MiLoPYP: self-supervised molecular pattern mining and particle localization in situ.

Cryo-electron tomography allows the routine visualization of cellular landscapes in three dimensions at nanometer-range resolutions. When combined with single-particle tomography, it is possible to obtain near-atomic resolution structures of frequently occurring macromolecules within their native environment. Two outstanding challenges associated with cryo-electron tomography/single-particle tomography are the automatic identification and localization of proteins, tasks that are hindered by the molecular crowding inside cells, imaging distortions characteristic of cryo-electron tomography tomograms and the sheer size of tomographic datasets.

Structures of trehalose-6-phosphate synthase, Tps1, from the fungal pathogen : 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.

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 can be aligned and averaged in 3D to improve resolution. While extensive work in 2D particle picking has been done in the context of single-particle cryo-EM, comparatively fewer strategies have been proposed to pick particles from 3D tomograms, in part due to the challenges associated with working with noisy 3D volumes affected by the missing wedge.

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 required for attaining high resolution structures. Increased data availability has provided a driving force for exciting improvements in cryo-ET data processing methodologies throughout the entire processing pipeline and the development of accessible graphical user interfaces (GUIs) that enable individuals inexperienced in computational fields to convert raw tilt series into 3D structures.

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 identification of proteins from these low signal-to-noise ratio (SNR) images is a critical task, as the detected positions serve as inputs for the downstream 3D structure determination process.

nextPYP: a comprehensive and scalable platform for characterizing protein variability in situ using single-particle cryo-electron tomography.

Single-particle cryo-electron tomography 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 of proteins at physiological concentrations, the complex data analysis pipeline and the demanding storage and computational requirements pose major barriers for the development and broader adoption of this technology. Here, we present a scalable, end-to-end framework for single-particle cryo-electron tomography data analysis from on-the-fly pre-processing of tilt series to high-resolution refinement and classification, which allows efficient analysis and visualization of datasets with hundreds of tilt series and hundreds of thousands of particles.

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 averaging.

Molecular architecture and conservation of an immature human endogenous retrovirus.

A significant part of the human genome consists of endogenous retroviruses sequences. Human endogenous retrovirus K (HERV-K) is the most recently acquired endogenous retrovirus, is activated and expressed in many cancers and amyotrophic lateral sclerosis and possibly contributes to the aging process. To understand the molecular architecture of endogenous retroviruses, we determined the structure of immature HERV-K from native virus-like particles (VLPs) using cryo-electron tomography and subtomogram averaging (cryoET STA).

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 affinity maturation process.

Structures of trehalose-6-phosphate synthase, Tps1, from the fungal pathogen : a target for novel antifungals.

Unlabelled: 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 novel drugs that target additional biosynthetic pathways that are absent from humans.

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 the signal. To overcome these low signal-to-noise ratios (SNRs), hundreds of thousands of particle projections are averaged to determine the three-dimensional structure of the molecule of interest.

Automated systematic evaluation of cryo-EM specimens with SmartScope.

Finding the conditions to stabilize a macromolecular target for imaging remains the most critical barrier to determining its structure by cryo-electron microscopy (cryo-EM). While automation has significantly increased the speed of data collection, specimens are still screened manually, a laborious and subjective task that often determines the success of a project. Here, we present SmartScope, the first framework to streamline, standardize, and automate specimen evaluation in cryo-EM.

High-resolution structure determination using high-throughput electron cryo-tomography.

Tomographic reconstruction of frozen-hydrated specimens followed by extraction and averaging of sub-tomograms has successfully been used to determine the structure of macromolecules in their native environment at resolutions that are high enough to reveal molecular level interactions. The low throughput characteristic of tomographic data acquisition combined with the complex data-analysis pipeline that is required to obtain high-resolution maps, however, 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 significantly accelerate the acquisition of tilt series without sacrificing image quality, was introduced.

Redox-sensitive E2 Rad6 controls cellular response to oxidative stress via K63-linked ubiquitination of ribosomes.

Protein ubiquitination is an essential process that rapidly regulates protein synthesis, function, and fate in dynamic environments. Within its non-proteolytic functions, we showed that K63-linked polyubiquitinated conjugates heavily accumulate in yeast cells exposed to oxidative stress, stalling ribosomes at elongation. K63-ubiquitinated conjugates accumulate mostly because of redox inhibition of the deubiquitinating enzyme Ubp2; however, the role and regulation of ubiquitin-conjugating enzymes (E2) in this pathway remained unclear.

Data-driven determination of number of discrete conformations in single-particle cryo-EM.

Background And Objective: One of the strengths of single-particle cryo-EM compared to other structural determination techniques is its ability to image heterogeneous samples containing multiple molecular species, different oligomeric states or distinct conformations. This is achieved using routines for in-silico 3D classification that are now well established in the field and have successfully been used to characterize the structural heterogeneity of important biomolecules. These techniques, however, rely on expert-user knowledge and trial-and-error experimentation to determine the correct number of conformations, making it a labor intensive, subjective, and difficult to reproduce procedure.

Structural basis of NPR1 in activating plant immunity.

NPR1 is a master regulator of the defence transcriptome induced by the plant immune signal salicylic acid. Despite the important role of NPR1 in plant immunity, understanding of its regulatory mechanisms has been hindered by a lack of structural information. Here we report cryo-electron microscopy and crystal structures of Arabidopsis NPR1 and its complex with the transcription factor TGA3.

Fab-dimerized glycan-reactive antibodies are a structural category of natural antibodies.

Natural antibodies (Abs) can target host glycans on the surface of pathogens. We studied the evolution of glycan-reactive B cells of rhesus macaques and humans using glycosylated HIV-1 envelope (Env) as a model antigen. 2G12 is a broadly neutralizing Ab (bnAb) that targets a conserved glycan patch on Env of geographically diverse HIV-1 strains using a unique heavy-chain (V) domain-swapped architecture that results in fragment antigen-binding (Fab) dimerization.

Beam image-shift accelerated data acquisition for near-atomic resolution single-particle cryo-electron tomography.

Tomographic reconstruction of cryopreserved specimens imaged in an electron microscope followed by extraction and averaging of sub-volumes has been successfully used to derive atomic models of macromolecules in their biological environment. Eliminating biochemical isolation steps required by other techniques, this method opens up the cell to in-situ structural studies. However, the need to compensate for errors in targeting introduced during mechanical navigation of the specimen significantly slows down tomographic data collection thus limiting its practical value.

Structural Basis for Virulence Activation of Francisella tularensis.

The bacterium Francisella tularensis (Ft) is one of the most infectious agents known. Ft virulence is controlled by a unique combination of transcription regulators: the MglA-SspA heterodimer, PigR, and the stress signal, ppGpp. MglA-SspA assembles with the σ-associated RNAP holoenzyme (RNAPσ), forming a virulence-specialized polymerase.

Disruption of the HIV-1 Envelope allosteric network blocks CD4-induced rearrangements.

The trimeric HIV-1 Envelope protein (Env) mediates viral-host cell fusion via a network of conformational transitions, with allosteric elements in each protomer orchestrating host receptor-induced exposure of the co-receptor binding site and fusion elements. To understand the molecular details of this allostery, here, we introduce Env mutations aimed to prevent CD4-induced rearrangements in the HIV-1 BG505 Env trimer. Binding analysis and single-molecule Förster Resonance Energy Transfer confirm that these mutations prevent CD4-induced transitions of the HIV-1 Env.

Single-particle cryo-EM structure of a voltage-activated potassium channel in lipid nanodiscs.

Voltage-activated potassium (Kv) channels open to conduct K ions in response to membrane depolarization, and subsequently enter non-conducting states through distinct mechanisms of inactivation. X-ray structures of detergent-solubilized Kv channels appear to have captured an open state even though a non-conducting C-type inactivated state would predominate in membranes in the absence of a transmembrane voltage. However, structures for a voltage-activated ion channel in a lipid bilayer environment have not yet been reported.