Ligand identification in CryoEM and X-ray maps using deep learning.

Motivation: Accurately identifying ligands plays a crucial role in the process of structure-guided drug design. Based on density maps from X-ray diffraction or cryogenic-sample electron microscopy (cryoEM), scientists verify whether small-molecule ligands bind to active sites of interest. However, the interpretation of density maps is challenging, and cognitive bias can sometimes mislead investigators into modeling fictitious compounds.

Ligand Identification in CryoEM and X-ray Maps Using Deep Learning.

Motivation: Accurately identifying ligands plays a crucial role in the process of structure-guided drug design. Based on density maps from X-ray diffraction or cryogenic-sample electron microscopy (cryoEM), scientists verify whether small-molecule ligands bind to active sites of interest. However, the interpretation of density maps is challenging, and cognitive bias can sometimes mislead investigators into modeling fictitious compounds.

Structure and topography of the synaptic V-ATPase-synaptophysin complex.

Synaptic vesicles are organelles with a precisely defined protein and lipid composition, yet the molecular mechanisms for the biogenesis of synaptic vesicles are mainly unknown. Here we discovered a well-defined interface between the synaptic vesicle V-ATPase and synaptophysin by in situ cryo-electron tomography and single-particle cryo-electron microscopy of functional synaptic vesicles isolated from mouse brains. The synaptic vesicle V-ATPase is an ATP-dependent proton pump that establishes the proton gradient across the synaptic vesicle, which in turn drives the uptake of neurotransmitters.

The SkillsCenter: Creating scalable research opportunities for STEM students.

Undergraduate students generally need laboratory skills and experience to be accepted into a position within an academic lab or a company. However, those settings are traditionally where students would develop that necessary expertise. We developed a laboratory course paradigm to equip students with the skills they need to access future opportunities.

Post-Translational Modifications Control Phase Transitions of Tau.

The self-assembly of Tau(297-391) into filaments, which mirror the structures observed in Alzheimer's disease (AD) brains, raises questions about the role of AD-specific post-translational modifications (PTMs) in the formation of paired helical filaments (PHFs). To investigate this, we developed a synthetic approach to produce Tau(291-391) featuring N-acetyllysine, phosphoserine, phosphotyrosine, and N-glycosylation at positions commonly modified in post-mortem AD brains, thus facilitating the study of their roles in Tau pathology. Using transmission electron microscopy (TEM), cryo-electron microscopy (cryo-EM), and a range of optical microscopy techniques, we discovered that these modifications generally hinder the assembly of Tau into PHFs.

A simple flash and freeze system for cryogenic time-resolved electron microscopy.

As the resolution revolution in CryoEM expands to encompass all manner of macromolecular complexes, an important new frontier is the implementation of cryogenic time resolved EM (cryoTREM). Biological macromolecular complexes are dynamic systems that undergo conformational changes on timescales from microseconds to minutes. Understanding the dynamic nature of biological changes is critical to understanding function.

Progress and gaps of extracellular vesicle-mediated intercellular cargo transfer in the central nervous system.

A fundamentally novel function proposed for extracellular vesicles (EVs) is to transfer bioactive molecules in intercellular signaling. In this minireview, we discuss recent progress on EV-mediated cargo transfer in the central nervous system (CNS) and major gaps in previous studies. We also suggest a set of experiments necessary for bridging the gaps and establishing the physiological roles of EV-mediated cargo transfer.

Electron Tomographic Methods for Studying Organelles of the Murine Chemical Synapse.

Electron tomography of the chemical synapse provides important architectural information regarding the organization of synaptic organelles including synaptic vesicles, Nissl bodies, and early endosomes. Here, we describe methods for the preparation of select murine brain regions for high-pressure freezing, freeze substitution, and EM tomographic analysis of synaptic structures. The method uses fresh brain slices prepared using a vibratome and biopsy punches to collect specific brain regions of interest suitable for subsequent preservation and EM tomographic imaging.

Multiomic Analysis Reveals Disruption of Cholesterol Homeostasis by Cannabidiol in Human Cell Lines.

The nonpsychoactive cannabinoid, cannabidiol (CBD), is Food and Dug Administration approved for treatment of two drug-resistant epileptic disorders and is seeing increased use among the general public, yet the mechanisms that underlie its therapeutic effects and side-effect profiles remain unclear. Here, we report a systems-level analysis of CBD action in human cell lines using temporal multiomic profiling. FRET-based biosensor screening revealed that CBD elicits a sharp rise in cytosolic calcium, and activation of AMP-activated protein kinase in human keratinocyte and neuroblastoma cell lines.

Synergistic activation of the insulin receptor via two distinct sites.

Insulin receptor (IR) signaling controls multiple facets of animal physiology. Maximally four insulins bind to IR at two distinct sites, termed site-1 and site-2. However, the precise functional roles of each binding event during IR activation remain unresolved.

Structural mechanism of muscle nicotinic receptor desensitization and block by curare.

Binding of the neurotransmitter acetylcholine to its receptors on muscle fibers depolarizes the membrane and thereby triggers muscle contraction. We sought to understand at the level of three-dimensional structure how agonists and antagonists alter nicotinic acetylcholine receptor conformation. We used the muscle-type receptor from the Torpedo ray to first define the structure of the receptor in a resting, activatable state.

Homotypic fibrillization of TMEM106B across diverse neurodegenerative diseases.

Misfolding and aggregation of disease-specific proteins, resulting in the formation of filamentous cellular inclusions, is a hallmark of neurodegenerative disease with characteristic filament structures, or conformers, defining each proteinopathy. Here we show that a previously unsolved amyloid fibril composed of a 135 amino acid C-terminal fragment of TMEM106B is a common finding in distinct human neurodegenerative diseases, including cases characterized by abnormal aggregation of TDP-43, tau, or α-synuclein protein. A combination of cryoelectron microscopy and mass spectrometry was used to solve the structures of TMEM106B fibrils at a resolution of 2.

Room for Two: The Synaptophysin/Synaptobrevin Complex.

Synaptic vesicle release is regulated by upwards of 30 proteins at the fusion complex alone, but disruptions in any one of these components can have devastating consequences for neuronal communication. Aberrant molecular responses to calcium signaling at the pre-synaptic terminal dramatically affect vesicle trafficking, docking, fusion, and release. At the organismal level, this is reflected in disorders such as epilepsy, depression, and neurodegeneration.

Purification of a native nicotinic receptor.

Nicotinic acetylcholine receptors are members of the Cys-loop superfamily of pentameric ligand-gated ion channels. The electric organ of the Torpedo ray is extraordinarily rich in an acetylcholine receptor that is homologous to the human nicotinic receptor found at the neuromuscular junction. Due to this abundant natural source in the fish and the relatively accessible preparation of the neuromuscular junction (compared to a central synapse), this muscle-type receptor and specifically the fish receptors have long been used as the prototype for study of nicotinic receptors.

SNARE Zippering Is Suppressed by a Conformational Constraint that Is Removed by v-SNARE Splitting.

Intracellular vesicle fusion is catalyzed by soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). Vesicle-anchored v-SNAREs pair with target membrane-associated t-SNAREs to form trans-SNARE complexes, releasing free energy to drive membrane fusion. However, trans-SNARE complexes are unable to assemble efficiently unless activated by Sec1/Munc18 (SM) proteins.

Pulse-Chase Proteomics of the App Knockin Mouse Models of Alzheimer's Disease Reveals that Synaptic Dysfunction Originates in Presynaptic Terminals.

Compromised protein homeostasis underlies accumulation of plaques and tangles in Alzheimer's disease (AD). To observe protein turnover at early stages of amyloid beta (Aβ) proteotoxicity, we performed pulse-chase proteomics on mouse brains in three genetic models of AD that knock in alleles of amyloid precursor protein (APP) prior to the accumulation of plaques and during disease progression. At initial stages of Aβ accumulation, the turnover of proteins associated with presynaptic terminals is selectively impaired.

On-Chip Acousto Thermal Shift Assay for Rapid and Sensitive Assessment of Protein Thermodynamic Stability.

Thermal shift assays (TSAs) have been extensively used to study thermodynamics of proteins and provide an efficient means to assess protein-ligand binding or protein-protein interactions. However, existing TSAs have limitations, such as being time consuming, labor intensive, or having low sensitivity. Herein, an acousto thermal shift assay (ATSA), the first ultrasound enabled TSA, is reported for real-time analysis of protein thermodynamic stability.

Structure of the Native Muscle-type Nicotinic Receptor and Inhibition by Snake Venom Toxins.

The nicotinic acetylcholine receptor, a pentameric ligand-gated ion channel, converts the free energy of binding of the neurotransmitter acetylcholine into opening of its central pore. Here we present the first high-resolution structure of the receptor type found in muscle-endplate membrane and in the muscle-derived electric tissues of fish. The native receptor was purified from Torpedo electric tissue and functionally reconstituted in lipids optimal for cryo-electron microscopy.

An isothermal shift assay for proteome scale drug-target identification.

Most small molecule drugs act on living systems by physically interacting with specific proteins and modulating target function. Identification of drug binding targets, within the complex milieu of the human proteome, remains a challenging task of paramount importance in drug discovery. Existing approaches for target identification employ complex workflows with limited throughput.

Intracellular Vesicle Fusion Requires a Membrane-Destabilizing Peptide Located at the Juxtamembrane Region of the v-SNARE.

Intracellular vesicle fusion is mediated by soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs) and Sec1/Munc18 (SM) proteins. It is generally accepted that membrane fusion occurs when the vesicle and target membranes are brought into close proximity by SNAREs and SM proteins. In this work, we demonstrate that, for fusion to occur, membrane bilayers must be destabilized by a conserved membrane-embedded motif located at the juxtamembrane region of the vesicle-anchored v-SNARE.

Flattening of Diluted Species Profile via Passive Geometry in a Microfluidic Device.

In recent years, microfluidic devices have become an important tool for use in lab-on-a-chip processes, including drug screening and delivery, bio-chemical reactions, sample preparation and analysis, chemotaxis, and separations. In many such processes, a flat cross-sectional concentration profile with uniform flow velocity across the channel is desired to achieve controlled and precise solute transport. This is often accommodated by the use of electroosmotic flow, however, it is not an ideal for many applications, particularly biomicrofluidics.

Synaptotagmin 17 controls neurite outgrowth and synaptic physiology via distinct cellular pathways.

The synaptotagmin (syt) proteins have been widely studied for their role in regulating fusion of intracellular vesicles with the plasma membrane. Here we report that syt-17, an unusual isoform of unknown function, plays no role in exocytosis, and instead plays multiple roles in intracellular membrane trafficking. Syt-17 is localized to the Golgi complex in hippocampal neurons, where it coordinates import of vesicles from the endoplasmic reticulum to support neurite outgrowth and facilitate axon regrowth after injury.

Cryo-EM structure of OSCA1.2 from elucidates the mechanical basis of potential membrane hyperosmolality gating.

Sensing and responding to environmental water deficiency and osmotic stresses are essential for the growth, development, and survival of plants. Recently, an osmolality-sensing ion channel called OSCA1 was discovered that functions in sensing hyperosmolality in Here, we report the cryo-electron microscopy (cryo-EM) structure and function of an OSCA1 homolog from rice (; OsOSCA1.2), leading to a model of how it could mediate hyperosmolality sensing and transport pathway gating.