X-ray crystal structure of a designed rigidified imaging scaffold in the ligand-free conformation.

Imaging scaffolds composed of designed protein cages fused to designed ankyrin repeat proteins (DARPins) have enabled the structure determination of small proteins by cryogenic electron microscopy (cryo-EM). One particularly well characterized scaffold type is a symmetric tetrahedral assembly composed of 24 subunits, 12 A and 12 B, which has three cargo-binding DARPins positioned on each vertex. Here, the X-ray crystal structure of a representative tetrahedral scaffold in the apo state is reported at 3.

AlphaFold-assisted structure determination of a bacterial protein of unknown function using X-ray and electron crystallography.

Macromolecular crystallography generally requires the recovery of missing phase information from diffraction data to reconstruct an electron-density map of the crystallized molecule. Most recent structures have been solved using molecular replacement as a phasing method, requiring an a priori structure that is closely related to the target protein to serve as a search model; when no such search model exists, molecular replacement is not possible. New advances in computational machine-learning methods, however, have resulted in major advances in protein structure predictions from sequence information.

Congenital diaphragmatic hernia: automatic lung and liver MRI segmentation with nnU-Net, reproducibility of pyradiomics features, and a machine learning application for the classification of liver herniation.

Prenatal assessment of lung size and liver position is essential to stratify congenital diaphragmatic hernia (CDH) fetuses in risk categories, guiding counseling, and patient management. Manual segmentation on fetal MRI provides a quantitative estimation of total lung volume and liver herniation. However, it is time-consuming and operator-dependent.

A Highly Ordered Nitroxide Side Chain for Distance Mapping and Monitoring Slow Structural Fluctuations in Proteins.

Unlabelled: Site-directed spin labeling electron paramagnetic resonance (SDSL-EPR) is an established tool for exploring protein structure and dynamics. Although nitroxide side chains attached to a single cysteine via a disulfide linkage are commonly employed in SDSL-EPR, their internal flexibility complicates applications to monitor slow internal motions in proteins and to structure determination by distance mapping. Moreover, the labile disulfide linkage prohibits the use of reducing agents often needed for protein stability.

Cryo-EM structures of the D290V mutant of the hnRNPA2 low-complexity domain suggests how D290V affects phase separation and aggregation.

Heterogeneous nuclear ribonucleoprotein A2 (hnRNPA2) is a human ribonucleoprotein that transports RNA to designated locations for translation via its ability to phase separate. Its mutated form, D290V, is implicated in multisystem proteinopathy known to afflict two families, mainly with myopathy and Paget's disease of bone. Here, we investigate this mutant form of hnRNPA2 by determining cryo-EM structures of the recombinant D290V low complexity domain.

Structure-based design of nanobodies that inhibit seeding of Alzheimer's patient-extracted tau fibrils.

Despite much effort, antibody therapies for Alzheimer's disease (AD) have shown limited efficacy. Challenges to the rational design of effective antibodies include the difficulty of achieving specific affinity to critical targets, poor expression, and antibody aggregation caused by buried charges and unstructured loops. To overcome these challenges, we grafted previously determined sequences of fibril-capping amyloid inhibitors onto a camel heavy chain antibody scaffold.

Automated Stabilization, Enhancement and Capillaries Segmentation in Videocapillaroscopy.

Oral capillaroscopy is a critical and non-invasive technique used to evaluate microcirculation. Its ability to observe small vessels in vivo has generated significant interest in the field. Capillaroscopy serves as an essential tool for diagnosing and prognosing various pathologies, with anatomic-pathological lesions playing a crucial role in their progression.

Development and atomic structure of a new fluorescence-based sensor to probe heme transfer in bacterial pathogens.

Heme is the most abundant source of iron in the human body and is actively scavenged by bacterial pathogens during infections. Corynebacterium diphtheriae and other species of actinobacteria scavenge heme using cell wall associated and secreted proteins that contain Conserved Region (CR) domains. Here we report the development of a fluorescent sensor to measure heme transfer from the C-terminal CR domain within the HtaA protein (CR2) to other hemoproteins within the heme-uptake system.

Cryo-EM structure determination of small therapeutic protein targets at 3 Å-resolution using a rigid imaging scaffold.

Cryoelectron microscopy (Cryo-EM) has enabled structural determination of proteins larger than about 50 kDa, including many intractable by any other method, but it has largely failed for smaller proteins. Here, we obtain structures of small proteins by binding them to a rigid molecular scaffold based on a designed protein cage, revealing atomic details at resolutions reaching 2.9 Å.

Window-Based Energy Selecting X-ray Imaging and Charge Sharing in Cadmium Zinc Telluride Linear Array Detectors for Contaminant Detection.

The spectroscopic and imaging performance of energy-resolved photon counting detectors, based on new sub-millimetre boron oxide encapsulated vertical Bridgman cadmium zinc telluride linear arrays, are presented in this work. The activities are in the framework of the AVATAR X project, planning the development of X-ray scanners for contaminant detection in food industry. The detectors, characterized by high spatial (250 µm) and energy (<3 keV) resolution, allow spectral X-ray imaging with interesting image quality improvements.

The Shr receptor from uses a cap and release mechanism to acquire heme-iron from human hemoglobin.

(group A ) is a clinically important microbial pathogen that requires iron in order to proliferate. During infections, uses the surface displayed Shr receptor to capture human hemoglobin (Hb) and acquires its iron-laden heme molecules. Through a poorly understood mechanism, Shr engages Hb via two structurally unique N-terminal Hb-interacting domains (HID1 and HID2) which facilitate heme transfer to proximal NEAr Transporter (NEAT) domains.

Micro-electron diffraction structure of the aggregation-driving N terminus of Drosophila neuronal protein Orb2A reveals amyloid-like β-sheets.

Amyloid protein aggregation is commonly associated with progressive neurodegenerative diseases, however not all amyloid fibrils are pathogenic. The neuronal cytoplasmic polyadenylation element binding protein is a regulator of synaptic mRNA translation and has been shown to form functional amyloid aggregates that stabilize long-term memory. In adult Drosophila neurons, the cytoplasmic polyadenylation element binding homolog Orb2 is expressed as 2 isoforms, of which the Orb2B isoform is far more abundant, but the rarer Orb2A isoform is required to initiate Orb2 aggregation.

De novo determination of mosquitocidal Cry11Aa and Cry11Ba structures from naturally-occurring nanocrystals.

Cry11Aa and Cry11Ba are the two most potent toxins produced by mosquitocidal Bacillus thuringiensis subsp. israelensis and jegathesan, respectively. The toxins naturally crystallize within the host; however, the crystals are too small for structure determination at synchrotron sources.

Recognizing the Emergent and Submerged Iceberg of the Celiac Disease: ITAMA Project-Global Strategy Protocol.

Coeliac disease (CD) is frequently underdiagnosed with a consequent heavy burden in terms of morbidity and health care costs. Diagnosis of CD is based on the evaluation of symptoms and anti-transglutaminase antibodies IgA (TGA-IgA) levels, with values above a tenfold increase being the basis of the biopsy-free diagnostic approach suggested by present guidelines. This study showcased the largest screening project for CD carried out to date in school children (n=20,000) aimed at assessing the diagnostic accuracy of minimally invasive finger prick point-of-care tests (POCT) which, combined with conventional celiac serology and the aid of an artificial intelligence-based system, may eliminate the need for intestinal biopsy.

Crystal structure of mevalonate 3,5-bisphosphate decarboxylase reveals insight into the evolution of decarboxylases in the mevalonate metabolic pathways.

Mevalonate 3,5-bisphosphate decarboxylase is involved in the recently discovered Thermoplasma-type mevalonate pathway. The enzyme catalyzes the elimination of the 3-phosphate group from mevalonate 3,5-bisphosphate as well as concomitant decarboxylation of the substrate. This entire reaction of the enzyme resembles the latter half-reactions of its homologs, diphosphomevalonate decarboxylase and phosphomevalonate decarboxylase, which also catalyze ATP-dependent phosphorylation of the 3-hydroxyl group of their substrates.

Cryo-EM structure of RNA-induced tau fibrils reveals a small C-terminal core that may nucleate fibril formation.

In neurodegenerative diseases including Alzheimer’s and amyotrophic lateral sclerosis, proteins that bind RNA are found in aggregated forms in autopsied brains. Evidence suggests that RNA aids nucleation of these pathological aggregates; however, the mechanism has not been investigated at the level of atomic structure. Here, we present the 3.

Incomplete Charge Collection at Inter-Pixel Gap in Low- and High-Flux Cadmium Zinc Telluride Pixel Detectors.

The success of cadmium zinc telluride (CZT) detectors in room-temperature spectroscopic X-ray imaging is now widely accepted. The most common CZT detectors are characterized by enhanced-charge transport properties of electrons, with mobility-lifetime products μeτe > 10 cm/V and μhτh > 10 cm/V. These materials, typically termed low-flux LF-CZT, are successfully used for thick electron-sensing detectors and in low-flux conditions.

The structure of the Clostridium thermocellum RsgI9 ectodomain provides insight into the mechanism of biomass sensing.

Clostridium thermocellum is actively being developed as a microbial platform to produce biofuels and chemicals from renewable plant biomass. An attractive feature of this bacterium is its ability to efficiently degrade lignocellulose using surface-displayed cellulosomes, large multi-protein complexes that house different types of cellulase enzymes. Clostridium thermocellum tailors the enzyme composition of its cellulosome using nine membrane-embedded anti-σ factors (RsgI1-9), which are thought to sense different types of extracellular carbohydrates and then elicit distinct gene expression programs via cytoplasmic σ factors.

Insight into the molecular basis of substrate recognition by the wall teichoic acid glycosyltransferase TagA.

Wall teichoic acid (WTA) polymers are covalently affixed to the Gram-positive bacterial cell wall and have important functions in cell elongation, cell morphology, biofilm formation, and β-lactam antibiotic resistance. The first committed step in WTA biosynthesis is catalyzed by the TagA glycosyltransferase (also called TarA), a peripheral membrane protein that produces the conserved linkage unit, which joins WTA to the cell wall peptidoglycan. TagA contains a conserved GT26 core domain followed by a C-terminal polypeptide tail that is important for catalysis and membrane binding.

A maChine and deep Learning Approach to predict pulmoNary hyperteNsIon in newbornS with congenital diaphragmatic Hernia (CLANNISH): Protocol for a retrospective study.

Introduction: Outcome predictions of patients with congenital diaphragmatic hernia (CDH) still have some limitations in the prenatal estimate of postnatal pulmonary hypertension (PH). We propose applying Machine Learning (ML), and Deep Learning (DL) approaches to fetuses and newborns with CDH to develop forecasting models in prenatal epoch, based on the integrated analysis of clinical data, to provide neonatal PH as the first outcome and, possibly: favorable response to fetal endoscopic tracheal occlusion (FETO), need for Extracorporeal Membrane Oxygenation (ECMO), survival to ECMO, and death. Moreover, we plan to produce a (semi)automatic fetus lung segmentation system in Magnetic Resonance Imaging (MRI), which will be useful during project implementation but will also be an important tool itself to standardize lung volume measures for CDH fetuses.

Prospecting for natural products by genome mining and microcrystal electron diffraction.

More than 60% of pharmaceuticals are related to natural products (NPs), chemicals produced by living organisms. Despite this, the rate of NP discovery has slowed over the past few decades. In many cases the rate-limiting step in NP discovery is structural characterization.

Structure of human factor VIIa-soluble tissue factor with calcium, magnesium and rubidium.

Coagulation factor VIIa (FVIIa) consists of a γ-carboxyglutamic acid (GLA) domain, two epidermal growth factor-like (EGF) domains and a protease domain. FVIIa binds three Mg ions and four Ca ions in the GLA domain, one Ca ion in the EGF1 domain and one Ca ion in the protease domain. Further, FVIIa contains an Na site in the protease domain.

Energy Recovery of Multiple Charge Sharing Events in Room Temperature Semiconductor Pixel Detectors.

Multiple coincidence events from charge-sharing and fluorescent cross-talk are typical drawbacks in room-temperature semiconductor pixel detectors. The mitigation of these distortions in the measured energy spectra, using charge-sharing discrimination (CSD) and charge-sharing addition (CSA) techniques, is always a trade-off between counting efficiency and energy resolution. The energy recovery of multiple coincidence events is still challenging due to the presence of charge losses after CSA.