Nanostars in Highly Si-Doped GaN.

Understanding the relation between surface morphology during epitaxy of GaN:Si and its electrical properties is important from both the fundamental and application perspectives. This work evidences the formation of nanostars in highly doped GaN:Si layers with doping level ranging from 5 × 1019 to 1 × 1020 cm-3 grown by plasma-assisted molecular beam epitaxy (PAMBE). Nanostars are 50-nm-wide platelets arranged in six-fold symmetry around the [0001] axis and have different electrical properties from the surrounding layer. Nanostars are formed in highly doped GaN:Si layers due to the enhanced growth rate along the a-direction ⟨112̅0⟩. Then, the hexagonal-shaped growth spirals, typically observed in GaN grown on GaN/sapphire templates, develop distinct arms that extend in the a-direction ⟨112̅0⟩. The nanostar surface morphology is reflected in the inhomogeneity of electrical properties at the nanoscale as evidenced in this work. Complementary techniques such as electrochemical etching (ECE), atomic force microscopy (AFM), and scanning spreading resistance microscopy (SSRM) are used to link the morphology and conductivity variations across the surface. Additionally, transmission electron microscopy (TEM) studies with high spatial resolution composition mapping by energy-dispersive X-ray spectroscopy (EDX) confirmed about 10% lower incorporation of Si in the hillock arms than in the layer. However, the lower Si content in the nanostars cannot solely be responsible for the fact that they are not etched in ECE. The compensation mechanism in the nanostars observed in GaN:Si is discussed to be an additional contribution to the local decrease in conductivity at the nanoscale.

Structural and functional properties of the transporter SLC26A6 reveal mechanism of coupled anion exchange.

Members of the SLC26 family constitute a conserved class of anion transport proteins, which encompasses uncoupled transporters with channel-like properties, coupled exchangers and motor proteins. Among the 10 functional paralogs in humans, several participate in the secretion of bicarbonate in exchange with chloride and thus play an important role in maintaining pH homeostasis. Previously, we have elucidated the structure of murine SLC26A9 and defined its function as an uncoupled chloride transporter (Walter et al., 2019). Here we have determined the structure of the closely related human transporter SLC26A6 and characterized it as a coupled exchanger of chloride with bicarbonate and presumably also oxalate. The structure defines an inward-facing conformation of the protein that generally resembles known structures of SLC26A9. The altered anion selectivity between both paralogs is a consequence of a remodeled ion binding site located in the center of a mobile unit of the membrane-inserted domain, which also accounts for differences in the coupling mechanism.

Structure of a volume-regulated heteromeric LRRC8A/C channel.

Volume-regulated anion channels (VRACs) participate in the cellular response to osmotic swelling. These membrane proteins consist of heteromeric assemblies of LRRC8 subunits, whose compositions determine permeation properties. Although structures of the obligatory LRRC8A, also referred to as SWELL1, have previously defined the architecture of VRACs, the organization of heteromeric channels has remained elusive. Here we have addressed this question by the structural characterization of murine LRRC8A/C channels. Like LRRC8A, these proteins assemble as hexamers. Despite 12 possible arrangements, we find a predominant organization with an A:C ratio of two. In this assembly, four LRRC8A subunits cluster in their preferred conformation observed in homomers, as pairs of closely interacting proteins that stabilize a closed state of the channel. In contrast, the two interacting LRRC8C subunits show a larger flexibility, underlining their role in the destabilization of the tightly packed A subunits, thereby enhancing the activation properties of the protein.

Role of Metallic Adlayer in Limiting Ge Incorporation into GaN.

Atomically thin metal adlayers are used as surfactants in semiconductor crystal growth. The role of the adlayer in the incorporation of dopants in GaN is completely unexplored, probably because n-type doping of GaN with Si is relatively straightforward and can be scaled up with available Si atomic flux in a wide range of dopant concentrations. However, a surprisingly different behavior of the Ge dopant is observed, and the presence of atomically thin gallium or an indium layer dramatically affects Ge incorporation, hindering the fabrication of GaN:Ge structures with abrupt doping profiles. Here, we show an experimental study presenting a striking improvement in sharpness of the Ge doping profile obtained for indium as compared to the gallium surfactant layer during GaN-plasma-assisted molecular beam epitaxy. We show that the atomically thin indium surfactant layer promotes the incorporation of Ge in contrast to the gallium surfactant layer, which promotes segregation of Ge to the surface and Ge crystallite formation. Understanding the role of the surfactant is essential to control GaN doping and to obtain extremely high n-type doped III-nitride layers using Ge, because doping levels >1020 cm−3 are not easily available with Si.

R-loop formation and conformational activation mechanisms of Cas9.

Cas9 is a CRISPR-associated endonuclease capable of RNA-guided, site-specific DNA cleavage1-3. The programmable activity of Cas9 has been widely utilized for genome editing applications4-6, yet its precise mechanisms of target DNA binding and off-target discrimination remain incompletely understood. Here we report a series of cryo-electron microscopy structures of Streptococcus pyogenes Cas9 capturing the directional process of target DNA hybridization. In the early phase of R-loop formation, the Cas9 REC2 and REC3 domains form a positively charged cleft that accommodates the distal end of the target DNA duplex. Guide-target hybridization past the seed region induces rearrangements of the REC2 and REC3 domains and relocation of the HNH nuclease domain to assume a catalytically incompetent checkpoint conformation. Completion of the guide-target heteroduplex triggers conformational activation of the HNH nuclease domain, enabled by distortion of the guide-target heteroduplex, and complementary REC2 and REC3 domain rearrangements. Together, these results establish a structural framework for target DNA-dependent activation of Cas9 that sheds light on its conformational checkpoint mechanism and may facilitate the development of novel Cas9 variants and guide RNA designs with enhanced specificity and activity.

Regulators of cell volume: The structural and functional properties of anion channels of the LRRC8 family.

Members of the LRRC8 family participate in the response of vertebrate cells to osmotic changes in their environment. These proteins form heteromeric assemblies composed of the obligatory subunit LRRC8A and at least one of the other four homologs, which together function as anion-selective channels with distinct properties that are activated upon cell-swelling. The hexameric complexes share a conserved architecture consisting of a membrane-inserted pore domain with an ion permeation path located at the axis of symmetry and cytoplasmic leucine-rich repeat domains that regulate the open probability of the channel. In this review, we summarize the current understanding of structure-function relationships of these unusual ion channels whose mechanisms are, despite their large physiological importance, still poorly understood.

Electrically pumped blue laser diodes with nanoporous bottom cladding.

We demonstrate electrically pumped III-nitride edge-emitting laser diodes (LDs) with nanoporous bottom cladding grown by plasma-assisted molecular beam epitaxy on c-plane (0001) GaN. After the epitaxy of the LD structure, highly doped 350 nm thick GaN:Si cladding layer with Si concentration of 6·1019 cm-3 was electrochemically etched to obtain porosity of 15 ± 3% with pore size of 20 ± 9 nm. The devices with nanoporous bottom cladding are compared to the reference structures. The pulse mode operation was obtained at 448.7 nm with a slope efficiency (SE) of 0.2 W/A while the reference device without etched cladding layer was lasing at 457 nm with SE of 0.56 W/A. The design of the LDs with porous bottom cladding was modelled theoretically. Performed calculations allowed to choose the optimum porosity and thickness of the cladding needed for the desired optical mode confinement and reduced the risk of light leakage to the substrate and to the top-metal contact. This demonstration opens new possibilities for the fabrication of III-nitride LDs.

Mineral Materials as a Neutralizing Agent Used on Soil Contaminated with Copper.

The aim of the investigation was to evaluate the response of plants, using black mustard (Brassica nigra L. Koch) as a model plant, to soil contamination with copper (0, 200, 400, 600 mg Cu kg-1 of soil), and to determine the effectiveness of the Cu immobilization with mineral neutralizing materials, such as lime, clay and zeolite. The plant yield depended on soil contamination and mineral amendments. In the series without neutralizing materials, the level of 600 mg Cu kg-1 reduced the yield and increased leaf greenness. Lime alleviated the toxicity of Cu in objects with 200 mg Cu kg-1. Zeolite slightly mitigated the harmful effects of Cu at the level of 400 and 600 mg kg-1. Zeolite lowered the SPAD index. In the chemical composition of plants, the content of Cu, K, Mg, Na and Ca in plants increased to 400 mg Cu kg-1, while the content of P decreased to 600 mg Cu kg-1. Among the materials, lime reduced the Cu accumulation in plants the most, followed by clay. Cu narrowed the majority of ratios and widened the Ca:P and K:Ca ratios in plants. The applied mineral materials, except lime, did not significantly affect the formation of these indicators.

Allosteric modulation of LRRC8 channels by targeting their cytoplasmic domains.

Members of the LRRC8 family form heteromeric assemblies, which function as volume-regulated anion channels. These modular proteins consist of a transmembrane pore and cytoplasmic leucine-rich repeat (LRR) domains. Despite their known molecular architecture, the mechanism of activation and the role of the LRR domains in this process has remained elusive. Here we address this question by generating synthetic nanobodies, termed sybodies, which target the LRR domain of the obligatory subunit LRRC8A. We use these binders to investigate their interaction with homomeric LRRC8A channels by cryo-electron microscopy and the consequent effect on channel activation by electrophysiology. The five identified sybodies either inhibit or enhance activity by binding to distinct epitopes of the LRR domain, thereby altering channel conformations. In combination, our work provides a set of specific modulators of LRRC8 proteins and reveals the role of their cytoplasmic domains as regulators of channel activity by allosteric mechanisms.

Cryo-EM structures of the TTYH family reveal a novel architecture for lipid interactions.

The Tweety homologs (TTYHs) are members of a conserved family of eukaryotic membrane proteins that are abundant in the brain. The three human paralogs were assigned to function as anion channels that are either activated by Ca2+ or cell swelling. To uncover their unknown architecture and its relationship to function, we have determined the structures of human TTYH1-3 by cryo-electron microscopy. All structures display equivalent features of a dimeric membrane protein that contains five transmembrane segments and an extended extracellular domain. As none of the proteins shows attributes reminiscent of an anion channel, we revisited functional experiments and did not find any indication of ion conduction. Instead, we find density in an extended hydrophobic pocket contained in the extracellular domain that emerges from the lipid bilayer, which suggests a role of TTYH proteins in the interaction with lipid-like compounds residing in the membrane.

Cryo-EM structures of the caspase-activated protein XKR9 involved in apoptotic lipid scrambling.

The exposure of the negatively charged lipid phosphatidylserine on the cell surface, catalyzed by lipid scramblases, is an important signal for the clearance of apoptotic cells by macrophages. The protein XKR9 is a member of a conserved family that has been associated with apoptotic lipid scrambling. Here, we describe structures of full-length and caspase-treated XKR9 from Rattus norvegicus in complex with a synthetic nanobody determined by cryo-electron microscopy. The 43 kDa monomeric membrane protein can be divided into two structurally related repeats, each containing four membrane-spanning segments and a helix that is partly inserted into the lipid bilayer. In the full-length protein, the C-terminus interacts with a hydrophobic pocket located at the intracellular side acting as an inhibitor of protein function. Cleavage by caspase-3 at a specific site releases 16 residues of the C-terminus, thus making the pocket accessible to the cytoplasm. Collectively, the work has revealed the unknown architecture of the XKR family and has provided initial insight into its activation by caspases.

Cryo-EM structures and functional properties of CALHM channels of the human placenta.

The transport of substances across the placenta is essential for the development of the fetus. Here, we were interested in the role of channels of the calcium homeostasis modulator (CALHM) family in the human placenta. By transcript analysis, we found the paralogs CALHM2, 4, and 6 to be highly expressed in this organ and upregulated during trophoblast differentiation. Based on electrophysiology, we observed that activation of these paralogs differs from the voltage- and calcium-gated channel CALHM1. Cryo-EM structures of CALHM4 display decameric and undecameric assemblies with large cylindrical pore, while in CALHM6 a conformational change has converted the pore shape into a conus that narrows at the intracellular side, thus describing distinct functional states of the channel. The pore geometry alters the distribution of lipids, which occupy the cylindrical pore of CALHM4 in a bilayer-like arrangement whereas they have redistributed in the conical pore of CALHM6 with potential functional consequences.

Revealing inhomogeneous Si incorporation into GaN at the nanometer scale by electrochemical etching.

Typical methods of doping quantification are based on spectroscopy or conductivity measurements. The spatial dopant distribution assessment with nanometer-scale precision is limited usually to one or two dimensions. Here we demonstrate an approach to detect three-dimensional dopant homogeneity in GaN:Si layers using electrochemical etching (ECE). GaN:Si layers are grown by plasma-assisted molecular beam epitaxy. Dopant incorporation is uniform when the growth front morphology is atomically flat. Non-uniform Si incorporation into GaN is observed when step-bunches are present on the surface during epitaxy. In this study we show that local Si concentration in the area of step-bunch is about three times higher than in the area between step-bunches. ECE spatial resolution in our experiment is estimated to be about 50 nm. This makes ECE a simple and quantitative probing tool for local three-dimensional conductivity homogeneity assessment. Our study proves that ECE could be important both for fundamental studies of crystal growth physics and impurity incorporation and for ion-implanted structures and post-processing device control.

Cryo-EM structures and functional characterization of murine Slc26a9 reveal mechanism of uncoupled chloride transport.

The epithelial anion transporter SLC26A9 contributes to airway surface hydration and gastric acid production. Colocalizing with CFTR, SLC26A9 has been proposed as a target for the treatment of cystic fibrosis. To provide molecular details of its transport mechanism, we present cryo-EM structures and a functional characterization of murine Slc26a9. These structures define the general architecture of eukaryotic SLC26 family members and reveal an unusual mode of oligomerization which relies predominantly on the cytosolic STAS domain. Our data illustrates conformational transitions of Slc26a9, supporting a rapid alternate-access mechanism which mediates uncoupled chloride transport with negligible bicarbonate or sulfate permeability. The characterization of structure-guided mutants illuminates the properties of the ion transport path, including a selective anion binding site located in the center of a mobile module within the transmembrane domain. This study thus provides a structural foundation for the understanding of the entire SLC26 family and potentially facilitates their therapeutic exploitation.

Structure of a volume-regulated anion channel of the LRRC8 family.

Volume-regulated anion channels are activated in response to hypotonic stress. These channels are composed of closely related paralogues of the leucine-rich repeat-containing protein 8 (LRRC8) family that co-assemble to form hexameric complexes. Here, using cryo-electron microscopy and X-ray crystallography, we determine the structure of a homomeric channel of the obligatory subunit LRRC8A. This protein conducts ions and has properties in common with endogenous heteromeric channels. Its modular structure consists of a transmembrane pore domain followed by a cytoplasmic leucine-rich repeat domain. The transmembrane domain, which is structurally related to connexin proteins, is wide towards the cytoplasm but constricted on the outside by a structural unit that acts as a selectivity filter. An excess of basic residues in the filter and throughout the pore attracts anions by electrostatic interaction. Our work reveals the previously unknown architecture of volume-regulated anion channels and their mechanism of selective anion conduction.

The application of 7H-indolo[1,2-a]quinolinium merocyanine as a new water sensor in organic solvents.

ABSTRACT: The 7H-indolo[1,2-a]quinolinium merocyanine was applied as a new water sensor in organic solvents such as ethanol, propane-1-ol, propane-2-ol, DMSO, and DMF. The spectral changes of the dye caused by the addition of increasing amount of water into an organic solvent were investigated. Based on the results, the calibration curves were found as a relation between the position of the absorption band of the dye and the water concentration ranging from about 0.05 to 11% (w/w). In case of ethanol, propane-1-ol and propane-2-ol the plots were linear, whereas in DMSO and DMF, better results were obtained with the use of a polynomial function. The method allowed to determine the water content in a fast and precise manner.

Single-Particle Electron Microscopy Analysis of DNA Repair Complexes.

DNA repair complexes play crucial roles in maintaining genome integrity, which is essential for the survival of an organism. The understanding of their modes of action is often obscure due to limited structural knowledge. Structural characterizations of these complexes are often challenging due to a poor protein production yield, a conformational flexibility, and a relatively high molecular mass. Single-particle electron microscopy (EM) has been successfully applied to study some of these complexes as it requires low amount of samples, is not limited by the high molecular mass of a protein or a complex, and can separate heterogeneous assemblies. Recently, near-atomic resolution structures have been obtained with EM owing to the advances in technology and image processing algorithms. In this chapter, we review the EM methodology of obtaining three-dimensional reconstructions of macromolecular complexes and provide a workflow that can be applied to DNA repair complex assemblies.

The Dimeric Architecture of Checkpoint Kinases Mec1ATR and Tel1ATM Reveal a Common Structural Organization.

The phosphatidylinositol 3-kinase-related protein kinases are key regulators controlling a wide range of cellular events. The yeast Tel1 and Mec1·Ddc2 complex (ATM and ATR-ATRIP in humans) play pivotal roles in DNA replication, DNA damage signaling, and repair. Here, we present the first structural insight for dimers of Mec1·Ddc2 and Tel1 using single-particle electron microscopy. Both kinases reveal a head to head dimer with one major dimeric interface through the N-terminal HEAT (named after Huntingtin, elongation factor 3, protein phosphatase 2A, and yeast kinase TOR1) repeat. Their dimeric interface is significantly distinct from the interface of mTOR complex 1 dimer, which oligomerizes through two spatially separate interfaces. We also observe different structural organizations of kinase domains of Mec1 and Tel1. The kinase domains in the Mec1·Ddc2 dimer are located in close proximity to each other. However, in the Tel1 dimer they are fully separated, providing potential access of substrates to this kinase, even in its dimeric form.

The structural basis for enhancer-dependent assembly and activation of the AAA transcriptional activator NorR.

σ(54)-dependent transcription controls a wide range of stress-related genes in bacteria and is tightly regulated. In contrast to σ(70), the σ(54)-RNA polymerase holoenzyme forms a stable closed complex at the promoter site that rarely isomerises into transcriptionally competent open complexes. The conversion into open complexes requires the ATPase activity of activator proteins that bind remotely upstream of the transcriptional start site. These activators belong to the large AAA protein family and the majority of them consist of an N-terminal regulatory domain, a central AAA domain and a C-terminal DNA binding domain. Here we use a functional variant of the NorR activator, a dedicated NO sensor, to provide the first structural and functional characterisation of a full length AAA activator in complex with its enhancer DNA. Our data suggest an inter-dependent and synergistic relationship of all three functional domains and provide an explanation for the dependence of NorR on enhancer DNA. Our results show that NorR readily assembles into higher order oligomers upon enhancer binding, independent of activating signals. Upon inducing signals, the N-terminal regulatory domain relocates to the periphery of the AAA ring. Together our data provide an assembly and activation mechanism for NorR.