Biology subjects

Scheres, S.

Publications and source records attributed to Scheres, S..

At least 19 recordsLinked to original sources

bioRxiv2026

Templated seeding induces the formation of Alzheimer paired helical tau filaments in human brain organoids

The prion concept postulates that seeded aggregation results in the recruitment of soluble protein monomers and their assembly into amyloid filaments with the same structures as those of the seeds. Here we show that this concept applies to tau filaments formed in cortical organoids obtained from human iPSCs of control subjects. Tau seeds were extracted from the frontal cortex of individuals with Alzheimer disease. When added to human cortical organoids expressing endogenous levels of three-repeat tau, they gave rise to filaments of three-repeat tau whose formation required endogenous tau expression. By cryo-EM, these seeded tau filaments were identical to the paired helical filaments of the seeds. This novel finding demonstrates the faithful templated seeding of tau in human nerve cells expressing endogenous levels of tau and opens the way to studying the mechanisms of paired helical filament formation and their downstream effects in a human system amenable to genetic modification.

neuroscience↗

bioRxiv2026

Dimerisation and twist reversal of the Lewy fold in α-synuclein mutants with Parkinson's disease and dementia

Dominantly inherited missense and gene dosage mutations in SNCA, the -synuclein gene, cause familial forms of Parkinsons disease and dementia. Here we report the structures of -synuclein filaments from the brains of such individuals. Pathogenic mutations A53T and G51D in SNCA give rise to singlets and doublets of the Lewy fold with a left-handed helical twist in the absence of a peptide-like density for island A. By contrast, filaments from the non-pathogenic variant H50Q consist of singlets of the right-handed Lewy fold with a density for island A, like filaments of wild-type -synuclein. The structures of filaments from homozygous mice transgenic for human mutant A53T -synuclein (line M83) are unlike those from human brains. They are more similar to the multiple system atrophy folds than to the Lewy fold of Parkinsons disease, Parkinsons disease dementia and dementia with Lewy bodies

biophysics↗

bioRxiv2026

Prion-like transmission of human tau strains in the mouse brain

Most neurodegenerative diseases are believed to spread through the brain by prion-like mechanisms, where filamentous protein assemblies self-propagate by templated seeding 1. Distinct conformations of amyloid filaments are thought to provide the physical basis for the strains that lead to different diseases 2. However, a central pillar of the prion hypothesis, that strains retain their structural identity upon transmission, has not been demonstrated. Here we show that the injection of tau filaments from the brains of individuals with Alzheimers disease or corticobasal degeneration into the brains of wildtype mice leads to the seeded assembly of amyloid filaments made of mouse tau with the same structures as those of the seeds. Thereby, we establish that, like prion strains, tau filaments propagate through templated seeding, and that the mouse is a suitable model to study the molecular mechanisms by which distinct tau folds drive disease-specific pathology in the brain.

neuroscience↗

bioRxiv2026

Cryo-EM image processing of amyloid filaments in RELION-5.1

We present new tools for the structure determination of amyloid filaments from electron cryo-microscopy (cryo-EM) images. We introduce a new algorithm for automated filament picking, based on their characteristic 4.75 [A] repeat signal; we implement the new auto-picker in a fully automated procedure for on-the-fly pre-processing of cryo-EM data sets of amyloid filaments; we present a graphical tool to select filament types based on bi-hierarchical clustering of filaments and 2D class assignments; and we introduce a denoising neural network for Blush regularisation that is re-trained on amyloid reconstructions. The implementation of these tools in release 5.1 of our open-software package RELION ensures broad applicability. We demonstrate their usefulness on two experimental data sets, including a previously described data set on recombinant human islet amyloid protein (hIAPP) with the S20G mutation for which we identify two new filament types.

biophysics↗

bioRxiv2026

The amyloid packing difference: a pairwise comparison metric for amyloid structures

Several proteins from the human proteome have been observed to adopt multiple distinct amyloid filaments, and specific protofilament folds are associated with different diseases. Thereby, it has become necessary to compare pairs of amyloid structures of a given protein. This paper describes the amyloid packing difference (APD), which quantifies the difference between such a pair as the percentage of residues that are involved in unique cross-{beta} packing interactions or that have different side chain orientations relative to the {beta}-strands. Clustering of -synuclein protofilament folds on pairwise APD values recapitulates previously reported clustering based on structural superpositions. Any pair of known protofilament folds of the prion protein, tau, -synuclein, TDP-43 or TAF15 from different neurodegenerative diseases have APD values above 20%, whereas all pairs of structures that have been associated with the same disease have APD values below 40%. Structures of antibody light chain from different individuals with systemic amyloidosis differ from each by APDs above 60%, whereas structures of transthyretin filaments from different individuals are strikingly similar, with APDs below 25%. These observations provide context for the interpretation of APD values in future structure comparisons.

molecular biology↗

bioRxiv2025

Serial amplification of tau filaments using Alzheimer's brain homogenates and C322A or C322S recombinant tau

The assembly of tau into amyloid filaments is a defining characteristic of Alzheimers disease (AD) and other tauopathies. Cryo-electron microscopy (cryo-EM) showed that specific tau folds characterise different diseases, and that in vitro models often yield filaments with folds that do not replicate those that form in disease. Here, we investigated the aggregation of full-length recombinant 0N3R tau, using wild-type, or mutations C322A or C322S and a real-time quaking-induced conversion (RT-QuIC) assay with brain homogenate seeding. The assembly of C322A 0N3R tau resulted in filaments with a structure resembling the AD fold in paired helical filaments (PHFs), but with a more open C-shaped core attributed to the C322A mutation. C322S 0N3R tau formed structurally more distinct filaments with respect to PHFs, with an ordered carboxy-terminal region. Both mutant filaments retained the ability to seed a second round of aggregation. Meanwhile, wild-type 0N3R tau exhibited poor reproducibility and formed predominantly unfolded aggregates. Our findings emphasise the need for optimised assembly conditions to obtain disease-relevant filament folds. Refining these methodologies could enhance our understanding of the molecular origins of tauopathies and facilitate the development of targeted therapeutic strategies for these conditions.

neuroscience↗

bioRxiv2024

An image processing pipeline for electron cryo-tomography in RELION-5

Electron tomography of frozen, hydrated samples allows structure determination of macromolecular complexes that are embedded in complex environments. Provided that the target complexes may be localised in noisy, three-dimensional tomographic reconstructions, averaging images of multiple instances of these molecules can lead to structures with sufficient resolution for de novo atomic modelling. Although many research groups have contributed image processing tools for these tasks, a lack of standardisation and inter-operability represents a barrier for newcomers to the field. Here, we present an image processing pipeline for electron tomography data in RELION-5, with functionality ranging from the import of unprocessed movies to the automated building of atomic models in the final maps. Our explicit definition of metadata items that describe the steps of our pipeline has been designed for inter-operability with other software tools and provides a framework for further standardisation.

biophysics↗

bioRxiv2023

DynaMight: estimating molecular motions with improved reconstruction from cryo-EM images

How to deal with continuously flexing molecules is one of the biggest outstanding challenges in single-particle analysis of proteins from cryo-EM images. Here, we present DynaMight, a new software tool that estimates a continuous space of conformations in a cryo-EM data set by learning 3D deformations of a Gaussian pseudo-atomic model of a consensus structure for every particle image. Inversion of the learnt deformations is then used to obtain an improved reconstruction of the consensus structure. We illustrate the performance of DynaMight for several experimental cryo-EM data sets. We also show how error estimates on the deformations may be obtained by independently training two variational autoencoders (VAEs) on half sets of the cryo-EM data, and how regularisation of the 3D deformations through the use of atomic models may lead to important artefacts due to model bias. DynaMight is distributed as free, open-source software, as part of RELION-5.

biophysics↗

bioRxiv2023

Cryo-EM structures of Tau filaments from the brains of mice transgenic for human mutant P301S Tau

Mice transgenic for human mutant P301S tau are widely used as models for human tauopathies. They develop neurodegeneration and abundant filamentous inclusions made of human mutant four-repeat tau. Here we used electron cryo-microscopy (cryo-EM) to determine the structures of tau filaments from the brains of Tg2541 and PS19 mice. Both lines express human P301S tau (0N4R for Tg2541 and 1N4R for PS19) on mixed genetic backgrounds and downstream of different promoters (murine Thy1 for Tg2541 and murine Prnp for PS19). The structures of tau filaments from Tg2541 and PS19 mice differ from each other and those of tau filaments from human brains. However, they share a substructure at the junction of repeats 2 and 3, which comprises residues I297-V312 of tau and includes the P301S mutation. The filament core from the brainstem of Tg2541 mice consists of residues K274-H329 of tau and two disconnected protein densities. Two non-proteinaceous densities are also in evidence. The filament core from the cerebral cortex of line PS19 extends from residues G271-P364 of tau. One strong non-proteinaceous density is also present. Unlike the tau filaments from human brains, the sequences following repeat 4 are missing from the cores of tau filaments from the brains of Tg2541 and PS19 mice.

neuroscience↗

bioRxiv2023

Automated model building and protein identification in cryo-EM maps

Interpreting electron cryo-microscopy (cryo-EM) maps with atomic models requires high levels of expertise and labour-intensive manual intervention. We present ModelAngelo, a machine-learning approach for automated atomic model building in cryo-EM maps. By combining information from the cryo-EM map with information from protein sequence and structure in a single graph neural network, ModelAngelo builds atomic models for proteins that are of similar quality as those generated by human experts. For nucleotides, ModelAngelo builds backbones with similar accuracy as humans. By using its predicted amino acid probabilities for each residue in hidden Markov model sequence searches, ModelAngelo outperforms human experts in the identification of proteins with unknown sequences. ModelAngelo will thus remove bottlenecks and increase objectivity in cryo-EM structure determination.

molecular biology↗

bioRxiv2023

Cryo-EM structures of tau filaments from SH-SY5Y cells seeded with brain extracts from cases of Alzheimer's disease and corticobasal degeneration

The formation of amyloid filaments through templated seeding is believed to underlie the propagation of pathology in most human neurodegenerative diseases. A widely used model system to study this process is to seed amyloid filament formation in cultured cells using human brain extracts. Here, we report the electron cryo-microscopy (cryo-EM) structures of tau filaments from undifferentiated seeded SH-SY5Y cells, that transiently expressed N-terminally HA-tagged 1N3R or 1N4R human tau, using brain extracts from individuals with AD or CBD. Although the resulting filament structures differed from those of the brain seeds, some degree of structural templating was observed. Studying templated seeding in cultured cells, and determining the structures of the resulting filaments, can thus provide insights into the cellular aspects underlying neurodegenerative diseases.

neuroscience↗

bioRxiv2023

Identical tau filaments in subacute sclerosing panencephalitis and chronic traumatic encephalopathy

Subacute sclerosing panencephalitis (SSPE) occurs in some individuals after measles infection, following a symptom-free period of several years. It resembles chronic traumatic encephalopathy (CTE), which happens after repetitive head impacts or exposure to blast waves, following a symptom-free period. As in CTE, when present, the neurofibrillary changes of SSPE are concentrated in superficial cortical layers. Here we used electron cryo-microscopy of tau filaments from two cases of SSPE to show that the tau folds of SSPE and CTE are identical. Two types of filaments were each made of two identical protofilaments with an extra density in the {beta}-helix region. Like in CTE, the vast majority of tau filaments were Type I, with a minority of Type II filaments. These findings suggest that the CTE tau fold can be caused by different environmental insults, which may be linked by inflammatory changes.

neuroscience↗

bioRxiv2022

Cryo-EM structures of chronic traumatic encephalopathy tau filaments with PET ligand flortaucipir

Positron emission tomography (PET) imaging allows monitoring the progression of amyloid aggregation in the living brain. [18F]-Flortaucipir is the only approved PET tracer compound for the visualisation of tau aggregation. Here, we describe cryo-EM experiments on tau filaments in the presence and absence of flortaucipir. We used tau filaments isolated from the brain of an individual with Alzheimers disease (AD), and from the brain of an individual with primary age-related tauopathy (PART) with a co-pathology of chronic traumatic encephalopathy (CTE). Unexpectedly, we were unable to visualise additional cryo-EM density for flortaucipir for AD paired helical or straight filaments (PHFs or SFs), but we did observe density for flortaucipir binding to CTE Type I filaments from the case with PART. In the latter, flortaucipir binds in a 1:1 molecular stoichiometry with tau, adjacent to lysine 353 and aspartate 358. By adopting a tilted geometry with respect to the helical axis, the 4.7 [A] distance between neighbouring tau monomers is reconciled with the 3.5 [A] distance consistent with {pi}-{pi}-stacking between neighbouring molecules of flortaucipir.

neuroscience↗

bioRxiv2022

Cryo-EM structures of α-synuclein filaments from Parkinson's disease and dementia with Lewy bodies

Parkinsons disease (PD) is the most common movement disorder, with resting tremor, rigidity, bradykinesia and postural instability being major symptoms (1). Neuropathologically, it is characterised by the presence of abundant filamentous inclusions of -synuclein in the form of Lewy bodies and Lewy neurites in some brain cells, including dopaminergic nerve cells of the substantia nigra (2). PD is increasingly recognised as a multisystem disorder, with cognitive decline being one of its most common non-motor symptoms. Many patients with PD develop dementia more than 10 years after diagnosis (3). PD dementia (PDD) is clinically and neuropathologically similar to dementia with Lewy bodies (DLB), which is diagnosed when cognitive impairment precedes parkinsonian motor signs or begins within one year from their onset (4). In PDD, cognitive impairment develops in the setting of well-established PD. Besides PD and DLB, multiple system atrophy (MSA) is the third major synucleinopathy (5). It is characterised by the presence of abundant filamentous -synuclein inclusions in brain cells, especially oligodendrocytes (Papp-Lantos bodies). We previously reported the electron cryo-microscopy (cryo-EM) structures of two types of -synuclein filaments extracted from the brains of individuals with MSA (6). Each filament type is made of two different protofilaments. Here we report that the cryo-EM structures of -synuclein filaments from the brains of individuals with PD, PDD and DLB are made of a single protofilament (Lewy fold) that is markedly different from the protofilaments of MSA. These findings establish the existence of distinct molecular conformers of assembled -synuclein in neurodegenerative disease.

neuroscience↗

bioRxiv2022

A Bayesian approach to single-particle electron cryo-tomography in RELION-4.0

We present a new approach for macromolecular structure determination from multiple particles in electron cryo-tomography (cryo-ET) data sets. Whereas existing subtomogram averaging approaches are based on 3D data models, we propose to optimise a regularised likelihood target that approximates a function of the 2D experimental images. In addition, analogous to Bayesian polishing and contrast transfer function (CTF) refinement in single-particle analysis, we describe approaches that exploit the increased signal-to-noise ratio in the averaged structure to optimise tilt series alignments, beam-induced motions of the particles throughout the tilt series acquisition, defoci of the individual particles, as well as higher-order optical aberrations of the microscope. Implementation of our approaches in the open-source software package RELION aims to facilitate their general use, in particular for those researchers who are already familiar with its single-particle analysis tools. We illustrate for three applications that our approaches allow structure determination from cryo-ET data to resolutions sufficient for de novo atomic modelling.

biophysics↗

bioRxiv2022

High-throughput cryo-EM structure determination of amyloids

The formation of amyloid filaments is characteristic of various degenerative diseases. Recent breakthroughs in electron cryo-microscopy (cryo-EM) have led to atomic structure determination of multiple amyloid filaments, both of filaments assembled in vitro from recombinant proteins, and of filaments extracted from diseased tissue. These observations revealed that a single protein may adopt multiple different amyloid folds, and that in vitro assembly does not necessarily lead to the same filaments as those observed in disease. In order to develop relevant model systems for disease, and ultimately to better understand the molecular mechanisms of disease, it will be important to determine which factors determine the formation of distinct amyloid folds. High-throughput cryo-EM, in which structure determination becomes a tool rather than a project in itself, will facilitate the screening of large numbers of in vitro assembly conditions. To this end, we describe a new filament picking algorithm based on the Topaz approach, and we outline image processing strategies in Relion that enable atomic structure determination of amyloids within days.

biophysics↗

bioRxiv2021

Assembly of recombinant tau into filaments identical to those of Alzheimer's disease and chronic traumatic encephalopathy

Abundant filamentous inclusions of tau are characteristic of more than 20 neurodegenerative diseases that are collectively termed tauopathies. Electron cryo-microscopy (cryo-EM) structures of tau amyloid filaments from human brain revealed that distinct tau folds characterise many different diseases. A lack of laboratory-based model systems to generate these structures has hampered efforts to uncover the molecular mechanisms that underlie tauopathies. Here, we report in vitro assembly conditions with recombinant tau that replicate the structures of filaments from both Alzheimers disease (AD) and chronic traumatic encephalopathy (CTE), as determined by cryo-EM. Our results suggest that post-translational modifications of tau modulate filament assembly, and that previously observed additional densities in AD and CTE filaments may arise from the presence of inorganic salts, like phosphates and sodium chloride. In vitro assembly of tau into disease-relevant filaments will facilitate studies to determine their roles in different diseases, as well as the development of compounds that specifically bind to these structures or prevent their formation.

neuroscience↗

bioRxiv2021

Age-dependent formation of TMEM106B amyloid filaments in human brain

Many age-dependent neurodegenerative diseases, like Alzheimers and Parkinsons, are characterised by abundant inclusions of amyloid filaments. Filamentous inclusions of the proteins tau, amyloid-{beta} (A{beta}), -synuclein and TDP-43 are the most common. Here, we used electron cryo-microscopy (cryo-EM) structure determination to show that residues 120-254 of the lysosomal type II transmembrane protein 106B (TMEM106B) also form amyloid filaments in the human brain. We solved cryo-EM structures of TMEM106B filaments from the brains of 22 individuals with neurodegenerative conditions, including sporadic and inherited tauopathies, A{beta}-amyloidoses, synucleinopathies and TDP-43opathies, as well as from the brains of two neurologically normal individuals. We observed three different TMEM106B folds, with no clear relationship between folds and diseases. The presence of TMEM106B filaments correlated with that of a 29 kDa sarkosyl-insoluble fragment of the protein on Western blots. The presence of TMEM106B filaments in the brains of older, but not younger, neurologically normal individuals indicates that they form in an age-dependent manner.

neuroscience↗