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Neuraminidase (NA) inhibitors are one of the two major classes of antivirals available for the treatment and prevention of influenza. X-ray crystal structure determination of NA head domains and their complexes with various inhibitors are of importance for the design and optimization of anti-influenza drugs. However, the globular tetrameric properties of NA head domains may produce crystals with pathological imperfections, lattice-translocation defects, making structure determination no longer straightforward. In this report, using a crystal of the NA head domain from the Wuhan Asiatic toad influenza virus as an example, the identification and solution of this type of crystal pathology are presented. Furthermore, its underlying mechanism of formation is explored.

Receptor dimerization of urokinase-type plasminogen activator receptor (uPAR) was previously identified at protein level and on the cell surface. Recently, a dimeric form of mouse uPAR isoform 2 was proposed, which induced kidney disease. Here, we report the crystal structure of human uPAR dimer at 2.96 Å. The structure reveals enormous conformational changes of the dimer compared to the monomeric structure: D1 of uPAR opens up into a large expanded loop that captures a β-hairpin loop of a neighboring uPAR to form an expanded β-sheet, leading to an elongated, highly intertwined dimeric uPAR. Based on the structure, we identify the E49P mutation promoting dimer formation. The mutation increases receptor binding to amino terminal fragment (ATF) of its primary ligand uPA, induces the receptor to distribute to the basal membrane, promotes cell proliferation, and alters cell morphology via the ERK activation of β1 integrin signaling. These results reveal the structural basis for uPAR dimerization, its effect on cell function, and provide new insight and tools to study this multifunctional receptor.

Significance Lysyl oxidases (LOXs) catalyze oxidative deamination of peptidyl lysines on collagen and elastin, generating a highly reactive aldehyde group to initiate intermolecular cross-linking. In humans, this reaction plays fundamental roles in the formation and repair of extracellular matrix fiber networks and the development of connective tissues. Moreover, a relationship between LOX proteins and tissue fibrosis and cancer has been established. Since the identification of prototypic LOX, the atomic structure of LOX protein has remained to be elucidated. Here, we present the high-resolution structure of human lysyl oxidase-like 2 protein. The zinc-bound precursor-state structure provides an important framework for understanding the structure–function relationship of the LOX family and for drug discovery that aims to block LTQ generation of LOX proteins., Lysyl oxidases (LOXs), a type of copper- and lysyl tyrosylquinone (LTQ) -dependent amine oxidase, catalyze the oxidative deamination of lysine residues of extracellular matrix (ECM) proteins such as elastins and collagens and generate aldehyde groups. The oxidative deamination of lysine represents the foundational step for the cross-linking of elastin and collagen and thus is crucial for ECM modeling. Despite their physiological significance, the structure of this important family of enzymes remains elusive. Here we report the crystal structure of human lysyl oxidase-like 2 (hLOXL2) at 2.4-Å resolution. Unexpectedly, the copper-binding site of hLOXL2 is occupied by zinc, which blocks LTQ generation and the enzymatic activity of hLOXL2 in our in vitro assay. Biochemical analysis confirms that copper loading robustly activates hLOXL2 and supports LTQ formation. Furthermore, the LTQ precursor residues in the structure are distanced by 16.6 Å, corroborating the notion that the present structure may represent a precursor state and that pronounced conformational rearrangements would be required for protein activation. The structure presented here establishes an important foundation for understanding the structure–function relationship of LOX proteins and will facilitate LOX-targeting drug discovery.

In this study, the effects of Al addition on the microstructure and hardness of CoCrFeNi3 high entropy alloy prepared by direct energy deposition (DED) were investigated. As Al content increased, the structure of alloy transformed from a face-centered cubic (FCC)-based crystal structure (including the ordered L12 phase) into a duplex FCC coexisting with body-centered cubic (BCC) structure. Besides, eutectic precipitations appeared when Al content in the alloy exceeded 12.5%. The increase in hardness of the alloy was mostly attributed to the solid solution hardening and precipitation hardening.

Four structurally novel transition-metal (TM)-bridging risedronate (Ris)-based polyoxomolybdates (Ris-based PODs), Cs4Na2[{MO(H2O)2}(Mo2O5L)]2·16H2O [M = Ni (1), Co (2)], Cs2Na2H[(CrO2){(Mo2O5)(HL)}2]·17H2O (3) Cs11Na4H7[MnO2{HL(Mo2O5)MnO2(H2O)(Mo2O4) L}2]2·26H2O (4) [L = {O3PC(O)(CH2-3-C5NH4)PO3}, HL = {O3PC(O)(CH2-3-C5NH5)PO3}], have been presented through conventional aqueous solution and characterized by single crystal X-ray diffraction and PXRD, SEM images and EDX analyses, IR and UV spectra, elemental analyses and X-ray photoelectron spectroscopy. Compared to the reported edge-sharing {MoV2O4}, the face-sharing building block {Mo2VIO5} is employed in compounds 1–4, which are uncommon in Ris-based PODs. Furthermore, preliminary variable-temperature magnetic susceptibility measurements indicate that 1 and 4 display weak ferromagnetic behaviors and antiferromagnetic interactions, respectively.

Iron is essential for life. Accessing iron from the environment can be a limiting factor that determines success in a given environmental niche. For bacteria, access of chelated iron from the environment is often mediated by TonB-dependent transporters (TBDTs), which are β-barrel proteins that form sophisticated channels in the outer membrane. Reports of iron-bearing proteins being used as a source of iron indicate specific protein import reactions across the bacterial outer membrane. The molecular mechanism by which a folded protein can be imported in this way had remained mysterious, as did the evolutionary process that could lead to such a protein import pathway. How does the bacterium evolve the specificity factors that would be required to select and import a protein encoded on another organism’s genome? We describe here a model whereby the plant iron–bearing protein ferredoxin can be imported across the outer membrane of the plant pathogen Pectobacterium by means of a Brownian ratchet mechanism, thereby liberating iron into the bacterium to enable its growth in plant tissues. This import pathway is facilitated by FusC, a member of the same protein family as the mitochondrial processing peptidase (MPP). The Brownian ratchet depends on binding sites discovered in crystal structures of FusC that engage a linear segment of the plant protein ferredoxin. Sequence relationships suggest that the bacterial gene encoding FusC has previously unappreciated homologues in plants and that the protein import mechanism employed by the bacterium is an evolutionary echo of the protein import pathway in plant mitochondria and plastids., Author summary Earth’s carbon cycle depends on saprophytic microbes to rot old or diseased plant matter and recycle carbon from that biomass. Some bacteria (phytopathogens) have evolved to cause disease and rot in even healthy plants and may have utility as biological control agents against noxious weeds. To understand the mechanisms driving each of these scenarios has significance in environmental engineering and agriculture. Access to iron is a limiting factor for bacteria-mediated plant rot. Here, we show how a plant-pathogenic bacteria has reevolved a mechanism, analogous to the protein import pathways that evolved in plant plastids and mitochondria, to import the plant iron–bearing protein ferredoxin from plant tissue. The study is based on structural and biophysical characterization of a key M16 family protease, FusC, resident inside the bacterial outer membrane.

P-type layered oxide is a promising cathode candidate for sodium-ion batteries (SIBs), but faces the challenge of simultaneously realizing high rate capability and long cycle life. Herein, Co-substituted Nax MnO2 nanosheets with tunable P2/P3 biphase structures are synthesized by a novel dealloying-annealing strategy. The optimized P2/P3-Na0.67 Mn0.64 Co0.30 Al0.06 O2 cathode delivers an excellent rate capability of 83 mA h g-1 at a high current density of 1700 mA g-1 (10 C), and an outstanding cycling stability over 500 cycles at 1000 mA g-1 . This excellent performance is attributed to the unique P2/P3 biphases with stable crystal structures and fast Na+ diffusion between open prismatic Na sites. Moreover, operando X-ray diffraction is applied to explore the structural evolution of Na0.67 Mn0.64 Co0.30 Al0.06 O2 during the Na+ extraction/insertion processes, and the P2-P2' phase transition is effectively suppressed. Operando Raman technique is utilized to explore the structural superiority of P2/P3 biphase cathode compared with pure P2 or P3 phase. This work highlights precisely tailoring the phase composition as an effective strategy to design advanced cathode materials for SIBs.

The essential step for epitaxial growth of tetragonal Mn3Ge films with high perpendicular magnetic anisotropy (PMA) is to choose suitable substrates with small lattice misfit. The exploration process has involved large efforts on depositing films on single crystalline substrates using buffer layers preferably formed from Cr or Pt, but they lacked a systematically comparative investigation for practical applications. This study investigates the structural, surface and magnetic properties of ultrathin Fe (2 nm) seed layer to induce homoepitaxial Mn3Ge films on MgO (0 0 1) substrates compared with that of the heteroepitaxial Mn3Ge films on three typical buffer layers, such as Cr (40 nm), Cr (20 nm)/Pt (10 nm), Fe (2 nm)/Pt (20 nm). Furthermore, a correlation between film strain and film quality has been established, which is critical for spintronics applications. More importantly, we attribute the homoepitaxial growth of Mn3Ge films on the ultrathin Fe seed layers to the Fe diffusion and formation of Fe-Mn-Ge alloy at the interface, and confirm this supposition with HAADF-STEM characterizations. The Fe-doped Mn3Ge interlayer can act as the gradual buffer layer, and lead to a high-quality crystal structure and extremely high magnetic squareness ratio of Mn3Ge films in a large range of thickness (100 ~ 400 nm). This result offers a new concept of high-quality growth of D022-Mn3Ge films, which may enhance the prospect for tetragonal Mn3Ge thin films in superior spintronics applications.

Quasi-racemic crystallography has been used to determine the X-ray structures of K27-linked ubiquitin (Ub) chains prepared through total chemical synthesis. Crystal structures of K27-linked di- and tri-ubiquitins reveal that the isopeptide linkages are confined in a unique buried conformation, which provides the molecular basis for the distinctive function of K27 linkage compared to the other seven Ub chains. K27-linked di- and triUb were found to adopt different structural conformations in the crystals, one being symmetric whereas the other triangular. Furthermore, bioactivity experiments showed that the ovarian tumor family de-ubiquitinase 2 significantly favors K27-linked triUb than K27-linked diUb. K27-linked triUb represents the so-far largest chemically synthesized protein (228 amino acids) that has been crystallized to afford a high-resolution X-ray structure.

The mechanistic target of rapamycin (mTOR) signal-transduction pathway plays a key role in regulating many aspects of metabolic processes. The central player of the mTOR signaling pathway, mTOR complex 1 (mTORC1), is recruited by the pentameric Ragulator complex and the heterodimeric Rag GTPase complex to the lysosomal membrane and thereafter activated. Here, we determined the crystal structure of the human Ragulator complex, which shows that Lamtor1 possesses a belt-like shape and wraps the other four subunits around. Extensive hydrophobic interactions occur between Lamtor1 and the Lamtor2-Lamtor3, Lamtor4-Lamtor5 roadblock domain protein pairs, while there is no substantial contact between Lamtor2-Lamtor3 and Lamtor4-Lamtor5 subcomplexes. Interestingly, an α-helix from Lamtor1 occupies each of the positions on Lamtor4 and Lamtor5 equivalent to the α3-helices of Lamtor2 and Lamtor3, thus stabilizing Lamtor4 and Lamtor5. Structural comparison between Ragulator and the yeast Ego1-Ego2-Ego3 ternary complex (Ego-TC) reveals that Ego-TC only corresponds to half of the Ragulator complex. Coupling with the fact that in the Ego-TC structure, Ego2 and Ego3 are lone roadblock domain proteins without another roadblock domain protein pairing with them, we suggest that additional components of the yeast Ego complex might exist.

Bacteria use vitamin C (L-ascorbic acid) as a carbon source under anaerobic conditions. The phosphoenolpyruvate-dependent phosphotransferase system (PTS), comprising a transporter (UlaA), a IIB-like enzyme (UlaB) and a IIA-like enzyme (UlaC), is required for the anaerobic uptake of vitamin C and its phosphorylation to L-ascorbate 6-phosphate. Here, we present the crystal structures of vitamin C-bound UlaA from Escherichia coli in two conformations at 1.65-Å and 2.35-Å resolution. UlaA forms a homodimer and exhibits a new fold. Each UlaA protomer consists of 11 transmembrane segments arranged into a 'V-motif' domain and a 'core' domain. The V motifs form the interface between the two protomers, and the core-domain residues coordinate vitamin C. The alternating access of the substrate from the opposite side of the cell membrane may be achieved through rigid-body rotation of the core relative to the V motif.

Racemic or quasi-racemic crystallography recently emerges as a useful technology for solution of the crystal structures of biomacromolecules. It remains unclear to what extent the biomacromolecules of opposite handedness can differ from each other in racemic or quasi-racemic crystallography. Here we report a finding that monomeric d-ubiquitin (Ub) has propensity to cocrystallize with different dimers, trimers, and even a tetramer of l-Ub. In these cocrystals the unconnected monomeric d-Ubs can self-assemble to form pseudomirror images of different oligomers of l-Ub. This monomer/oligomer cocrystallization phenomenon expands the concept of racemic crystallography. Using the monomer/oligomer cocrystallization technology we obtained, for the first time the X-ray structures of linear M1-linked tri- and tetra-Ubs and a K11/K63-branched tri-Ub.

Hexagonal Zn3(OH)2V2O7·2H2O nanoplates have been successfully synthesized via a facile and template-free hydrothermal method. The nanocrystals have a hexagonal shape with 650–750 nm in diameter and 120–140 nm in thickness. The possible mechanism of forming such hexagonal Zn3(OH)2V2O7·2H2O nanoplates may be due to its inherent anisotropic crystal structure. Magnetic hysteresis measurement indicates that the as-synthesized hexagonal Zn3(OH)2V2O7·2H2O nanoplates have weak ferromagnetic property at room temperature. Compared to the floriated-like nanostructured Zn3V2O7(OH)2(H2O)2 synthesized by a hydrothermal route, the as-prepared hexagonal Zn3(OH)2V2O7·2H2O nanoplates exhibited a significant increase in the methylene blue (MB) photodegradation rate under UV irradiation.

Nano-scale Tb-incorporated apatite (nano-Tb-AP) particles with different Tb contents (Tb/(Tb+Ca)) of 0%, 5%, 10% and 20% were synthesized through a simple wet chemical method in this study. The crystal structure, thermal stabilities, chemical groups, crystal morphologies and crystal sizes of the nano-Tb-AP particles were characterized by X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), and transmission electron microscopy (TEM), respectively. It was found that lattice constants, particle sizes, crystalline and thermal stability varied with the doped Tb contents. With the increasing of Tb content, the lattice constants, particle size, length/diameter ratio, crystalline and thermal stability of nano-Tb-AP gradually decrease. Especially, almost all the 20%Tb-AP nano particles had been decomposed at 1200 °C while only a few of the decomposed products (β-TCP) were detected in the Tb-free nano apatite powders. This kind of nano-scale Tb-incorporated apatite exhibits an extremely potential clinic application because it integrates both the excellent biological functions of Tb element and apatite in human body.

Phasing of the crystal structures of four standard proteins (lysozyme, trypsin, glucose isomerase and thaumatin) and a novel 69 kDa protein from Thermus thermophilus, TT0570, was performed using the single-wavelength anomalous diffraction of S atoms intrinsically present within the native protein molecules. To utilize the sulfur anomalous diffraction, the data sets were collected using the loopless data-collection method with chromium Kalpha X-rays of wavelength 2.29 A. Three phasing methods, MLPHARE, SHARP and OASIS-2004, were tested in combination with the DM or SOLOMON density-modification method. The results showed that the solvent contents are still an important factor for phasing with the S-SAD method, even when longer wavelength Cr Kalpha radiation is used. Of the three procedures, the improved direct phasing of OASIS-2004 with its implemented fragment feedback to the direct-method probability calculation gave the best results in determining the initial phases. For all five proteins, almost the entire models could be built automatically.

Keeping the Inflammasome in Check Nucleotide-binding and oligomerization domain (NOD)–like receptors (NLRs) play an important role in the detection of pathogens by cells of the innate immune system. For several NLR family members, activation results in relief from autoinhibition, oligomerization, and the recruitment of signaling components that together make up the inflammasome, a large multiprotein complex. The inflammasome protects the host by inducing cell death and cytokine secretion. The specific molecular mechanisms that regulate NLR activation and inhibition, however, are not well understood. Hu et al. (p. 172 , published online 13 June) report the crystal structure of autoinhibited NLR family member NLRC4, which reveals the domains that are critical for interaction with adenosine diphosphate to keep NLRC4 in its inactive state and the domains that mediate oligomerization of the protein upon activation.

The crystal structure of triclinic hen egg-white lysozyme (HEWL) has been refined against diffraction data extending to 0.65 A resolution measured at 100 K using synchrotron radiation. Refinement with anisotropic displacement parameters and with the removal of stereochemical restraints for the well ordered parts of the structure converged with a conventional R factor of 8.39% and an R(free) of 9.52%. The use of full-matrix refinement provided an estimate of the variances in the derived parameters. In addition to the 129-residue protein, a total of 170 water molecules, nine nitrate ions, one acetate ion and three ethylene glycol molecules were located in the electron-density map. Eight sections of the main chain and many side chains were modeled with alternate conformations. The occupancies of the water sites were refined and this step is meaningful when assessed by use of the free R factor. A detailed description and comparison of the structure are made with reference to the previously reported triclinic HEWL structures refined at 0.925 A (at the low temperature of 120 K) and at 0.95 A resolution (at room temperature).

TNFAIP8-like 2 (TIPE2) has an essential role in immune homeostasis, yet the underlying mechanism remains enigmatic. The high-resolution crystal structure of TIPE2 reveals a previously uncharacterized fold that is different from the predicted fold of a death effector domain (DED). Strikingly, TIPE2 contains a large, hydrophobic central cavity that is poised for cofactor binding. These structural features will be important for understanding the functions of TIPE2 and other TNFAIP8 family proteins.