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 cryogenic-electron microscopy (cryo-EM) images. Here, we present DynaMight, a software tool that estimates a continuous space of conformations in a cryo-EM dataset by learning three-dimensional deformations of a Gaussian pseudo-atomic model of a consensus structure for every particle image. Inversion of the learned deformations is then used to obtain an improved reconstruction of the consensus structure. We illustrate the performance of DynaMight for several experimental cryo-EM datasets. We also show how error estimates on the deformations may be obtained by independently training two variational autoencoders on half sets of the cryo-EM data, and how regularization of the three-dimensional deformations through the use of atomic models may lead to important artifacts due to model bias. DynaMight is distributed as free, open-source software, as part of RELION-5.

Novel tau filament folds in individuals with MAPT mutations P301L and P301T.

Mutations in MAPT, the microtubule-associated protein tau gene, give rise to cases of frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) with abundant filamentous tau inclusions in brain cells. Individuals with pathological MAPT variants exhibit behavioural changes, cognitive impairment and signs of parkinsonism. Missense mutations of residue P301, which are the most common MAPT mutations associated with FTDP-17, give rise to the assembly of mutant four-repeat tau into filamentous inclusions, in the absence of extracellular deposits. Here we report the cryo-EM structures of tau filaments from five individuals belonging to three unrelated families with mutation P301L and from one individual belonging to a family with mutation P301T. A novel three-lobed tau fold resembling the two-layered tau fold of Pick's disease was present in all cases with the P301L tau mutation. Two different tau folds were found in the case with mutation P301T, the less abundant of which was a variant of the three-lobed fold. The major P301T tau fold was V-shaped, with partial similarity to the four-layered tau folds of corticobasal degeneration and argyrophilic grain disease. These findings suggest that FTDP-17 with mutations in P301 should be considered distinct inherited tauopathies and that model systems with these mutations should be used with caution in the study of sporadic tauopathies.

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 interoperability 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 interoperability with other software tools and provides a framework for further standardisation.

Data-driven regularization lowers the size barrier of cryo-EM structure determination.

Macromolecular structure determination by electron cryo-microscopy (cryo-EM) is limited by the alignment of noisy images of individual particles. Because smaller particles have weaker signals, alignment errors impose size limitations on its applicability. Here, we explore how image alignment is improved by the application of deep learning to exploit prior knowledge about biological macromolecular structures that would otherwise be difficult to express mathematically. We train a denoising convolutional neural network on pairs of half-set reconstructions from the electron microscopy data bank (EMDB) and use this denoiser as an alternative to a commonly used smoothness prior. We demonstrate that this approach, which we call Blush regularization, yields better reconstructions than do existing algorithms, in particular for data with low signal-to-noise ratios. The reconstruction of a protein-nucleic acid complex with a molecular weight of 40 kDa, which was previously intractable, illustrates that denoising neural networks will expand the applicability of cryo-EM structure determination for a wide range of biological macromolecules.

Cleaved TMEM106B forms amyloid aggregates in central and peripheral nervous systems.

Filaments made of residues 120-254 of transmembrane protein 106B (TMEM106B) form in an age-dependent manner and can be extracted from the brains of neurologically normal individuals and those of subjects with a variety of neurodegenerative diseases. TMEM106B filament formation requires cleavage at residue 120 of the 274 amino acid protein; at present, it is not known if residues 255-274 form the fuzzy coat of TMEM106B filaments. Here we show that a second cleavage appears likely, based on staining with an antibody raised against residues 263-274 of TMEM106B. We also show that besides the brain TMEM106B inclusions form in dorsal root ganglia and spinal cord, where they were mostly found in non-neuronal cells. We confirm that in the brain, inclusions were most abundant in astrocytes. No inclusions were detected in heart, liver, spleen or hilar lymph nodes. Based on their staining with luminescent conjugated oligothiophenes, we confirm that TMEM106B inclusions are amyloids. By in situ immunoelectron microscopy, TMEM106B assemblies were often found in structures resembling endosomes and lysosomes.

Tau filaments with the chronic traumatic encephalopathy fold in a case of vacuolar tauopathy with VCP mutation D395G.

Dominantly inherited mutation D395G in the gene encoding valosin-containing protein causes vacuolar tauopathy, a type of behavioural-variant frontotemporal dementia, with marked vacuolation and abundant filamentous tau inclusions made of all six brain isoforms. Here we report that tau inclusions were concentrated in layers II/III of the frontotemporal cortex in a case of vacuolar tauopathy. By electron cryomicroscopy, tau filaments had the chronic traumatic encephalopathy (CTE) fold. Tau inclusions of vacuolar tauopathy share this cortical location and the tau fold with CTE, subacute sclerosing panencephalitis and amyotrophic lateral sclerosis/parkinsonism-dementia complex, which are believed to be environmentally induced. Vacuolar tauopathy is the first inherited disease with the CTE tau fold.

Tau filaments with the Alzheimer fold in cases with MAPT mutations V337M and R406W.

Frontotemporal dementia (FTD) and Alzheimer's disease are the most common forms of early-onset dementia. Dominantly inherited mutations in MAPT, the microtubule-associated protein tau gene, cause FTD and parkinsonism linked to chromosome 17 (FTDP-17). Individuals with FTDP-17 develop abundant filamentous tau inclusions in brain cells. Here we used electron cryo-microscopy to determine the structures of tau filaments from the brains of individuals with MAPT mutations V337M and R406W. Both mutations gave rise to tau filaments with the Alzheimer fold, which consisted of paired helical filaments in all V337M and R406W cases and of straight filaments in two V337M cases. We also identified a new assembly of the Alzheimer fold into triple tau filaments in a V337M case. Filaments assembled from recombinant tau(297-391) with mutation V337M had the Alzheimer fold and showed an increased rate of assembly.

Community recommendations on cryoEM data archiving and validation.

In January 2020, a workshop was held at EMBL-EBI (Hinxton, UK) to discuss data requirements for the deposition and validation of cryoEM structures, with a focus on single-particle analysis. The meeting was attended by 47 experts in data processing, model building and refinement, validation, and archiving of such structures. This report describes the workshop's motivation and history, the topics discussed, and the resulting consensus recommendations. Some challenges for future methods-development efforts in this area are also highlighted, as is the implementation to date of some of the recommendations.

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 in three-dimensional computer graphics programs1,2. Here 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 to those generated by human experts. For nucleotides, ModelAngelo builds backbones with similar accuracy to those built by 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 therefore remove bottlenecks and increase objectivity in cryo-EM structure determination.

Tau and neurodegeneration.

First identified in 1975, tau was implicated in Alzheimer's disease 10 years later. Filamentous tangle inclusions were known to be made of hyperphosphorylated tau by 1991, with similar inclusions gaining recognition for being associated with other neurodegenerative diseases. In 1998, mutations in MAPT, the gene that encodes tau, were identified as the cause of a dominantly inherited form of frontotemporal dementia with abundant filamentous tau inclusions. While this result indicated that assembly of tau into aberrant filaments is sufficient to drive neurodegeneration and dementia, most cases of tauopathy are sporadic. More recent work in experimental systems showed that filamentous assemblies of tau may first form in one brain area, and then spread to others in a prion-like fashion. Beginning in 2017, work on human brains using high-resolution techniques has led to a structure-based classification of tauopathies, which has opened the door to a better understanding of the significance of tau filament formation.

Community recommendations on cryoEM data archiving and validation: Outcomes of a wwPDB/EMDB workshop on cryoEM data management, deposition and validation.

In January 2020, a workshop was held at EMBL-EBI (Hinxton, UK) to discuss data requirements for deposition and validation of cryoEM structures, with a focus on single-particle analysis. The meeting was attended by 47 experts in data processing, model building and refinement, validation, and archiving of such structures. This report describes the workshop's motivation and history, the topics discussed, and consensus recommendations resulting from the workshop. Some challenges for future methods-development efforts in this area are also highlighted, as is the implementation to date of some of the recommendations.

Disease-specific tau filaments assemble via polymorphic intermediates.

Intermediate species in the assembly of amyloid filaments are believed to play a central role in neurodegenerative diseases and may constitute important targets for therapeutic intervention1,2. However, structural information about intermediate species has been scarce and the molecular mechanisms by which amyloids assemble remain largely unknown. Here we use time-resolved cryogenic electron microscopy to study the in vitro assembly of recombinant truncated tau (amino acid residues 297-391) into paired helical filaments of Alzheimer's disease or into filaments of chronic traumatic encephalopathy3. We report the formation of a shared first intermediate amyloid filament, with an ordered core comprising residues 302-316. Nuclear magnetic resonance indicates that the same residues adopt rigid, ß-strand-like conformations in monomeric tau. At later time points, the first intermediate amyloid disappears and we observe many different intermediate amyloid filaments, with structures that depend on the reaction conditions. At the end of both assembly reactions, most intermediate amyloids disappear and filaments with the same ordered cores as those from human brains remain. Our results provide structural insights into the processes of primary and secondary nucleation of amyloid assembly, with implications for the design of new therapies.

Cryo-EM structures of Aß40 filaments from the leptomeninges of individuals with Alzheimer's disease and cerebral amyloid angiopathy.

We used electron cryo-microscopy (cryo-EM) to determine the structures of Aß40 filaments from the leptomeninges of individuals with Alzheimer's disease and cerebral amyloid angiopathy. In agreement with previously reported structures, which were solved to a resolution of 4.4 Å, we found three types of filaments. However, our new structures, solved to a resolution of 2.4 Å, revealed differences in the sequence assignment that redefine the fold of Aß40 peptides and their interactions. Filaments are made of pairs of protofilaments, the ordered core of which comprises D1-G38. The different filament types comprise one, two or three protofilament pairs. In each pair, residues H14-G37 of both protofilaments adopt an extended conformation and pack against each other in an anti-parallel fashion, held together by hydrophobic interactions and hydrogen bonds between main chains and side chains. Residues D1-H13 fold back on the adjacent parts of their own chains through both polar and non-polar interactions. There are also several additional densities of unknown identity. Sarkosyl extraction and aqueous extraction gave the same structures. By cryo-EM, parenchymal deposits of Aß42 and blood vessel deposits of Aß40 have distinct structures, supporting the view that Alzheimer's disease and cerebral amyloid angiopathy are different Aß proteinopathies.

Tau filaments from amyotrophic lateral sclerosis/parkinsonism-dementia complex adopt the CTE fold.

The amyotrophic lateral sclerosis/parkinsonism-dementia complex (ALS/PDC) of the island of Guam and the Kii peninsula of Japan is a fatal neurodegenerative disease of unknown cause that is characterized by the presence of abundant filamentous tau inclusions in brains and spinal cords. Here, we used electron cryo-microscopy to determine the structures of tau filaments from the cerebral cortex of three cases of ALS/PDC from Guam and eight cases from Kii, as well as from the spinal cord of two of the Guam cases. Tau filaments had the chronic traumatic encephalopathy (CTE) fold, with variable amounts of Type I and Type II filaments. Paired helical tau filaments were also found in three Kii cases and tau filaments with the corticobasal degeneration fold in one Kii case. We identified a new Type III CTE tau filament, where protofilaments pack against each other in an antiparallel fashion. ALS/PDC is the third known tauopathy with CTE-type filaments and abundant tau inclusions in cortical layers II/III, the others being CTE and subacute sclerosing panencephalitis. Because these tauopathies are believed to have environmental causes, our findings support the hypothesis that ALS/PDC is caused by exogenous factors.

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 wild-type tau filaments from human brains. The structures of tau filaments from the brains of humans with mutations P301L, P301S or P301T in MAPT are not known. Filaments from the brains of Tg2541 and PS19 mice 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.

Molecular pathology of neurodegenerative diseases by cryo-EM of amyloids.

Abnormal assembly of tau, α-synuclein, TDP-43 and amyloid-ß proteins into amyloid filaments defines most human neurodegenerative diseases. Genetics provides a direct link between filament formation and the causes of disease. Developments in cryo-electron microscopy (cryo-EM) have made it possible to determine the atomic structures of amyloids from postmortem human brains. Here we review the structures of brain-derived amyloid filaments that have been determined so far and discuss their impact on research into neurodegeneration. Whereas a given protein can adopt many different filament structures, specific amyloid folds define distinct diseases. Amyloid structures thus provide a description of neuropathology at the atomic level and a basis for studying disease. Future research should focus on model systems that replicate the structures observed in disease to better understand the molecular mechanisms of disease and develop improved diagnostics and therapies.

Cryo-EM structure of the complete inner kinetochore of the budding yeast point centromere.

The point centromere of budding yeast specifies assembly of the large kinetochore complex to mediate chromatid segregation. Kinetochores comprise the centromere-associated inner kinetochore (CCAN) complex and the microtubule-binding outer kinetochore KNL1-MIS12-NDC80 (KMN) network. The budding yeast inner kinetochore also contains the DNA binding centromere-binding factor 1 (CBF1) and CBF3 complexes. We determined the cryo-electron microscopy structure of the yeast inner kinetochore assembled onto the centromere-specific centromere protein A nucleosomes (CENP-ANuc). This revealed a central CENP-ANuc with extensively unwrapped DNA ends. These free DNA duplexes bind two CCAN protomers, one of which entraps DNA topologically, positioned on the centromere DNA element I (CDEI) motif by CBF1. The two CCAN protomers are linked through CBF3 forming an arch-like configuration. With a structural mechanism for how CENP-ANuc can also be linked to KMN involving only CENP-QU, we present a model for inner kinetochore assembly onto a point centromere and how it organizes the outer kinetochore for chromosome attachment to the mitotic spindle.