network, the kinetoplast DNA (kDNA). The replicated kDNA is segregated during cell division by the tripartite attachment complex (TAC), a multi-protein bridge that physically links each daughter kDNA to a basal body (BB). BB movements drive kDNA segregation prior to cell division. How the TAC accommodates constant BB movements while maintaining a stable kDNA anchor throughout the cell cycle has remained unclear. Here we used cryo-electron tomography to image the cytoplasmic part of the TAC in its native context. We resolved the BB, the mitochondrial membranes, and the exclusion zone filaments (EZFs) connecting the BB and pro-BB to the outer mitochondrial membrane (OMM) and quantified the geometry of the region across many cells. EZF lengths spanned 230 to 625 nm in zoid cells and up to 874 nm in NP40-treated cells, and the BB occupied a wide range of positions and orientations relative to the OMM, while the pro-BB sat closer and more constrained. Building on these observations and prior evidence that p197 alone defines the BB-OMM distance, we propose that p197 is a length-variable connector: the length of a tandem array of -helical repeats depends on their relative orientation, and filaments of different lengths coexist at one basal body. How that orientation is set remains open, but such a connector reconciles stable kDNA anchoring with the mechanical demands of BB movement during the cell cycle.

cell biology↗

bioRxiv2022

Morphofunctional changes at the active zone during synaptic vesicle exocytosis

The fusion of synaptic vesicles (SVs) with the plasma membrane (PM) proceeds through intermediate steps that remain poorly resolved. Additionally, the effect of persistent high or low exocytosis activity on intermediate steps remains unknown. Through time-resolved cryo-electron tomography, we ordered events into a sequence. Following stimulation, proximal tethered SVs rapidly form additional tethers with the PM. Simultaneously, fusion initiation occurs by membrane curvature ( buckling) of the SV and PM. It is followed by the formation of a fusion pore, and the collapse of SV membrane. At this time, membrane-proximal, but not membrane-distal, vesicles lose their interconnections, allowing them to move towards the PM. Two mutations of SNAP-25 that arrests or disinhibit spontaneous release, respectively, both caused a loss of interconnectors, while the disinhibiting mutant also caused a loss of membrane proximal multiple-tethered SVs. Overall, tether formation and connector dissolution is triggered by stimulation and respond to the spontaneous fusion rate. These morphological observations likely correspond to the transition of SVs from one functional pool to another.

neuroscience↗

bioRxiv2020

SARS-CoV-2 Assembly and Egress Pathway Revealed by Correlative Multi-modal Multi-scale Cryo-imaging

Since the outbreak of the SARS-CoV-2 pandemic, there have been intense structural studies on purified recombinant viral components and inactivated viruses. However, investigation of the SARS-CoV-2 infection in the native cellular context is scarce, and there is a lack of comprehensive knowledge on SARS-CoV-2 replicative cycle. Understanding the genome replication, assembly and egress of SARS-CoV-2, a multistage process that involves different cellular compartments and the activity of many viral and cellular proteins, is critically important as it bears the means of medical intervention to stop infection. Here, we investigated SARS-CoV-2 replication in Vero cells under the near-native frozen-hydrated condition using a unique correlative multi-modal, multi-scale cryo-imaging approach combining soft X-ray cryo-tomography and serial cryoFIB/SEM volume imaging of the entire SARS-CoV-2 infected cell with cryo-electron tomography (cryoET) of cellular lamellae and cell periphery, as well as structure determination of viral components by subtomogram averaging. Our results reveal at the whole cell level profound cytopathic effects of SARS-CoV-2 infection, exemplified by a large amount of heterogeneous vesicles in the cytoplasm for RNA synthesis and virus assembly, formation of membrane tunnels through which viruses exit, and drastic cytoplasm invasion into nucleus. Furthermore, cryoET of cell lamellae reveals how viral RNAs are transported from double-membrane vesicles where they are synthesized to viral assembly sites; how viral spikes and RNPs assist in virus assembly and budding; and how fully assembled virus particles exit the cell, thus stablishing a model of SARS-CoV-2 genome replication, virus assembly and egress pathways.

microbiology↗

bioRxiv2020

The SARS-CoV-2 Spike harbours a lipid binding pocket which modulates stability of the prefusion trimer

Large trimeric Spikes decorate SARS-CoV-2 and bind host cells via receptor binding domains (RBDs). We report a conformation in which the trimer is locked into a compact well-ordered form. This differs from previous structures where the RBD can flip up to recognise the receptor. In the locked form regions associated with fusion transitions are stabilised and the RBD harbours curved lipids. The acyl chains bind a hydrophobic pocket in one RBD whilst the polar headgroups attach to an adjacent RBD of the trimer. By functional analogy with enteroviral pocket factors loss of the lipid would destabilise the locked form facilitating receptor attachment, conversion to the postfusion state and virus infection. The nature of lipids available at the site of infection might affect the antigenicity/pathogenicity of released virus. These results reveal a potentially druggable pocket and suggest that the natural prefusion state occludes neutralising RBD epitopes, achieving conformational shielding from antibodies. HighlightsO_LISARS-CoV-2 Spike can adopt a locked conformation with all receptor binding domains (RBDs) down, likely to represent the prefusion resting state C_LIO_LIThis locked conformation is compact and stable, braced by lipid bound within a potentially druggable pocket C_LIO_LIKey neutralization epitopes are shielded in the locked form C_LIO_LILoss of lipid may trigger a cascade of events that lead to cell entry analogous to the role of lipids in enterovirus cell entry C_LI

microbiology↗

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↗

bioRxiv2020

Neutralization of SARS-CoV-2 by destruction of the prefusion Spike

There are as yet no licenced therapeutics for the COVID-19 pandemic. The causal coronavirus (SARS-CoV-2) binds host cells via a trimeric Spike whose receptor binding domain (RBD) recognizes angiotensin-converting enzyme 2 (ACE2), initiating conformational changes that drive membrane fusion. We find that monoclonal antibody CR3022 binds the RBD tightly, neutralising SARS-CoV-2 and report the crystal structure at 2.4 [A] of the Fab/RBD complex. Some crystals are suitable for screening for entry-blocking inhibitors. The highly conserved, structure-stabilising, CR3022 epitope is inaccessible in the prefusion Spike, suggesting that CR3022 binding would facilitate conversion to the fusion-incompetent post-fusion state. Cryo-EM analysis confirms that incubation of Spike with CR3022 Fab leads to destruction of the prefusion trimer. Presentation of this cryptic epitope in an RBD-based vaccine might advantageously focus immune responses. Binders at this epitope may be useful therapeutically, possibly in synergy with an antibody blocking receptor attachment. HighlightsO_LICR3022 neutralises SARS-CoV-2 C_LIO_LINeutralisation is by destroying the prefusion SPIKE conformation C_LIO_LIThis antibody may have therapeutic potential alone or with one blocking receptor attachment C_LI

microbiology↗

bioRxiv2017

High contrast staining for serial block face scanning electron microscopy without uranyl acetate

1Serial block face scanning electron microscopy (SBFSEM) is an increasingly popular method for investigating the three-dimensional ultrastructure of large biological samples. Prior to imaging, samples are typically chemically fixed, stained with osmium and uranyl acetate, and subsequently embedded in resin. The purpose of staining is to provide image contrast and reduce specimen charging under the electron beam, which is detrimental to the quality of imaging. Obtaining, using, and disposing of uranyl acetate is getting increasingly cumbersome in many countries due to new regulations on the handling of radioactive substances. Therefore, we developed an alternative staining procedure that does not rely on the use of uranium or any other radioactive substance. This procedure provides excellent contrast and efficiently reduces specimen charging.

cell biology↗