CryoDRGN-ET: deep reconstructing generative networks for visualizing dynamic biomolecules inside cells.

Advances in cryo-electron tomography (cryo-ET) have produced new opportunities to visualize the structures of dynamic macromolecules in native cellular environments. While cryo-ET can reveal structures at molecular resolution, image processing algorithms remain a bottleneck in resolving the heterogeneity of biomolecular structures in situ. Here, we introduce cryoDRGN-ET for heterogeneous reconstruction of cryo-ET subtomograms. CryoDRGN-ET learns a deep generative model of three-dimensional density maps directly from subtomogram tilt-series images and can capture states diverse in both composition and conformation. We validate this approach by recovering the known translational states in Mycoplasma pneumoniae ribosomes in situ. We then perform cryo-ET on cryogenic focused ion beam-milled Saccharomyces cerevisiae cells. CryoDRGN-ET reveals the structural landscape of S. cerevisiae ribosomes during translation and captures continuous motions of fatty acid synthase complexes inside cells. This method is openly available in the cryoDRGN software.

Structural and quantum chemical basis for OCP-mediated quenching of phycobilisomes.

Cyanobacteria use large antenna complexes called phycobilisomes (PBSs) for light harvesting. However, intense light triggers non-photochemical quenching, where the orange carotenoid protein (OCP) binds to PBS, dissipating excess energy as heat. The mechanism of efficiently transferring energy from phycocyanobilins in PBS to canthaxanthin in OCP remains insufficiently understood. Using cryo-electron microscopy, we unveiled the OCP-PBS complex structure at 1.6- to 2.1-angstrom resolution, showcasing its inherent flexibility. Using multiscale quantum chemistry, we disclosed the quenching mechanism. Identifying key protein residues, we clarified how canthaxanthin's transition dipole moment in its lowest-energy dark state becomes large enough for efficient energy transfer from phycocyanobilins. Our energy transfer model offers a detailed understanding of the atomic determinants of light harvesting regulation and antenna architecture in cyanobacteria.

Functional and structural insights into activation of TRPV2 by weak acids.

Transient receptor potential (TRP) ion channels are involved in the surveillance or regulation of the acid-base balance. Here, we demonstrate that weak carbonic acids, including acetic acid, lactic acid, and CO2 activate and sensitize TRPV2 through a mechanism requiring permeation through the cell membrane. TRPV2 channels in cell-free inside-out patches maintain weak acid-sensitivity, but protons applied on either side of the membrane do not induce channel activation or sensitization. The involvement of proton modulation sites for weak acid-sensitivity was supported by the identification of titratable extracellular (Glu495, Glu561) and intracellular (His521) residues on a cryo-EM structure of rat TRPV2 (rTRPV2) treated with acetic acid. Molecular dynamics simulations as well as patch clamp experiments on mutant rTRPV2 constructs confirmed that these residues are critical for weak acid-sensitivity. We also demonstrate that the pore residue Glu609 dictates an inhibition of weak acid-induced currents by extracellular calcium. Finally, TRPV2-expression in HEK293 cells is associated with an increased weak acid-induced cytotoxicity. Together, our data provide new insights into weak acids as endogenous modulators of TRPV2.

High-resolution cryo-EM of the human CDK-activating kinase for structure-based drug design.

Rational design of next-generation therapeutics can be facilitated by high-resolution structures of drug targets bound to small-molecule inhibitors. However, application of structure-based methods to macromolecules refractory to crystallization has been hampered by the often-limiting resolution and throughput of cryogenic electron microscopy (cryo-EM). Here, we use high-resolution cryo-EM to determine structures of the CDK-activating kinase, a master regulator of cell growth and division, in its free and nucleotide-bound states and in complex with 15 inhibitors at up to 1.8 Å resolution. Our structures provide detailed insight into inhibitor interactions and networks of water molecules in the active site of cyclin-dependent kinase 7 and provide insights into the mechanisms contributing to inhibitor selectivity, thereby providing the basis for rational design of next-generation therapeutics. These results establish a methodological framework for the use of high-resolution cryo-EM in structure-based drug design.

Cryo-EM structure of SRP68/72 reveals an extended dimerization domain with RNA-binding activity.

The signal recognition particle (SRP) is a critical component in protein sorting pathways in all domains of life. Human SRP contains six proteins bound to the 7S RNA and their structures and functions have been mostly elucidated. The SRP68/72 dimer is the largest SRP component and is essential for SRP function. Although the structures of the SRP68/72 RNA binding and dimerization domains have been previously reported, the structure and function of large portions of the SRP68/72 dimer remain unknown. Here, we analyse full-length SRP68/72 using cryo-EM and report that SRP68/72 depend on each other for stability and form an extended dimerization domain. This newly observed dimerization domain is both a protein- and RNA-binding domain. Comparative analysis with current structural models suggests that this dimerization domain undergoes dramatic translocation upon SRP docking onto SRP receptor and eventually comes close to the Alu domain. We propose that the SRP68/72 dimerization domain functions by binding and detaching the Alu domain and SRP9/14 from the ribosomal surface, thus releasing elongation arrest upon docking onto the ER membrane.

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 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 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 Alzheimer's disease or corticobasal degeneration. Although the resulting filament structures differed from those of the brain seeds, some degrees of structural templating were 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.

New SNCA mutation and structures of α-synuclein filaments from juvenile-onset synucleinopathy.

A 21-nucleotide duplication in one allele of SNCA was identified in a previously described disease with abundant α-synuclein inclusions that we now call juvenile-onset synucleinopathy (JOS). This mutation translates into the insertion of MAAAEKT after residue 22 of α-synuclein, resulting in a protein of 147 amino acids. Both wild-type and mutant proteins were present in sarkosyl-insoluble material that was extracted from frontal cortex of the individual with JOS and examined by electron cryo-microscopy. The structures of JOS filaments, comprising either a single protofilament, or a pair of protofilaments, revealed a new α-synuclein fold that differs from the folds of Lewy body diseases and multiple system atrophy (MSA). The JOS fold consists of a compact core, the sequence of which (residues 36-100 of wild-type α-synuclein) is unaffected by the mutation, and two disconnected density islands (A and B) of mixed sequences. There is a non-proteinaceous cofactor bound between the core and island A. The JOS fold resembles the common substructure of MSA Type I and Type II dimeric filaments, with its core segment approximating the C-terminal body of MSA protofilaments B and its islands mimicking the N-terminal arm of MSA protofilaments A. The partial similarity of JOS and MSA folds extends to the locations of their cofactor-binding sites. In vitro assembly of recombinant wild-type α-synuclein, its insertion mutant and their mixture yielded structures that were distinct from those of JOS filaments. Our findings provide insight into a possible mechanism of JOS fibrillation in which mutant α-synuclein of 147 amino acids forms a nucleus with the JOS fold, around which wild-type and mutant proteins assemble during elongation.

Visualization of translation and protein biogenesis at the ER membrane.

The dynamic ribosome-translocon complex, which resides at the endoplasmic reticulum (ER) membrane, produces a major fraction of the human proteome1,2. It governs the synthesis, translocation, membrane insertion, N-glycosylation, folding and disulfide-bond formation of nascent proteins. Although individual components of this machinery have been studied at high resolution in isolation3-7, insights into their interplay in the native membrane remain limited. Here we use cryo-electron tomography, extensive classification and molecular modelling to capture snapshots of mRNA translation and protein maturation at the ER membrane at molecular resolution. We identify a highly abundant classical pre-translocation intermediate with eukaryotic elongation factor 1a (eEF1a) in an extended conformation, suggesting that eEF1a may remain associated with the ribosome after GTP hydrolysis during proofreading. At the ER membrane, distinct polysomes bind to different ER translocons specialized in the synthesis of proteins with signal peptides or multipass transmembrane proteins with the translocon-associated protein complex (TRAP) present in both. The near-complete atomic model of the most abundant ER translocon variant comprising the protein-conducting channel SEC61, TRAP and the oligosaccharyltransferase complex A (OSTA) reveals specific interactions of TRAP with other translocon components. We observe stoichiometric and sub-stoichiometric cofactors associated with OSTA, which are likely to include protein isomerases. In sum, we visualize ER-bound polysomes with their coordinated downstream machinery.

Cryo-EM at ACA 2022.

The 2022 meeting of the American Crystallographic Association in Portland was an inspiring event, addressing a range of both conventional and emerging themes in structural biology. The increasing emphasis at the conference on methods outside the conventional envelope of crystallography, especially cryo-electron microscopy, is discussed.

Structure of SARS-CoV-2 M protein in lipid nanodiscs.

SARS-CoV-2 encodes four structural proteins incorporated into virions, spike (S), envelope (E), nucleocapsid (N), and membrane (M). M plays an essential role in viral assembly by organizing other structural proteins through physical interactions and directing them to sites of viral budding. As the most abundant protein in the viral envelope and a target of patient antibodies, M is a compelling target for vaccines and therapeutics. Still, the structure of M and molecular basis for its role in virion formation are unknown. Here, we present the cryo-EM structure of SARS-CoV-2 M in lipid nanodiscs to 3.5 Å resolution. M forms a 50 kDa homodimer that is structurally related to the SARS-CoV-2 ORF3a viroporin, suggesting a shared ancestral origin. Structural comparisons reveal how intersubunit gaps create a small, enclosed pocket in M and large open cavity in ORF3a, consistent with a structural role and ion channel activity, respectively. M displays a strikingly electropositive cytosolic surface that may be important for interactions with N, S, and viral RNA. Molecular dynamics simulations show a high degree of structural rigidity in a simple lipid bilayer and support a role for M homodimers in scaffolding viral assembly. Together, these results provide insight into roles for M in coronavirus assembly and structure.

Structures of α-synuclein filaments from human brains with Lewy pathology.

Parkinson's disease (PD) is the most common movement disorder, with resting tremor, rigidity, bradykinesia and postural instability being major symptoms1. Neuropathologically, it is characterized by the presence of abundant filamentous inclusions of α-synuclein in the form of Lewy bodies and Lewy neurites in some brain cells, including dopaminergic nerve cells of the substantia nigra2. 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 diagnosis3. 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 onset4. In PDD, cognitive impairment develops in the setting of well-established PD. Besides PD and DLB, multiple system atrophy (MSA) is the third major synucleinopathy5. It is characterized by the presence of abundant filamentous α-synuclein inclusions in brain cells, especially oligodendrocytes (Papp-Lantos bodies). We previously reported the electron cryo-microscopy structures of two types of α-synuclein filament extracted from the brains of individuals with MSA6. Each filament type is made of two different protofilaments. Here we report that the cryo-electron microscopy structures of α-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 α-synuclein in neurodegenerative disease.

Routine Collection of High-Resolution cryo-EM Datasets Using 200 KV Transmission Electron Microscope.

Cryo-electron microscopy (cryo-EM) has been established as a routine method for protein structure determination during the past decade, taking an ever-increasing share of published structural data. Recent advances in TEM technology and automation have boosted both the speed of data collection and quality of acquired images while simultaneously decreasing the required level of expertise for obtaining cryo-EM maps at sub-3 Å resolutions. While most of such high-resolution structures have been obtained using state-of-the-art 300 kV cryo-TEM systems, high-resolution structures can be also obtained with 200 kV cryo-TEM systems, especially when equipped with an energy filter. Additionally, automation of microscope alignments and data collection with real-time image quality assessment reduces system complexity and assures optimal microscope settings, resulting in increased yield of high-quality images and overall throughput of data collection. This protocol demonstrates the implementation of recent technological advances and automation features on a 200 kV cryo-transmission electron microscope and shows how to collect data for the reconstruction of 3D maps that are sufficient for de novo atomic model building. We focus on best practices, critical variables, and common issues that must be considered to enable the routine collection of such high-resolution cryo-EM datasets. Particularly the following essential topics are reviewed in detail: i) automation of microscope alignments, ii) selection of suitable areas for data acquisition, iii) optimal optical parameters for high-quality, high-throughput data collection, iv) energy filter tuning for zero-loss imaging, and v) data management and quality assessment. Application of the best practices and improvement of achievable resolution using an energy filter will be demonstrated on the example of apo-ferritin that was reconstructed to 1.6 Å, and Thermoplasma acidophilum 20S proteasome reconstructed to 2.1-Å resolution using a 200 kV TEM equipped with an energy filter and a direct electron detector.

Age-dependent formation of TMEM106B amyloid filaments in human brains.

Many age-dependent neurodegenerative diseases, such as Alzheimer's and Parkinson's, are characterized by abundant inclusions of amyloid filaments. Filamentous inclusions of the proteins tau, amyloid-ß, α-synuclein and transactive response DNA-binding protein (TARDBP; also known as TDP-43) are the most common1,2. Here we used structure determination by cryogenic electron microscopy to show that residues 120-254 of the lysosomal type II transmembrane protein 106B (TMEM106B) also form amyloid filaments in human brains. We determined the structures of TMEM106B filaments from a number of brain regions of 22 individuals with abundant amyloid deposits, including those resulting from sporadic and inherited tauopathies, amyloid-ß amyloidoses, synucleinopathies and TDP-43 proteinopathies, as well as from the frontal cortex of 3 individuals with normal neurology and no or only a few amyloid deposits. We observed three TMEM106B folds, with no clear relationships between folds and diseases. TMEM106B filaments correlated with the presence of a 29-kDa sarkosyl-insoluble fragment and globular cytoplasmic inclusions, as detected by an antibody specific to the carboxy-terminal region of TMEM106B. The identification of TMEM106B filaments in the brains of older, but not younger, individuals with normal neurology indicates that they form in an age-dependent manner.