A realistic phantom dataset for benchmarking cryo-ET data annotation.

Cryo-electron tomography (cryo-ET) is a powerful technique for imaging molecular complexes in their native cellular environments. However, identifying the vast majority of molecular species in cellular tomograms remains prohibitively difficult. Machine learning (ML) methods provide an opportunity to automate the annotation process, but algorithm development has been hindered by the lack of large, standardized datasets.

Real-space heterogeneous reconstruction, refinement, and disentanglement of CryoEM conformational states with HetSIREN.

Single-particle analysis by Cryo-electron microscopy (CryoEM) provides direct access to the conformations of macromolecules. Traditional methods assume discrete conformations, while newer algorithms estimate conformational landscapes representing the different structural states a biomolecule explores. This work presents HetSIREN, a deep learning-based method that can fully reconstruct or refine a CryoEM volume in real space based on the structural information summarized in a conformational latent space.

Deconvolution to restore cryo-EM maps with anisotropic resolution.

With technological advancements in recent years, single particle cryogenic electron microscopy (cryo-EM) has become a major methodology for structural biology. Structure determination by single particle cryo-EM is premised on randomly orientated particles embedded in thin layer of vitreous ice to resolve high-resolution structural information in all directions. Otherwise, preferentially distributed particle orientations will lead to anisotropic resolution of the structure.

The structure of basal body inner junctions from Tetrahymena revealed by electron cryo-tomography.

The cilium is a microtubule-based eukaryotic organelle critical for many cellular functions. Its assembly initiates at a basal body and continues as an axoneme that projects out of the cell to form a functional cilium. This assembly process is tightly regulated.

A novel lncRNA FAM151B-DT regulates autophagy and degradation of aggregation prone proteins.

Neurodegenerative diseases share common features of protein aggregation along with other pleiotropic traits, including shifts in transcriptional patterns, neuroinflammation, disruptions in synaptic signaling, mitochondrial dysfunction, oxidative stress, and impaired clearance mechanisms like autophagy. However, key regulators of these pleotropic traits have yet to be identified. Here, we discovered a novel long non-coding RNA (lncRNA), , that is reduced in a stem cell model of frontotemporal dementia with tau inclusions (FTLD-tau) and in brains from FTLD-tau, progressive supranuclear palsy, Alzheimer's disease, and Parkinson's disease patients.

The Structure of Cilium Inner Junctions Revealed by Electron Cryo-tomography.

The cilium is a microtubule-based organelle critical for many cellular functions. Its assembly initiates at a basal body and continues as an axoneme that projects out of the cell to form a functional cilium. This assembly process is tightly regulated.

Poc1 bridges basal body inner junctions to promote triplet microtubule integrity and connections.

Basal bodies (BBs) are conserved eukaryotic structures that organize cilia. They are comprised of nine, cylindrically arranged, triplet microtubules (TMTs) connected to each other by inter-TMT linkages which stabilize the structure. Poc1 is a conserved protein important for BB structural integrity in the face of ciliary forces transmitted to BBs.

Functionalized graphene-oxide grids enable high-resolution cryo-EM structures of the SNF2h-nucleosome complex without crosslinking.

Single-particle cryo-EM is widely used to determine enzyme-nucleosome complex structures. However, cryo-EM sample preparation remains challenging and inconsistent due to complex denaturation at the air-water interface (AWI). Here, to address this issue, we develop graphene-oxide-coated EM grids functionalized with either single-stranded DNA (ssDNA) or thiol-poly(acrylic acid-co-styrene) (TAASTY) co-polymer.

Poc1 is a basal body inner junction protein that promotes triplet microtubule integrity and interconnections.

Basal bodies (BBs) are conserved eukaryotic structures that organize motile and primary cilia. The BB is comprised of nine, cylindrically arranged, triplet microtubules (TMTs) that are connected to each other by inter-TMT linkages which maintain BB structure. During ciliary beating, forces transmitted to the BB must be resisted to prevent BB disassembly.

Cryo-EM reveals how Hsp90 and FKBP immunophilins co-regulate the glucocorticoid receptor.

Hsp90 is an essential molecular chaperone responsible for the folding and activation of hundreds of 'client' proteins, including the glucocorticoid receptor (GR). Previously, we revealed that Hsp70 and Hsp90 remodel the conformation of GR to regulate ligand binding, aided by co-chaperones. In vivo, the co-chaperones FKBP51 and FKBP52 antagonistically regulate GR activity, but a molecular understanding is lacking.

De novo protein identification in mammalian sperm using in situ cryoelectron tomography and AlphaFold2 docking.

To understand the molecular mechanisms of cellular pathways, contemporary workflows typically require multiple techniques to identify proteins, track their localization, and determine their structures in vitro. Here, we combined cellular cryoelectron tomography (cryo-ET) and AlphaFold2 modeling to address these questions and understand how mammalian sperm are built in situ. Our cellular cryo-ET and subtomogram averaging provided 6.

Functionalized graphene-oxide grids enable high-resolution cryo-EM structures of the SNF2h-nucleosome complex without crosslinking.

Single-particle cryo-EM is widely used to determine enzyme-nucleosome complex structures. However, cryo-EM sample preparation remains challenging and inconsistent due to complex denaturation at the air-water interface (AWI). To address this issue, we developed graphene-oxide-coated EM grids functionalized with either single-stranded DNA (ssDNA) or thiol-poly(acrylic acid-co-styrene) (TAASTY) co-polymer.

Hsp90 provides a platform for kinase dephosphorylation by PP5.

The Hsp90 molecular chaperone collaborates with the phosphorylated Cdc37 cochaperone for the folding and activation of its many client kinases. As with many kinases, the Hsp90 client kinase CRaf is activated by phosphorylation at specific regulatory sites. The cochaperone phosphatase PP5 dephosphorylates CRaf and Cdc37 in an Hsp90-dependent manner.

The ϕPA3 phage nucleus is enclosed by a self-assembling 2D crystalline lattice.

To protect themselves from host attack, numerous jumbo bacteriophages establish a phage nucleus-a micron-scale, proteinaceous structure encompassing the replicating phage DNA. Bacteriophage and host proteins associated with replication and transcription are concentrated inside the phage nucleus while other phage and host proteins are excluded, including CRISPR-Cas and restriction endonuclease host defense systems. Here, we show that nucleus fragments isolated from ϕPA3 infected Pseudomonas aeruginosa form a 2-dimensional lattice, having p2 or p4 symmetry.

Cryo-EM reveals how Hsp90 and FKBP immunophilins co-regulate the Glucocorticoid Receptor.

Hsp90 is an essential molecular chaperone responsible for the folding and activation of hundreds of 'client' proteins, including the glucocorticoid receptor (GR). Previously, we revealed that GR ligand binding activity is inhibited by Hsp70 and restored by Hsp90, aided by co-chaperones. We then presented cryo-EM structures mechanistically detailing how Hsp70 and Hsp90 remodel the conformation of GR to regulate ligand binding.

Solution of the protein structure prediction problem at last: crucial innovations and next frontiers.

The protein structure prediction problem is solved, at last, thanks in large part to the use of artificial intelligence. The structures predicted by AlphaFold and RoseTTAFold are becoming the requisite starting point for many protein scientists. New frontiers, such as the conformational sampling of intrinsically disordered proteins, are emerging.

In situ cryo-electron tomography reveals the asymmetric architecture of mammalian sperm axonemes.

The flagella of mammalian sperm display non-planar, asymmetric beating, in contrast to the planar, symmetric beating of flagella from sea urchin sperm and unicellular organisms. The molecular basis of this difference is unclear. Here, we perform in situ cryo-electron tomography of mouse and human sperm, providing the highest-resolution structural information to date.

AreTomo: An integrated software package for automated marker-free, motion-corrected cryo-electron tomographic alignment and reconstruction.

AreTomo, an abbreviation for Alignment and Reconstruction for Electron Tomography, is a GPU accelerated software package that fully automates motion-corrected marker-free tomographic alignment and reconstruction in a single package. By correcting in-plane rotation, translation, and importantly, the local motion resulting from beam-induced motion from tilt to tilt, AreTomo can produce tomograms with sufficient accuracy to be directly used for subtomogram averaging. Another major application is the on-the-fly reconstruction of tomograms in parallel with tilt series collection to provide users with real-time feedback of sample quality allowing users to make any necessary adjustments of collection parameters.

Electron cryo-tomography structure of axonemal doublet microtubule from .

Doublet microtubules (DMTs) provide a scaffold for axoneme assembly in motile cilia. Aside from α/β tubulins, the DMT comprises a large number of non-tubulin proteins in the luminal wall of DMTs, collectively named the microtubule inner proteins (MIPs). We used cryoET to study axoneme DMT isolated from We present the structures of DMT at nanometer and sub-nanometer resolution.

Structure of Hsp90-Hsp70-Hop-GR reveals the Hsp90 client-loading mechanism.

Maintaining a healthy proteome is fundamental for the survival of all organisms. Integral to this are Hsp90 and Hsp70, molecular chaperones that together facilitate the folding, remodelling and maturation of the many 'client proteins' of Hsp90. The glucocorticoid receptor (GR) is a model client protein that is strictly dependent on Hsp90 and Hsp70 for activity.

Structure of Hsp90-p23-GR reveals the Hsp90 client-remodelling mechanism.

Hsp90 is a conserved and essential molecular chaperone responsible for the folding and activation of hundreds of 'client' proteins. The glucocorticoid receptor (GR) is a model client that constantly depends on Hsp90 for activity. GR ligand binding was previously shown to nr inhibited by Hsp70 and restored by Hsp90, aided by the co-chaperone p23.