NCOA4 initiates ferritinophagy by binding GATE16 using two highly avid short linear interaction motifs.

Cells carefully regulate cytosolic iron, which is a vital enzymatic cofactor, yet is toxic in excess. In mammalian cells, surplus iron is sequestered in ferritin cages that, in iron limiting conditions, are degraded through the selective autophagy pathway ferritinophagy to liberate free iron. Prior work identified the ferritinophagy receptor protein NCOA4, which links ferritin and LC3/GABARAP-family member GATE16, effectively tethering ferritin to the autophagic machinery. Here, we elucidate the molecular mechanism underlying this interaction, discovering two short linear motifs in NCOA4 that each bind GATE16 with weak affinity. These binding motifs are highly avid and, in concert, support high-affinity NCOA4•GATE16 complex formation. We further find the minimal NCOA4383-522 fragment bearing these motifs is sufficient for ferritinophagy and that both motifs are necessary for this activity. This work suggests a general mechanism wherein selective autophagy receptors can distinguish between the inactive soluble pools of LC3/GABARAPs and the active membrane-conjugated forms that drive autophagy. Finally, we find that iron decreases NCOA4383-522's affinity for GATE16, providing a plausible mechanism for iron-dependent regulation of ferritinophagy.

Rapid structural analysis of bacterial ribosomes in situ.

Rapid structural analysis of purified proteins and their complexes has become increasingly common thanks to key methodological advances in cryo-electron microscopy (cryo-EM) and associated data processing software packages. In contrast, analogous structural analysis in cells via cryo-electron tomography (cryo-ET) remains challenging due to critical technical bottlenecks, including low-throughput sample preparation and imaging, and laborious data processing methods. Here, we describe the development of a rapid in situ cryo-ET sample preparation and data analysis workflow that results in the routine determination of sub-nm resolution ribosomal structures. We apply this workflow to E. coli, producing a 5.8 Å structure of the 70S ribosome from cells in less than 10 days, and we expect this workflow will be widely applicable to related bacterial samples.

Subventricular zone stem cell niche injury is associated with intestinal perforation in preterm infants and predicts future motor impairment.

Brain injury is highly associated with preterm birth. Complications of prematurity, including spontaneous or necrotizing enterocolitis (NEC)-associated intestinal perforations, are linked to lifelong neurologic impairment, yet the mechanisms are poorly understood. Early diagnosis of preterm brain injuries remains a significant challenge. Here, we identified subventricular zone echogenicity (SVE) on cranial ultrasound in preterm infants following intestinal perforations. The development of SVE was significantly associated with motor impairment at 2 years. SVE was replicated in a neonatal mouse model of intestinal perforation. Examination of the murine echogenic subventricular zone (SVZ) revealed NLRP3-inflammasome assembly in multiciliated FoxJ1+ ependymal cells and a loss of the ependymal border in this postnatal stem cell niche. These data suggest a mechanism of preterm brain injury localized to the SVZ that has not been adequately considered. Ultrasound detection of SVE may serve as an early biomarker for neurodevelopmental impairment after inflammatory disease in preterm infants.

Learning structural heterogeneity from cryo-electron sub-tomograms with tomoDRGN.

Cryo-electron tomography (cryo-ET) enables observation of macromolecular complexes in their native, spatially contextualized cellular environment. Cryo-ET processing software to visualize such complexes at nanometer resolution via iterative alignment and averaging are well developed but rely upon assumptions of structural homogeneity among the complexes of interest. Recently developed tools allow for some assessment of structural diversity but have limited capacity to represent highly heterogeneous structures, including those undergoing continuous conformational changes. Here we extend the highly expressive cryoDRGN (Deep Reconstructing Generative Networks) deep learning architecture, originally created for single-particle cryo-electron microscopy analysis, to cryo-ET. Our new tool, tomoDRGN, learns a continuous low-dimensional representation of structural heterogeneity in cryo-ET datasets while also learning to reconstruct heterogeneous structural ensembles supported by the underlying data. Using simulated and experimental data, we describe and benchmark architectural choices within tomoDRGN that are uniquely necessitated and enabled by cryo-ET. We additionally illustrate tomoDRGN's efficacy in analyzing diverse datasets, using it to reveal high-level organization of human immunodeficiency virus (HIV) capsid complexes assembled in virus-like particles and to resolve extensive structural heterogeneity among ribosomes imaged in situ.

A closed translocation channel in the substrate-free AAA+ ClpXP protease diminishes rogue degradation.

AAA+ proteases degrade intracellular proteins in a highly specific manner. E. coli ClpXP, for example, relies on a C-terminal ssrA tag or other terminal degron sequences to recognize proteins, which are then unfolded by ClpX and subsequently translocated through its axial channel and into the degradation chamber of ClpP for proteolysis. Prior cryo-EM structures reveal that the ssrA tag initially binds to a ClpX conformation in which the axial channel is closed by a pore-2 loop. Here, we show that substrate-free ClpXP has a nearly identical closed-channel conformation. We destabilize this closed-channel conformation by deleting residues from the ClpX pore-2 loop. Strikingly, open-channel ClpXP variants degrade non-native proteins lacking degrons faster than the parental enzymes in vitro but degraded GFP-ssrA more slowly. When expressed in E. coli, these open channel variants behave similarly to the wild-type enzyme in assays of filamentation and phage-Mu plating but resulted in reduced growth phenotypes at elevated temperatures or when cells were exposed to sub-lethal antibiotic concentrations. Thus, channel closure is an important determinant of ClpXP degradation specificity.

Application of Monolayer Graphene to Cryo-Electron Microscopy Grids for High-resolution Structure Determination.

In cryogenic electron microscopy (cryoEM), purified macromolecules are applied to a grid bearing a holey carbon foil; the molecules are then blotted to remove excess liquid and rapidly frozen in a roughly 20-100 nm thick layer of vitreous ice, suspended across roughly 1 µm wide foil holes. The resulting sample is imaged using cryogenic transmission electron microscopy, and after image processing using suitable software, near-atomic resolution structures can be determined. Despite cryoEM's widespread adoption, sample preparation remains a severe bottleneck in cryoEM workflows, with users often encountering challenges related to samples behaving poorly in the suspended vitreous ice. Recently, methods have been developed to modify cryoEM grids with a single continuous layer of graphene, which acts as a support surface that often increases particle density in the imaged area and can reduce interactions between particles and the air-water interface. Here, we provide detailed protocols for the application of graphene to cryoEM grids and for rapidly assessing the relative hydrophilicity of the resulting grids. Additionally, we describe an EM-based method to confirm the presence of graphene by visualizing its characteristic diffraction pattern. Finally, we demonstrate the utility of these graphene supports by rapidly reconstructing a 2.7 Å resolution density map of a Cas9 complex using a pure sample at a relatively low concentration.

Imaging structurally dynamic ribosomes with cryogenic electron microscopy.

Throughout the history of electron microscopy, ribosomes have served as an ideal subject for imaging and technological development, which in turn has driven our understanding of ribosomal biology. Here, we provide a historical perspective at the intersection of electron microscopy technology development and ribosome biology and reflect on how this technique has shed light on each stage of the life cycle of this dynamic macromolecular machine. With an emphasis on prokaryotic systems, we specifically describe how pairing cryo-EM with clever experimental design, time-resolved techniques, and next-generation heterogeneous structural analysis has afforded insights into the modular nature of assembly, the roles of the many transient biogenesis and translation co-factors, and the subtle variations in structure and function between strains and species. The work concludes with a prospective outlook on the field, highlighting the pivotal role cryogenic electron tomography is playing in adding cellular context to our understanding of ribosomal life cycles, and noting how this exciting technology promises to bridge the gap between cellular and structural biology.

KsgA facilitates ribosomal small subunit maturation by proofreading a key structural lesion.

Ribosome assembly is orchestrated by many assembly factors, including ribosomal RNA methyltransferases, whose precise role is poorly understood. Here, we leverage the power of cryo-EM and machine learning to discover that the E. coli methyltransferase KsgA performs a 'proofreading' function in the assembly of the small ribosomal subunit by recognizing and partially disassembling particles that have matured but are not competent for translation. We propose that this activity allows inactive particles an opportunity to reassemble into an active state, thereby increasing overall assembly fidelity. Detailed structural quantifications in our datasets additionally enabled the expansion of the Nomura assembly map to highlight rRNA helix and r-protein interdependencies, detailing how the binding and docking of these elements are tightly coupled. These results have wide-ranging implications for our understanding of the quality-control mechanisms governing ribosome biogenesis and showcase the power of heterogeneity analysis in cryo-EM to unveil functionally relevant information in biological systems.

Application of monolayer graphene to cryo-electron microscopy grids for high-resolution structure determination.

In cryogenic electron microscopy (cryo-EM), purified macromolecules are typically applied to a grid bearing a holey carbon foil, blotted to remove excess liquid and rapidly frozen in a roughly 20-100 nm thick layer of vitreous ice that is suspended across roughly 1 µm-wide foil holes. The resulting sample is then imaged using cryogenic transmission electron microscopy and, after substantial image processing, near-atomic resolution structures can be determined. Despite cryo-EM's widespread adoption, sample preparation remains a severe bottleneck in cryo-EM workflows, with users often encountering challenges related to samples behaving poorly in the suspended vitreous ice. Recently, methods have been developed to modify cryo-EM grids with a single continuous layer of graphene, which acts as a support surface that often increases particle density in the imaged area and can reduce interactions between particles and the air-water interface. Here, we provide detailed protocols for the application of graphene to cryo-EM grids, and for rapidly assessing the relative hydrophilicity of the resulting grids. Additionally, we describe an EM-based method to confirm the presence of graphene by visualizing its characteristic diffraction pattern. Finally, we demonstrate the utility of these graphene supports by rapidly reconstructing a 2.7 Å resolution density map of an exemplar Cas9 complex using a highly pure sample at a relatively low concentration.

Learning structural heterogeneity from cryo-electron sub-tomograms with tomoDRGN.

Cryo-electron tomography (cryo-ET) allows one to observe macromolecular complexes in their native, spatially contextualized environment. Tools to visualize such complexes at nanometer resolution via iterative alignment and averaging are well-developed but rely on assumptions of structural homogeneity among the complexes under consideration. Recently developed downstream analysis tools allow for some assessment of macromolecular diversity but have limited capacity to represent highly heterogeneous macromolecules, including those undergoing continuous conformational changes. Here, we extend the highly expressive cryoDRGN deep learning architecture, originally created for cryo-electron microscopy single particle analysis, to sub-tomograms. Our new tool, tomoDRGN, learns a continuous low-dimensional representation of structural heterogeneity in cryo-ET datasets while also learning to reconstruct a large, heterogeneous ensemble of structures supported by the underlying data. Using simulated and experimental data, we describe and benchmark architectural choices within tomoDRGN that are uniquely necessitated and enabled by cryo-ET data. We additionally illustrate tomoDRGN's efficacy in analyzing an exemplar dataset, using it to reveal extensive structural heterogeneity among ribosomes imaged in situ.

Sulfur oxidizing bacteria in agro ecosystem and its role in plant productivity-a review.

Sulfur (S) deficiency is becoming more common in agro-ecosystems worldwide due to factors such as agronomic practices, high biomass production, reduced sulfur emissions, and the use of non-sulfur fertilizers. This review explores the natural occurrence and commercial exploitation of sulfur pools in nature, the mineralization and immobilization of sulfur, the physiological role of sulfur in plants, and its deficiency symptoms. Additionally, the organic and inorganic forms of sulfur in soil, their transformations, and the process of microbiological oxidation of sulfur are discussed. The review also addresses the diversity of sulfur-oxidizing bacteria (SOB) and the various biochemical mechanisms involved in their role in plant productivity and soil reclamation. The measurement of S oxidation rate in soil and the variables that influence the process are also examined. Typically, the rate of oxidation of added elemental S is around 40%-51%, which is available for plant uptake. These characteristics of SOB demonstrate their potential as bioinoculants for increasing plant growth, indicating their use as biofertilizers for sustainable crop production in agro-ecosystems.

Ameliorating the drought stress tolerance of a susceptible soybean cultivar, MAUS 2 through dual inoculation with selected rhizobia and AM fungus.

BACKGROUND: Drought stress is currently the primary abiotic stress factor for crop loss worldwide. Although drought stress reduces the crop yield significantly, species and genotypes differ in their stress response; some tolerate the stress effect while others not. In several systems, it has been shown that, some of the beneficial soil microbes ameliorate the stress effect and thereby, minimizing yield losses under stress conditions. Realizing the importance of beneficial soil microbes, a field experiment was conducted to study the effect of selected microbial inoculants namely, N-fixing bacteria, Bradyrhizobium liaoningense and P-supplying arbuscular mycorrhizal fungus, Ambispora leptoticha on growth and performance of a drought susceptible and high yielding soybean cultivar, MAUS 2 under drought condition. RESULTS: Drought stress imposed during flowering and pod filling stages showed that, dual inoculation consisting of B. liaoningense and A. leptoticha improved the physiological and biometric characteristics including nutrient uptake and yield under drought conditions. Inoculated plants showed an increased number of pods and pod weight per plant by 19% and 34% respectively, while the number of seeds and seed weight per plant increased by 17% and 32% respectively over un-inoculated plants under drought stress condition. Further, the inoculated plants showed higher chlorophyll and osmolyte content, higher detoxifying enzyme activity, and higher cell viability because of less membrane damage compared to un-inoculated plants under stress condition. In addition, they also showed higher water use efficiency coupled with more nutrients accumulation besides exhibiting higher load of beneficial microbes. CONCLUSION: Dual inoculation of soybean plants with beneficial microbes would alleviate the drought stress effects, thereby allowing normal plants' growth under stress condition. The study therefore, infers that AM fungal and rhizobia inoculation seems to be necessary when soybean is to be cultivated under drought or water limiting conditions.

The SspB adaptor drives structural changes in the AAA+ ClpXP protease during ssrA-tagged substrate delivery.

Energy-dependent protein degradation by the AAA+ ClpXP protease helps maintain protein homeostasis in bacteria and eukaryotic organelles of bacterial origin. In Escherichia coli and many other proteobacteria, the SspB adaptor assists ClpXP in degrading ssrA-tagged polypeptides produced as a consequence of tmRNA-mediated ribosome rescue. By tethering these incomplete ssrA-tagged proteins to ClpXP, SspB facilitates their efficient degradation at low substrate concentrations. How this process occurs structurally is unknown. Here, we present a cryo-EM structure of the SspB adaptor bound to a GFP-ssrA substrate and to ClpXP. This structure provides evidence for simultaneous contacts of SspB and ClpX with the ssrA tag within the tethering complex, allowing direct substrate handoff concomitant with the initiation of substrate translocation. Furthermore, our structure reveals that binding of the substrate·adaptor complex induces unexpected conformational changes within the spiral structure of the AAA+ ClpX hexamer and its interaction with the ClpP tetradecamer.

Uncovering structural ensembles from single-particle cryo-EM data using cryoDRGN.

Single-particle cryogenic electron microscopy (cryo-EM) has emerged as a powerful technique to visualize the structural landscape sampled by a protein complex. However, algorithmic and computational bottlenecks in analyzing heterogeneous cryo-EM datasets have prevented the full realization of this potential. CryoDRGN is a machine learning system for heterogeneous cryo-EM reconstruction of proteins and protein complexes from single-particle cryo-EM data. Central to this approach is a deep generative model for heterogeneous cryo-EM density maps, which we empirically find is effective in modeling both discrete and continuous forms of structural variability. Once trained, cryoDRGN is capable of generating an arbitrary number of 3D density maps, and thus interpreting the resulting ensemble is a challenge. Here, we showcase interactive and automated processing approaches for analyzing cryoDRGN results. Specifically, we detail a step-by-step protocol for the analysis of an existing assembling 50S ribosome dataset, including preparation of inputs, network training and visualization of the resulting ensemble of density maps. Additionally, we describe and implement methods to comprehensively analyze and interpret the distribution of volumes with the assistance of an associated atomic model. This protocol is appropriate for structural biologists familiar with processing single-particle cryo-EM datasets and with moderate experience navigating Python and Jupyter notebooks. It requires 3-4 days to complete. CryoDRGN is open source software that is freely available.

A proteolytic AAA+ machine poised to unfold a protein substrate.

AAA+ proteolytic machines unfold proteins prior to degradation. Cryo-EM of a ClpXP-substrate complex reveals a postulated but heretofore unseen intermediate in substrate unfolding/degradation. The natively folded substrate is drawn tightly against the ClpX channel by interactions between axial pore loops and the substrate degron tail, and by contacts with the native substrate that are, in part, enabled by movement of one ClpX subunit out of the typically observed hexameric spiral.

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.

The Management of Traumatic Abdominal Wall Flank Hernia Along the Spigelian Aponeurosis Using Component Separation, Synthetic, and Biological Mesh.

Blunt abdominal trauma is associated with a variety of medical complications. Traumatic abdominal wall hernias (TAWHs) are a rare sequela of blunt trauma. Of the various forms of TAWH, a rare subtype described as a "spontaneous lateral ventral hernia" or flank hernia occurs in less than 1% of all blunt abdominal traumas. We present a case of a 39-year-old male with a past medical history of epilepsy who was involved in a rollover motor vehicle collision. It was reported that the patient had a seizure while driving. On physical exam, the patient had a large left lower flank contusion. Computed tomography revealed a complex TAWH with complete avulsion of the abdominal wall musculature from the iliac crest and near to total disruption of the internal oblique. To address this, we used a biological mesh inlay, reinforced with a synthetic Ventralight™ mesh secured to the iliac crest. In this article, we describe the patient's experience and management of a complex TAWH.

Pharmacy Practice Standards for Outpatient Nephrology Settings.

Patients with kidney disease represent a medically complex group of patients with high medication burdens that could benefit from clinical pharmacy services as part of the interdisciplinary care team to optimize medication use. The "Advancing American Kidney Health" executive order includes new value-based reimbursement models to be tested by the Center for Medicare and Medicaid Innovation beginning January 2021 and January 2022. Advancing American Kidney Health executive order poses opportunities for the inclusion of comprehensive medication management. Following an iterative process integrating input from a diverse expert panel, published standards, clinical practice guidelines, peer review, and stakeholder feedback, our group developed practice standards for pharmacists caring for patients with kidney disease in health care settings. The standards focus on activities that are part of direct patient care and also include activities related to public health and advocacy, population health, leadership and management, and teaching, education and dissemination of knowledge. These standards are intended to be used by a variety of professionals, from pharmacists starting new practices to practice managers looking to add a pharmacist to the clinical team, to create standardization in services provided.

Conformational landscape of the yeast SAGA complex as revealed by cryo-EM.

Spt-Ada-Gcn5-Acetyltransferase (SAGA) is a conserved multi-subunit complex that activates RNA polymerase II-mediated transcription by acetylating and deubiquitinating nucleosomal histones and by recruiting TATA box binding protein (TBP) to DNA. The prototypical yeast Saccharomyces cerevisiae SAGA contains 19 subunits that are organized into Tra1, core, histone acetyltransferase, and deubiquitination modules. Recent cryo-electron microscopy studies have generated high-resolution structural information on the Tra1 and core modules of yeast SAGA. However, the two catalytical modules were poorly resolved due to conformational flexibility of the full assembly. Furthermore, the high sample requirement created a formidable barrier to further structural investigations of SAGA. Here, we report a workflow for isolating/stabilizing yeast SAGA and preparing cryo-EM specimens at low protein concentration using a graphene oxide support layer. With this procedure, we were able to determine a cryo-EM reconstruction of yeast SAGA at 3.1 Å resolution and examine its conformational landscape with the neural network-based algorithm cryoDRGN. Our analysis revealed that SAGA adopts a range of conformations with its HAT module and central core in different orientations relative to Tra1.