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The Receptor Binding Domain (RBD) of SARS-CoV-2 is the primary target of neutralizing antibodies. We designed a trimeric, highly thermotolerant glycan engineered RBD by fusion to a heterologous, poorly immunogenic disulfide linked trimerization domain derived from cartilage matrix protein. The protein expressed at a yield of ∼80-100 mg/liter in transiently transfected Expi293 cells, as well as CHO and HEK293 stable cell lines and formed homogeneous disulfide-linked trimers. When lyophilized, these possessed remarkable functional stability to transient thermal stress of upto 100 °C and were stable to long term storage of over 4 weeks at 37 °C unlike an alternative RBD-trimer with a different trimerization domain. Two intramuscular immunizations with a human-compatible SWE adjuvanted formulation, elicited antibodies with pseudoviral neutralizing titers in guinea pigs and mice that were 25-250 fold higher than corresponding values in human convalescent sera. Against the beta (B.1.351) variant of concern (VOC), pseudoviral neutralization titers for RBD trimer were ∼ three-fold lower than against wildtype B.1 virus. RBD was also displayed on a designed ferritin-like Msdps2 nanoparticle. This showed decreased yield and immunogenicity relative to trimeric RBD. Replicative virus neutralization assays using mouse sera demonstrated that antibodies induced by the trimers neutralized all four VOC to date, namely B.1.1.7, B.1.351, P.1 and B.1.617.2 without significant differences. Trimeric RBD immunized hamsters were protected from viral challenge. The excellent immunogenicity, thermotolerance, and high yield of these immunogens suggest that they are a promising modality to combat COVID-19, including all SARS-CoV-2 VOC to date.

Uracil is not always a mistakenly occurring base in DNA. Uracils in DNA genomes are known to be important in the life cycles of Bacillus subtilis phages (PBS1/2) and the malarial parasite, Plasmodium falciparum; and have been implicated in the development of fruit fly and antibody maturation in B-lymphocytes. Availability of a sensitive, specific and robust technique for the detection uracils in genes/genomes is essential to understand its varied biological roles. Mycobacterium smegmatis UdgX (MsmUdgX), identified and characterised in our laboratory, forms covalent complexes with the uracil sites in DNA in a specific manner. MsmUdgX cleaves the glycosidic bond between uracil and the deoxyribose sugar in DNA to produce uracilate and oxocarbenium ions. The oxocarbenium ion is then captured into a covalent complex by the nucleophilic attack of a histidine side chain of MsmUdgX. Here, we describe the use of a fusion protein, mCherry tagged MsmUdgX (mChUdgX), which combines the property of MsmUdgX to covalently and specifically bind the uracil sites in the genome, with the sensitivity of fluorescent detection of mCherry as a reporter. We show that both the purified mChUdgX and the Escherichia coli cell-extracts overexpressing mChUdgX provide high sensitivity and specificity of detecting uracils in DNA.

In this manuscript, we report the application of graphene oxide (GO) in the preparation of cryo-electron microscopy (cryo-EM) and transmission electron microscopy (TEM) grids. We treated GO with water and organic solvents, such as, methanol, ethanol and isopropanol separately to isolate significantly large GO monolayer flake to fabricate the grids for cryo-EM and TEM study. We implemented a simplified approach to isolate flakes of GO monolayer for constructing the TEM grids, independent of expensive heavy equipment (Langmuir–Blodgett trough, glow-discharge system, carbon-evaporator or plasma-cleaner or peristaltic pumps). We employed confocal microscopy, SEM and TEM to characterize the flake size, stability and transparency of the GO monolayer and atomic force microscopy (AFM) to probe the depth of GO coated grids. Additionally, GO grids are visualized at cryogenic condition for suitability of GO monolayer for cryo-EM study. In addition, GO-Met-H2O grids reduce the effect of preferred orientation of biological macromolecules within the amorphous ice. The power-spectrum and contrast-transfer-function unequivocally suggest that GO-Met-H2O fabricated holey grids have excellent potential for application in high-resolution structural characterization of biomolecules. Furthermore, only 200 movies and ~8000 70S ribosome particles are selected on GO-coated grids for cryo-EM reconstruction to achieve high-resolution structure.

Binding to the von Willebrand factor (VWF) D′D3 domains protects factor VIII (FVIII) from rapid clearance. We performed single-particle electron microscopy (EM) analysis of negatively stained specimens to examine the architecture of D′D3 alone and in complex with FVIII. The D′D3 dimer ([D′D3]2) comprises 2 antiparallel D3 monomers with flexibly attached protrusions of D′. FVIII-VWF association is primarily established between the FVIII C1 domain and the VWF D′ domain, whereas weaker interactions appear to be mediated between both FVIII C domains and the VWF D3 core. Modeling the FVIII structure into the three-dimensional EM reconstructions of [D′D3]2-FVIII ternary and quaternary complexes indicates conformational rearrangements of the FVIII C domains compared with their disposition in the unbound state. These results illustrate the cooperative plasticity between VWF and FVIII that coordinate their high-affinity interaction.

Polyketide natural products constitute a broad class of compounds with diverse structural features and biological activities. Their biosynthetic machinery, represented by type I polyketide synthases (PKSs), has an architecture in which successive modules catalyse two-carbon linear extensions and keto-group processing reactions on intermediates covalently tethered to carrier domains. Here we used electron cryo-microscopy to determine sub-nanometre-resolution three-dimensional reconstructions of a full-length PKS module from the bacterium Streptomyces venezuelae that revealed an unexpectedly different architecture compared to the homologous dimeric mammalian fatty acid synthase. A single reaction chamber provides access to all catalytic sites for the intramodule carrier domain. In contrast, the carrier from the preceding module uses a separate entrance outside the reaction chamber to deliver the upstream polyketide intermediate for subsequent extension and modification. This study reveals for the first time, to our knowledge, the structural basis for both intramodule and intermodule substrate transfer in polyketide synthases, and establishes a new model for molecular dissection of these multifunctional enzyme systems. Polyketide synthases are multidomain enzymes that produce polyketides, which form the basis of many therapeutic agents; here, electron cryo-microscopy is used to establish the structure of a bacterial full-length module, and to elucidate the structural basis of both intramodule and intermodule substrate transfer. Polyketide synthases (PKSs) are multi-domain enzyme complexes producing polyketides, a large class of secondary metabolites — in other words, natural products. Two papers from Georgios Skiniotis and colleagues use cryo-electron microscopy to probe the structure of an intact module of a full-length multienzyme PKS module involved in pikromycin biosynthesis in the bacterium Streptomyces venezuelae in different functional states. The structures reveal how the ketosynthase, acyltransferase, ketoreductase and acyl carrier protein (ACP) domains interact during the catalytic cycle. In each state the ACP is differentially positioned to facilitate intermediate transfer for the next catalytic step and for transfer to the next module.

Polyketide synthases (PKSs) are multidomain enzymes that produce polyketides, which form the basis of many therapeutic agents; here, electron cryo-microscopy is used to probe the structure of an intact module of a multi-enzyme PKS in different functional states. Polyketide synthases (PKSs) are multi-domain enzyme complexes producing polyketides, a large class of secondary metabolites — in other words, natural products. Two papers from Georgios Skiniotis and colleagues use cryo-electron microscopy to probe the structure of an intact module of a full-length multienzyme PKS module involved in pikromycin biosynthesis in the bacterium Streptomyces venezuelae in different functional states. The structures reveal how the ketosynthase, acyltransferase, ketoreductase and acyl carrier protein (ACP) domains interact during the catalytic cycle. In each state the ACP is differentially positioned to facilitate intermediate transfer for the next catalytic step and for transfer to the next module. The polyketide synthase (PKS) mega-enzyme assembly line uses a modular architecture to synthesize diverse and bioactive natural products that often constitute the core structures or complete chemical entities for many clinically approved therapeutic agents1. The architecture of a full-length PKS module from the pikromycin pathway of Streptomyces venezuelae creates a reaction chamber for the intramodule acyl carrier protein (ACP) domain that carries building blocks and intermediates between acyltransferase, ketosynthase and ketoreductase active sites (see accompanying paper2). Here we determine electron cryo-microscopy structures of a full-length pikromycin PKS module in three key biochemical states of its catalytic cycle. Each biochemical state was confirmed by bottom-up liquid chromatography/Fourier transform ion cyclotron resonance mass spectrometry. The ACP domain is differentially and precisely positioned after polyketide chain substrate loading on the active site of the ketosynthase, after extension to the β-keto intermediate, and after β-hydroxy product generation. The structures reveal the ACP dynamics for sequential interactions with catalytic domains within the reaction chamber, and for transferring the elongated and processed polyketide substrate to the next module in the PKS pathway. During the enzymatic cycle the ketoreductase domain undergoes dramatic conformational rearrangements that enable optimal positioning for reductive processing of the ACP-bound polyketide chain elongation intermediate. These findings have crucial implications for the design of functional PKS modules, and for the engineering of pathways to generate pharmacologically relevant molecules.

A broad survey of both the simple and double decomposition modes of transition metal alkyls has been undertaken. From the two main processes of C–M bond rupture, homolysis, and β-metal hydride elimination, the following modes of decomposition and types of useful reagents generated can be distinguished: 1) reductive decomposition of RnMEm–n to yield subvalent, carbene- or free-radical-like MEm–n, which can cleave π- or σ-bonds by direct epimetallation; 2) β-metal hydride elimination to produce hydrometallating agents; 3) combined reductive and β-metal hydride eliminations to produce anionic, and hence more nucleophilic, epimetallating agents; 4) α-metal hydride elimination thought to be involved in the transition state leading to C–C bond coupling from (RR'HC)2ME2 derivatives; 5) α,μ-elimination from lithiated RHC(Li)–MEn–1, which produces alkylidenes, RHC=MEn–2, capable of ROMP catalysis; 6) metal–hydrogen metathesis, whereby sufficiently acidic C–H bonds become C–M bonds; 7) metal–metal metathesis, whereby RR'ME2 undergoes an equilibrating redistribution reaction to form R2ME2 and R'2ME2; 8) transfer epimetallation, in which epimetallation (point 1) is achieved with R2ME2 in one step by transfer of ME2 to the substrate with the concomitant loss of R groups; and 9) transfer epimetallation of olefins by R2TiE2 in hydrocarbons to produce titana(IV)cyclopropanes, which serve as active sites in Ziegler–Natta polymerization. In each situation, the reaction-mechanistic features are analyzed in terms of existing experimental data and evidence. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2005)

The mechanism of the rearrangement of diorganylzirconium dialkoxides, such as R1(R2CH2)Zr(OEt)2, into organylzirconium hydride alkoxides of the type R1R2CHZr(OEt)2H, has been investigated as a function of the R1 and R2 substituents and been found to involve optimally an intramolecular migration of R1 to an sp3-hybridized C−H center. A remarkably rapid transfer hydrozirconation between Bun2Zr(OEt)2 and the olefin RCHCH2 now permits the synthesis of (RCH2CH2)2Zr(OEt)2, whose rearrangement offers a route to the linear olefin dimer R(CH2)4R.

Vibrio cholerae hemolysin (HlyA) is a 65 kDa pore-forming toxin which causes lysis of target eukaryotic cells by forming heptameric channels in the plasma membrane. Deletion of the 15 kDa C-terminus β-prism carbohydrate-binding domain generates a 50 kDa truncated variant (HlyA50) with 1000-fold-reduced pore-forming activity. Previously, we showed by cryo-electron microscopy that the two toxin oligomers have central channels, but the 65 kDa toxin oligomer is a seven-fold symmetric structure with bowl-, ring-, and arm-like domains, whereas the 50 kDa oligomer is an asymmetric jar-like heptamer. In the present study, we determined three-dimensional(3D) structures of HlyA and HlyA50 in presence of erythrocyte stroma and observed that interaction of the 65 kDa toxin with the stroma induced a significant decrease in the height of the β-barrel oligomer with a change in conformation of the ring- and arm-like domains of HlyA. These features were absent in interaction of HlyA50 with stroma. We propose that this conformational transition is critical for membrane-insertion of the toxin.

Benzylic hydrocarbons of the types Ar−CH3 and Ar2CH2 undergo zirconation at their benzylic C−H bonds by Bun2Zr(OEt)2 at room temperature in Lewis basic solvents and catalyzed by lithium ethoxide. Upon heating, such benzylic organozirconium derivatives can undergo C−C bond formation, either intermolecularly or intramolecularly, with the elimination of zirconium(IV) hydrides of the type H2Zr(OEt)2. Such oxidative dimerizations or cyclizations are exemplified by the conversion of toluene into trans-stilbene or the formation of 9,10-dihydrophenanthrene from 2,2 ‘-dimethylbibenzyl.

The complexes [VIVO(L)(bpy)] and [VVO(L)(OMe)(OHMe)] (H2L  Schiff base condensate of salicylaldehyde and an α-amino acid and bpy = 2,2′-bipyridine) have been synthesized. The [VIVO(L)(OMe)(OHMe)]− species have been electrogenerated in solutions. The VO 3+ − VO 2+ E 1 2 values of [VO(L)(OHMe)] are lower than those of [VO(L)(bpy)] by ≈750 mV. Upon exposure to air [VO(L)(OMe)(OHMe)]− is spontaneously oxidized to [VO(L)(OMe)(OHMe)]. The [VO(L)(bpy)] and [VO(L)(OMe) (OHMe)]− complexes display dxy1 type axial EPR spectra. The 51V hyperfine constants follow the order [VO(L)(OMe)(OHMe)]− > [VO(L)(bpy)]. The dxy → dxz, dyz transition in these complexes appear in the region 700–720 nm.

The complexes [VO(L1)(hquin)] and [VO(L2)(hquin)] incorporating N-salicylidene-glycinate and -L-phenylalaninate respectively have been synthesised in excellent yields from [VIVO(L)(H2O)] and quinolin-8-ol (Hhquin) in air which acts as the oxidant. The crystal structure of [VO(L2)(hquin)] revealed tridentate ONO and bidentate ON binding by [L2]2– and hquin– respectively. The V–O (carboxylate) bond length is longer than V–O (phenolate) by ≈0.1 A. The CD spectrum of [VO(L2)(hquin)] in the visible region revealed the composite nature of the phenolato→vanadium charge-transfer band. The absolute configuration of [VO(L2)(hquin)] is CS both in the crystalline state and in solution. In CDCl3 solution the 1H NMR spectrum of the complex shows that the amino acid side chain has the same conformation as that in the crystal lattice. The complexes display the quasi-reversible one-electron couple [VO(L)(hquin)]–[VO(L)(hquin)]– near 0.0 V vs. saturated calomel electrode. Electrogenerated solutions of [VO(L)(hquin)]– are EPR-active corresponding to a dxy1 configuration.

Water soluble manganese(III) complexes of sulphonated azophenol and salicylaldimine ligands 3–6, have been isolated: Na3[Mn(LS)2]·nH2O and Na5[Mn(LSS)2]·nH2O (n = 4 or 5) where H3LS and H4LSS represent mono- and di-sulphonated ligands, respectively. The coordination sphere of the complexes are of the type Mn(ONO)2 where the O and N atoms are of phenolic and azo/azomethine type. The complexes are uniformly high-spin (t23e1) in character (∼5 μB). The manganese(IV)-manganese(III) couple in water has cyclic voltammetric E 1 2 values in the range 0.30–0.50 V vs SCE and it is estimated that the potentials increase by 70 mV for each sulphonate substitution. The manganese(IV) complexes [Mn(LS)2]2− and [Mn(LSS)2]4− have been quantitatively generated in solution both chemically and electrochemically. Their X-band EPR spectra consist of a strong resonance near g = 2. In frozen water-ethylene glycol glass (77 K) the g = 2 resonance shows 55Mn hyperfine structure (A, 94–97 G). Forbidden lines are also resolved and with their help the axial zero-field splitting parameter D is estimated to be 0.012 cm−1. It is small compared to the X-band quantum, 0.31 cm−1.

The reaction of bis(acetylacetonato)oxovanadium(IV) with a mixture of a tridentate ONO-co-ordinating hydrazone H2L1–H2L4(general abbreviation H2L; condensates of benzoylhydrazine with benzoylacetone, salicylaldehyde, 2-hydroxy-1-naphthaldehyde and 2-hydroxybutyrophenone respectively) and a bidentate compound 2,2′-bipyridine (bipy), 1,10-phenanthroline (phen) or quinolin-8-ol (Hquin) has afforded the complexes [VIVO(L)(bipy)]. [VIVO(L)(phen)] and [VVO(L)(quin)] in high yields. The crystal structure of [VO(L1)(quin)] has been determined, revealing the distorted-octahedral VO(ONO)(ON) co-ordination sphere with the L1 ligand spanning meridionally. The VO distance is 1.592(3)A and the atom lying trans to VO is the quin nitrogen. There is a large decrease (≈ 700 mV) in the VO3+–VO2+E½ values between [VO(L)(bipy)]{or [VO(L)(phen)]} and [VO(L)(quin)]. The complex [VIVO(L)(quin)]– was electrogenerated in solution but spontaneously reoxidized to [VVO(L)(quin)] in air. For a given bidentate ligand, the VO3+–VO2+E½ values follow the order L1 < L4 < L2 < L3. The ESR spectra of the VO2+ complexes correspond to an axially compressed dxy1 configuration. The 51V hyperfine constants are slightly larger for [VO(L)(quin)]– than for [VO(L)-(bipy)] and [VO(L)(phen)]. The dxy→ dxz,dyz transition of the tetravalent complexes occurs in the region 700–950 nm.

The aerial reaction of 2,2'-dihydroxyazobenzene (H 2 L) with bis(acetylacetonato)oxovanadium(IV) in methanol affords binuclear V v 2 O 3 L 2 . In the presence of KOH, KV v O 2 L.1.5H 2 O is isolated. Dilute acids convert VO 2 L. to V 2 O 3 L 2 via V v O(OH)L. The electronic and IR spectra of the complexesare described. In crystalline KVO 2 L.1.5H 2 O, the vanadium(V) atom has square pyramidal geometry. The base is formed by coordinated ONO atoma of L 2- and an oxo oxygen atom

The reaction of the tris(arylazo oximato)iron(II) anion, Fe(RL) 3 - (R=Me, Ph, p-MeC 6 H 4 ), with Cu(MeCN) 4 + in dichloromethane under air affords the trinuclear complex Cu II Fe II 2 (RL) 6 . In the presence of PPh 3 in the reaction mixture, the binuclear complex Cu I Fe II (RL) 3 (PPh 3 ) is isolated. The X-ray structures of CuFe(MeL) 3 (PPh 3 ) and CuFe 2 (PhL) 6 .2CH 2 Cl 2 are reported. In both complexes the Fe(RL) 3 - fragment acts as a facially disposed tridentate oximato-O 3 ligand

In the title families the ONO ligands are deprotonated forms of N-(2-hydroxyphenyl)salicylaldimine (H 2 amp), 2,2'-dihydroxyazobenzene (H 2 azp), N-(2-carboxyphenyl)salicylaldimine (H 2 amc), or 2-hydroxy-2'-carboxy-5-methylazobenzene (H 2 azc). The ON ligand is deprotonated 8-quinolinol (Hhq), and the NN ligands are 2,2'- bipyridine (bpy) or 1,10-phenanthroline (phen). Reaction of VO(acac) 2 with a mixture of H 2 amp and bpy or Hhq in methanol affords V IV O(amp)(bpy) or V V O(amp)(hq)

Vibrio cholerae hemolysin (HlyA) is a 65-kDa water-soluble pore-forming toxin that causes lysis of eukaryotic cells by destroying selective permeability of the plasma membrane bilayer. The HlyA monomer self-assembles on the target cell surface to the more stable β-barrel amphipathic heptamer, which inserts into the membrane bilayer to form a diffusion channel. Deletion of the 15-kDa β-prism lectin domain at the C terminus generates a 50-kDa hemolysin variant (HlyA50) with an ∼1,000-fold decrease in hemolytic activity. Because functional differences are eventually dictated by structural differences, we determined three-dimensional structures of 65- and 50-kDa HlyA oligomers, using cryo-electron microscopy and single-particle methods. Our study clearly shows that the HlyA oligomer has sevenfold symmetry but that the HlyA50 oligomer is an asymmetric molecule. The HlyA oligomer has bowl-like, arm-like, and ring-like domains. The bowl-like domain is coupled with the ring-like domain, and seven side openings are present just beneath the ring-like domain. Although a central channel is present in both HlyA and HlyA50 oligomers, they differ in pore size as well as in shape of the molecules and channel. These structural differences may be relevant to the striking difference in efficiencies of functional channel formation by the two toxin forms.

The reaction of [VIVO(L)(H2O)] with ethane-1,2-diol (H2ed) in methanol in air afforded complexes of type [VVO(L)(Hed)] in very good yields (H2L1=N-salicylideneglycine, H2L2=N-salicylidene-L-phenylalanine). The crystal structure of [VO(L2)(Hed)] has revealed the presence of tridentate [L2]2– and bidentate Hed–. In the latter the alcoholic oxygen lies trans to the oxo atom. In the VO5N co-ordination sphere the five vanadium–oxygen lengths follow the order: oxo < alkoxidic < phenoxidic < carboxylic < alcoholic. The absolute configuration of the complex is CS. The lattice consists of hydrogen-bonded (between alcoholic and carboxylic oxygen atoms) dimers having two-fold symmetry. The CD spectrum of [VO(L2)(Hed)] has revealed the composite nature of the O(alkoxide)→ V charge-transfer band in the visible region. In the 1H NMR spectra of the complexes the four methylene protons of co-ordinated Hed– are inequivalent. The [VVO(L)(Hed)] complexes represent authentic examples of the very rare mononuclear oxovanadium species incorporating ethane-1,2-diol as a ligand.

In the title complex [VOL1L2] derived from the tridentate 2-hydroxy-2′-carboxy-5-methylazobenzene (H2L1) and the bidentate quinolin-8-ol (HL2) the V–O(carboxylate) and V–N(azo) distances are respectively 1.892(4) and 2.098(5)A; frozen solution (77 K) EPR parameters of electrogenerated [VOL1L2]– lie close to those of reduced bromoperoxidase (low pH).