Co-flow injection for serial crystallography at X-ray free-electron lasers

Serial femtosecond crystallography (SFX) is a powerful technique that exploits X-ray free-electron lasers to determine the structure of macromolecules at room temperature. Despite the impressive exposition of structural details with this novel crystallographic approach, the methods currently available to introduce crystals into the path of the X-ray beam sometimes exhibit serious drawbacks. Samples requiring liquid injection of crystal slurries consume large quantities of crystals (at times up to a gram of protein per data set), may not be compatible with vacuum configurations on beamlines or provide a high background due to additional sheathing liquids present during the injection. Proposed and characterized here is the use of an immiscible inert oil phase to supplement the flow of sample in a hybrid microfluidic 3D-printed co-flow device. Co-flow generation is reported with sample and oil phases flowing in parallel, resulting in stable injection conditions for two different resin materials experimentally. A numerical model is presented that adequately predicts these flow-rate conditions. The co-flow generating devices reduce crystal clogging effects, have the potential to conserve protein crystal samples up to 95% and will allow degradation-free light-induced time-resolved SFX.

Cryo-EM reconstruction of the human 40S ribosomal subunit at 2.15 Å resolution

The chemical modification of ribosomal RNA and proteins is critical for ribosome assembly, for protein synthesis and may drive ribosome specialization in development and disease. However, the inability to accurately visualize these modifications has limited mechanistic understanding of the role of these modifications in ribosome function. Here we report the 2.15 Å resolution cryo-EM reconstruction of the human 40S ribosomal subunit. We directly visualize post-transcriptional modifications within the 18S rRNA and post-translational modifications at the N-termini of two ribosomal proteins. Additionally, we interpret the solvation shells in the core regions of the 40S ribosomal subunit and reveal how potassium and magnesium ions establish both universally conserved and eukaryote-specific coordination to promote the stabilization and folding of key ribosomal elements. This work provides unprecedented structural details for the human 40S ribosomal subunit that will serve as an important reference for unraveling the functional role of ribosomal RNA modifications.

Crystallography relevant to Mars and Galilean icy moons: crystal behavior of kieserite-type monohydrate sulfates at extraterrestrial conditions down to 15 K

Monohydrate sulfate kieserites (M 2+SO4·H2O) and their solid solutions are essential constituents on the surface of Mars and most likely also on Galilean icy moons in our solar system. Phase stabilities of end-member representatives (M 2+ = Mg, Fe, Co, Ni) have been examined crystallographically using single-crystal X-ray diffraction at 1 bar and temperatures down to 15 K, by means of applying open He cryojet techniques at in-house laboratory instrumentation. All four representative phases show a comparable, highly anisotropic thermal expansion behavior with a remarkable negative thermal expansion along the monoclinic b axis and a pronounced anisotropic expansion perpendicular to it. The lattice changes down to 15 K correspond to an `inverse thermal pressure' of approximately 0.7 GPa, which is far below the critical pressures of transition under hydrostatic compression (Pc ≥ 2.40 GPa). Consequently, no equivalent structural phase transition was observed for any compound, and neither dehydration nor rearrangements of the hydrogen bonding schemes have been observed. The M 2+SO4·H2O (M 2+ = Mg, Fe, Co, Ni) end-member phases preserve the kieserite-type C2/c symmetry; hydrogen bonds and other structural details were found to vary smoothly down to the lowest experimental temperature. These findings serve as an important basis for the assignment of sulfate-related signals in remote-sensing data obtained from orbiters at celestial bodies, as well as for thermodynamic considerations and modeling of properties of kieserite-type sulfate monohydrates relevant to extraterrestrial sulfate associations at very low temperatures.

Neutron crystallography reveals mechanisms used by Pseudomonas aeruginosa for host-cell binding

The opportunistic pathogen Pseudomonas aeruginosa, a major cause of nosocomial infections, uses carbohydrate-binding proteins (lectins) as part of its binding to host cells. The fucose-binding lectin, LecB, displays a unique carbohydrate-binding site that incorporates two closely located calcium ions bridging between the ligand and protein, providing specificity and unusually high affinity. Here, we investigate the mechanisms involved in binding based on neutron crystallography studies of a fully deuterated LecB/fucose/calcium complex. The neutron structure, which includes the positions of all the hydrogen atoms, reveals that the high affinity of binding may be related to the occurrence of a low-barrier hydrogen bond induced by the proximity of the two calcium ions, the presence of coordination rings between the sugar, calcium and LecB, and the dynamic behaviour of bridging water molecules at room temperature. These key structural details may assist in the design of anti-adhesive compounds to combat multi-resistance bacterial infections.

Integrative Geophysical Studies at the Novaya Kurya-1 Cemetery in the Kulunda Steppe

We outline the results of prospection studies at the Novaya Kurya-1 cemetery in the south of Western Siberia, using remote sensing methods such as aerial photography, ground-based magnetometry, high-precision aeromagnetic survey, electromagnetic profi ling, and electrotomography. Original techniques were used to construct relative relief maps, and an inversion of data from ground-based magnetic survey at various altitudes was carried out. The fi rst technique reduces the effect of natural relief, and highlights anthropogenic altitudinal anomalies, making the analysis of digital elevation models more effi cient. The second technique is helpful for assessing the thickness and depth of anomalous magnetic bodies or horizons, not only providing planigraphic information but enabling us to evaluate two- and threedimensional geometric properties of the detected objects. As a result of the analyses, at least 14 kurgans were identifi ed at the cemetery, six of which lack salient outward features. Structural details suggest that most of them date to the Early Scythian time (800–400 BC). On the basis of the interpretation of the results of highly effi cient prospection analyses using the UAV platform, offering the possibility of surveying a large area (about 25 ha), the boundaries of the site were determined. Several features were detected. To identify these, further studies are needed.

Potential Role of Flavivirus NS2B-NS3 Proteases in Viral Pathogenesis and Anti-flavivirus Drug Discovery Employing Animal Cells and Models: A Review

Flaviviruses are known to cause a variety of diseases in humans in different parts of the world. There are very limited numbers of antivirals to combat flavivirus infection, and therefore new drug targets must be explored. The flavivirus NS2B-NS3 proteases are responsible for the cleavage of the flavivirus polyprotein, which is necessary for productive viral infection and for causing clinical infections; therefore, they are a promising drug target for devising novel drugs against different flaviviruses. This review highlights the structural details of the NS2B-NS3 proteases of different flaviviruses, and also describes potential antiviral drugs that can interfere with the viral protease activity, as determined by various studies. Moreover, optimized in vitro reaction conditions for studying the NS2B-NS3 proteases of different flaviviruses may vary and have been incorporated in this review. The increasing availability of the in silico and crystallographic/structural details of flavivirus NS2B-NS3 proteases in free and drug-bound states can pave the path for the development of promising antiflavivirus drugs to be used in clinics. However, there is a paucity of information available on using animal cells and models for studying flavivirus NS2B-NS3 proteases, as well as on the testing of the antiviral drug efficacy against NS2B-NS3 proteases. Therefore, on the basis of recent studies, an effort has also been made to propose potential cellular and animal models for the study of flavivirus NS2B-NS3 proteases for the purposes of exploring flavivirus pathogenesis and for testing the efficacy of possible drugs targets, in vitro and in vivo.

Insights into Background of Microbial Aggregations (Acinetobacter baumannii): A Century of Challenges

: In recent years, Acinetobacter baumannii has attracted the research community’s attention since they are turned into the leading cause of both community- and hospital-acquired infections. The emergence of MDR-Acinetobacter baumannii strains threatens hospitalized patients since antibiotics fail to withdraw the bacterial infectious agents. Despite its worldwide distribution, health settings fail to combat limitations in therapeutic regions against Acinetobacter baumannii. The capability of biofilm formation in Acinetobacter baumannii strengthens their virulence and also survival. Understanding the fundamental virulence mechanisms beyond the microbial aggregations leads to exploring alternative drug targets such as signaling molecules and Quorum sensing systems to block bacterial communication and antimicrobial resistance. The significance of examining the biofilm's structural details and the relationship between Quorum sensing networks and related signaling molecules has been explicitly highlighted. Accordingly, this review study aimed to explain the general biofilm structure, the mechanisms beyond biofilm formation, quorum sensing system, and the generation of signaling molecules in Acinetobacter baumannii.

Supramolecular assembly of chloroplast NAD(P)H dehydrogenase-like complex with photosystem I from Arabidopsis thaliana

Cyclic electron transport/flow (CET/CEF) in chloroplasts is a regulatory mechanism crucial for optimization of plant photosynthetic efficiency. CET is catalyzed by a membrane-embedded NAD(P)H dehydrogenase-like (NDH) complex containing at least 29 protein subunits and associating with photosystem I (PSI) to form the NDH-PSI supercomplex. Here we report the 3.9 angstrom resolution structure of Arabidopsis thaliana NDH-PSI (AtNDH-PSI) supercomplex. We have constructed structural models for 26 AtNDH subunits, among which 11 subunits are unique to chloroplast and stabilize the core part of NDH complex. In the supercomplex, one NDH can bind up to two PSI-LHCI complexes at both sides of its membrane arm. Two minor LHCIs, Lhca5 and Lhca6, each present in one PSI-LHCI, interact with NDH and contribute to the supercomplex formation and stabilization. Our results showed structural details of the supercomplex assembly and provide molecular basis for further investigation of the regulatory mechanism of CEF in plants.

Preoperative Evaluation and Surgical Simulation for Osteochondritis Dissecans of the Elbow Using Three-Dimensional MRI-CT Image Fusion Images

We used our novel three-dimensional magnetic resonance imaging-computed tomography fusion images (3D MRI-CT fusion images; MCFIs) for detailed preoperative lesion evaluation and surgical simulation in osteochondritis dissecans (OCD) of the elbow. Herein, we introduce our procedure and report the findings of the assessment of its utility. We enrolled 16 men (mean age: 14.0 years) and performed preoperative MRI using 7 kg axial traction with a 3-Tesla imager and CT. Three-dimensional-MRI models of the humerus and articular cartilage and a 3D-CT model of the humerus were constructed. We created MCFIs using both models. We validated the findings obtained from the MCFIs and intraoperative findings using the following items: articular cartilage fissures and defects, articular surface deformities, vertical and horizontal lesion diameters, the International Cartilage Repair Society (ICRS) classification, and surgical procedures. The MCFIs accurately reproduced the lesions and correctly matched the ICRS classification in 93.5% of cases. Surgery was performed as simulated in all cases. Preoperatively measured lesion diameters exhibited no significant differences compared to the intraoperative measurements. MCFIs were useful in the evaluation of OCD lesions and detailed preoperative surgical simulation through accurate reproduction of 3D structural details of the lesions.