Mouse α-synuclein fibrils are structurally and functionally distinct from human fibrils associated with Lewy body diseases The intricate process of α-synuclein aggregation and fibrillization holds pivotal roles in Parkinson’s disease (PD) and multiple system atrophy (MSA). While mouse α-synuclein can fibrillize in vitro, whether these fibrils commonly used in research to induce this process or form can reproduce structures…
MiLoPYP: self-supervised molecular pattern mining and particle localization in situ Cryo-electron tomography (CET) allows the routine visualization of cellular landscapes in three dimensions at nanometer-range resolutions. When combined with single-particle tomography (SPT), it is possible to obtain near-atomic resolution structures of frequently occurring macromolecules within their native environment. Two outstanding challenges associated with CET/SPT are the automatic identification and…
Structures of trehalose-6-phosphate synthase, Tps1, from the fungal pathogen Cryptococcus neoformans: A target for antifungals Invasive fungal diseases are a major threat to human health, resulting in more than 1.5 million annual deaths worldwide. The arsenal of antifungal therapeutics remains limited and is in dire need of drugs that target additional biosynthetic pathways that are absent from humans. One…
Advances in cryo-ET data processing: meeting the demands of visual proteomics Cryogenic electron tomography (cryo-ET), a method that enables the viewing of biomolecules in near-native environments at high resolution, is rising in accessibility and applicability. Over the past several years, once slow sample preparation and data collection procedures have seen innovations which enable rapid collection of the large datasets…
Accurate size-based protein localization from cryo-ET tomograms Cryo-electron tomography (cryo-ET) combined with sub-tomogram averaging (STA) allows the determination of protein structures imaged within the native context of the cell at near-atomic resolution. Particle picking is an essential step in the cryo-ET/STA image analysis pipeline that consists in locating the position of proteins within crowded cellular tomograms so that they…
Joint micrograph denoising and protein localization in cryo-electron microscopy Cryo-electron microscopy (cryo-EM) is an imaging technique that allows the visualization of proteins and macromolecular complexes at near-atomic resolution. The low electron doses used to prevent radiation damage to the biological samples result in images where the power of noise is 100 times stronger than that of the signal. Accurate…
nextPYP: a comprehensive and scalable platform for characterizing protein variability in situ using single-particle cryo-ET Single-particle cryo-electron tomography (SP-CET) is an emerging technique capable of determining the structure of proteins imaged within the native context of cells at molecular resolution. While high-throughput techniques for sample preparation and tilt-series acquisition are beginning to provide sufficient data to allow structural studies…
Multiple-image super-resolution of cryo-electron micrographs based on deep internal learning Single-particle cryo-electron microscopy (cryo-EM) is a powerful imaging modality capable of visualizing proteins and macromolecular complexes at near-atomic resolution. The low electron-doses used to prevent radiation damage to the biological samples, however, result in images where the power of the noise is 100 times greater than the power of…
Molecular architecture and conservation of an immature human endogenous retrovirus The human endogenous retrovirus K (HERV-K) is the most recently acquired endogenous retrovirus in the human genome and is activated and expressed in many cancers and amyotrophic lateral sclerosis. We present the immature HERV-K capsid structure at 3.2 Å resolution determined from native virus-like particles using cryo-electron tomography and subtomogram…
Structural basis for breadth development in the HIV-1 V3-glycan targeting DH270 antibody clonal lineage Antibody affinity maturation enables adaptive immune responses to a wide range of pathogens. In some individuals broadly neutralizing antibodies develop to recognize rapidly mutating pathogens with extensive sequence diversity. Vaccine design for pathogens such as HIV-1 and influenza has therefore focused on recapitulating the natural…
Structure and dynamics of the Arabidopsis O-fucosyltransferase SPINDLY SPINDLY (SPY) in Arabidopsis thaliana is a novel nucleocytoplasmic protein O-fucosyltransferase (POFUT), which regulates diverse developmental processes. Sequence analysis indicates that SPY is distinct from ER-localized POFUTs and contains N-terminal tetratricopeptide repeats (TPRs) and a C-terminal catalytic domain resembling the O-linked-N-acetylglucosamine (GlcNAc) transferases (OGTs). However, the structural feature that determines the…
Accurate Detection of Proteins in Cryo-Electron Tomograms from Sparse Labels Cryo-electron tomography (CET) combined with sub-volume averaging (SVA), is currently the only imaging technique capable of determining the structure of proteins imaged inside cells at molecular resolution. To obtain high-resolution reconstructions, sub-volumes containing randomly distributed copies of the protein of interest need be identified, extracted and subjected to SVA,…
High-resolution structure determination using high-throughput electron cryo-tomography The low throughput characteristic of tomographic data acquisition combined with the complex data-analysis pipeline that is required to obtain high-resolution maps, has limited the applicability of this technique to favorable samples or to resolutions that are too low to provide useful mechanistic information. Recently, beam image-shift electron cryo-tomography (BISECT), a strategy to…
Redox-sensitive E2 Rad6 controls cellular response to oxidative stress via K63-linked ubiquitination of ribosomes In this study, we set out to investigate the key role of Rad6 in regulating cellular response to stress in budding yeast as part of the RTU. Rad6 is small (20 kDa), highly conserved, and a multifunctional E2 involved in DNA repair and in the…
Data-driven determination of number of discrete conformations in single-particle cryo-EM Single-particle cryo-EM can be used to image heterogeneous samples containing multiple molecular species, different oligomeric states or distinct conformations. This, however, requires expert-user knowledge and trial-and-error experimentation to determine the correct number of conformations present in a mixture. Here, we propose an approach to address the problem of automatically…
Structural basis of NPR1 in activating plant immunity We report cryo-electron microscopy and crystal structures of Arabidopsis NPR1 and its complex with the transcription factor TGA3. Cryo-electron microscopy analysis reveals that NPR1 is a bird-shaped homodimer comprising a central Broad-complex, Tramtrack and Bric-à-brac (BTB) domain, a BTB and carboxyterminal Kelch helix bundle, four ankyrin repeats and a disordered salicylic-acid-binding…
