Single particle cryo-electron microscopy with an enhanced 200 kV cryo-TEM configuration achieves near-atomic resolution.

Single particle cryogenic electron microscopy (cryo-EM) as a structural biology methodology has become increasingly attractive and accessible to investigators in both academia and industry as this ever-advancing technology enables successful structural determination of a wide range of protein and nucleic acid targets. Although data for many high resolution cryo-EM structures are still obtained using a 300 kV cryogenic transmission electron microscope (cryo-TEM), a modern 200 kV cryo-TEM equipped with an advanced direct electron detector and energy filter is a cost-effective choice for most single particle applications, routinely achieving sub 3 angstrom (Å) resolution. Here, we systematically evaluate performance of one such high-end configuration - a 200 kV Glacios microscope coupled with a Falcon 4 direct electron detector and Selectris energy filter (Glacios-F4-S). First, we evaluated data quality on the standard benchmarking sample, rabbit muscle aldolase, using three of the most frequently used cryo-EM data collection software: SerialEM, Leginon and EPU, and found that - despite sample heterogeneity - all final reconstructions yield same overall resolutions of 2.6 Å and map quality when using either of the three software. Furthermore, comparison between Glacios-F4-S and a 300 kV cryo-TEM (Titan Krios with Falcon 4) revealed nominal resolution differences in overall reconstructions of a reconstituted human nucleosome core particle, achieving 2.8 and 2.5 Å, respectively. Finally, we performed comparative data analysis on the human RAD51 paralog complex, BCDX2, a four-protein complex of approximately 150 kilodaltons, and found that a small dataset (≤1,000 micrographs) was sufficient to generate a 3.3 Å reconstruction, with sufficient detail to resolve co-bound ligands, AMP-PNP and Mg +2 . In summary, this study provides evidence that the Glacios-F4-S operates equally well with all standard data collection software, and is sufficient to obtain high resolution structural information of novel macromolecular complexes, readily acquiring single particle data rivaling that of 300 kV cryo-TEMs.

Cryo-EM structures of Uba7 reveal the molecular basis for ISG15 activation and E1-E2 thioester transfer.

ISG15 plays a crucial role in the innate immune response and has been well-studied due to its antiviral activity and regulation of signal transduction, apoptosis, and autophagy. ISG15 is a ubiquitin-like protein that is activated by an E1 enzyme (Uba7) and transferred to a cognate E2 enzyme (UBE2L6) to form a UBE2L6-ISG15 intermediate that functions with E3 ligases that catalyze conjugation of ISG15 to target proteins. Despite its biological importance, the molecular basis by which Uba7 catalyzes ISG15 activation and transfer to UBE2L6 is unknown as there is no available structure of Uba7. Here, we present cryo-EM structures of human Uba7 in complex with UBE2L6, ISG15 adenylate, and ISG15 thioester intermediate that are poised for catalysis of Uba7-UBE2L6-ISG15 thioester transfer. Our structures reveal a unique overall architecture of the complex compared to structures from the ubiquitin conjugation pathway, particularly with respect to the location of ISG15 thioester intermediate. Our structures also illuminate the molecular basis for Uba7 activities and for its exquisite specificity for ISG15 and UBE2L6. Altogether, our structural, biochemical, and human cell-based data provide significant insights into the functions of Uba7, UBE2L6, and ISG15 in cells.

Genomic Surveillance of SARS-CoV-2 Variants That Emerged in South and Southeast Asia during Early 2022.

The continuously emerging new variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have made the global coronavirus disease 2019 (COVID-19) pandemic unpredictable. Since the beginning of the pandemic, densely populated South and Southeast Asia have suffered great losses due to multiple COVID-19 surges because of vaccine and other medical resource shortages. Therefore, it is crucial to closely monitor the SARS-CoV-2 epidemic and to understand the evolutionary and transmission characteristics of SARS-CoV-2 in these regions. Here, we document the evolution of epidemic strains in the Philippines, Pakistan, and Malaysia from late 2021 to early 2022. Our results confirmed the circulation of at least five SARS-CoV-2 genotypes in these countries in January 2022, when Omicron BA.2, with a detection rate of 69.11%, replaced Delta B.1.617 as the dominant strain. Single-nucleotide polymorphism analysis indicated the distinct evolutionary directions of the Omicron and Delta isolates, with S, Nsp1, and Nsp6 genes potentially playing a significant role in the host adaptation of the Omicron strain. These findings are able to provide insights for predicting the evolutionary direction of SARS-CoV-2 in terms of variant competition, developing multi-part vaccines, and to support the evaluation and adjustment of current surveillance, prevention, and control strategies in South and Southeast Asia.

Structural insights into BCDX2 complex function in homologous recombination.

Homologous recombination (HR) fulfils a pivotal role in the repair of DNA double-strand breaks and collapsed replication forks1. HR depends on the products of several paralogues of RAD51, including the tetrameric complex of RAD51B, RAD51C, RAD51D and XRCC2 (BCDX2)2. BCDX2 functions as a mediator of nucleoprotein filament assembly by RAD51 and single-stranded DNA (ssDNA) during HR, but its mechanism remains undefined. Here we report cryogenic electron microscopy reconstructions of human BCDX2 in apo and ssDNA-bound states. The structures reveal how the amino-terminal domains of RAD51B, RAD51C and RAD51D participate in inter-subunit interactions that underpin complex formation and ssDNA-binding specificity. Single-molecule DNA curtain analysis yields insights into how BCDX2 enhances RAD51-ssDNA nucleoprotein filament assembly. Moreover, our cryogenic electron microscopy and functional analyses explain how RAD51C alterations found in patients with cancer3-6 inactivate DNA binding and the HR mediator activity of BCDX2. Our findings shed light on the role of BCDX2 in HR and provide a foundation for understanding how pathogenic alterations in BCDX2 impact genome repair.

Crystal structures reveal catalytic and regulatory mechanisms of the dual-specificity ubiquitin/FAT10 E1 enzyme Uba6.

The E1 enzyme Uba6 initiates signal transduction by activating ubiquitin and the ubiquitin-like protein FAT10 in a two-step process involving sequential catalysis of adenylation and thioester bond formation. To gain mechanistic insights into these processes, we determined the crystal structure of a human Uba6/ubiquitin complex. Two distinct architectures of the complex are observed: one in which Uba6 adopts an open conformation with the active site configured for catalysis of adenylation, and a second drastically different closed conformation in which the adenylation active site is disassembled and reconfigured for catalysis of thioester bond formation. Surprisingly, an inositol hexakisphosphate (InsP6) molecule binds to a previously unidentified allosteric site on Uba6. Our structural, biochemical, and biophysical data indicate that InsP6 allosterically inhibits Uba6 activity by altering interconversion of the open and closed conformations of Uba6 while also enhancing its stability. In addition to revealing the molecular mechanisms of catalysis by Uba6 and allosteric regulation of its activities, our structures provide a framework for developing Uba6-specific inhibitors and raise the possibility of allosteric regulation of other E1s by naturally occurring cellular metabolites.

Circulation, genomic characteristics, and evolutionary dynamics of class I Newcastle disease virus in China.

Newcastle disease caused by Newcastle disease virus (NDV) is one of the most serious threats to chickens and has two clinical forms, typical and atypical, caused by velogenic and lentogenic strains, respectively. To control the epidemic, many vaccines against velogenic class II NDVs have been introduced worldwide, but this has led to accelerated mutation of class II viruses under immune pressure and, on the other hand, to non-vaccine targeting class I NDVs becoming the dominant population in poultry. In this context, this study provided the first large-scale genomic epidemiological and quasispecies dynamic analysis of class I NDVs in China, and found that class I viruses that first appeared in East and South China have spread to central China and become the dominant class with an average evolutionary rate of 1.797 × 10-3. In addition, single nucleotide polymorphism and intra-host single nucleotide variation analyses show that HN and P genes have high mutation rates and may act as front-runners for NDV to expand their host range and enhance their virulence. This study also found that the class I NDV population has accumulated a number of mutations under positive selection and that six isolates with shortened C-terminal extensions of the HN protein are evolving toward increased virulence. These results not only enrich the research resources but also help us to better understand the dynamic evolution and mutational trends of NDV at the genomic level, which is crucial for monitoring, early warning, and controlling the outbreak of Newcastle disease.

Suppression and Activation of Intracellular Immune Response in Initial Severe Acute Respiratory Syndrome Coronavirus 2 Infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is currently the most important emerging pathogen worldwide, but its early transcriptional dynamics and host immune response remain unclear. Herein, the expression profiles of viral interactions with different types of hosts were comprehensively dissected to shed light on the early infection strategy of SARS-CoV-2 and the host immune response against infection. SARS-CoV-2 was found to exhibit a two-stage transcriptional strategy within the first 24 h of infection, comprising a lag phase that ends with the virus being paused and a log phase that starts when the viral load increases rapidly. Interestingly, the host innate immune response was found not to be activated (latent period) until the virus entered the log stage. Noteworthy, when intracellular immunity is suppressed, SARS-CoV-2 shows a correlation with dysregulation of metal ion homeostasis. Herein, the inhibitory activity of copper ions against SARS-CoV-2 was further validated in in vitro experiments. Coronavirus disease 2019-related genes (including CD38, PTX3, and TCN1) were also identified, which may serve as candidate host-restricted factors for interventional therapy. Collectively, these results confirm that the two-stage strategy of SARS-CoV-2 effectively aids its survival in early infection by regulating the host intracellular immunity, highlighting the key role of interferon in viral infection and potential therapeutic candidates for further investigations on antiviral strategies.

Targeting SARS-CoV-2 Proteases for COVID-19 Antiviral Development.

The emergence of severe acute respiratory syndrome (SARS-CoV-2) in 2019 marked the third occurrence of a highly pathogenic coronavirus in the human population since 2003. As the death toll surpasses 5 million globally and economic losses continue, designing drugs that could curtail infection and disease progression is critical. In the US, three highly effective Food and Drug Administration (FDA)-authorized vaccines are currently available, and Remdesivir is approved for the treatment of hospitalized patients. However, moderate vaccination rates and the sustained evolution of new viral variants necessitate the ongoing search for new antivirals. Several viral proteins have been prioritized as SARS-CoV-2 antiviral drug targets, among them the papain-like protease (PLpro) and the main protease (Mpro). Inhibition of these proteases would target viral replication, viral maturation, and suppression of host innate immune responses. Knowledge of inhibitors and assays for viruses were quickly adopted for SARS-CoV-2 protease research. Potential candidates have been identified to show inhibitory effects against PLpro and Mpro, both in biochemical assays and viral replication in cells. These results encourage further optimizations to improve prophylactic and therapeutic efficacy. In this review, we examine the latest developments of potential small-molecule inhibitors and peptide inhibitors for PLpro and Mpro, and how structural biology greatly facilitates this process.

Phylogeography, Transmission, and Viral Proteins of Nipah Virus.

Nipah virus (NiV), a zoonotic paramyxovirus belonging to the genus Henipavirus, is classified as a Biosafety Level-4 pathogen based on its high pathogenicity in humans and the lack of available vaccines or therapeutics. Since its initial emergence in 1998 in Malaysia, this virus has become a great threat to domestic animals and humans. Sporadic outbreaks and person-to-person transmission over the past two decades have resulted in hundreds of human fatalities. Epidemiological surveys have shown that NiV is distributed in Asia, Africa, and the South Pacific Ocean, and is transmitted by its natural reservoir, Pteropid bats. Numerous efforts have been made to analyze viral protein function and structure to develop feasible strategies for drug design. Increasing surveillance and preventative measures for the viral infectious disease are urgently needed.

Global QTL Analysis Identifies Genomic Regions on Chromosomes 4A and 4B Harboring Stable Loci for Yield-Related Traits Across Different Environments in Wheat (Triticum aestivum L.).

Major advances in wheat production are needed to address global food insecurity under future climate conditions, such as high temperatures. The grain yield of bread wheat (Triticum aestivum L.) is a quantitatively inherited complex trait that is strongly influenced by interacting genetic and environmental factors. Here, we conducted global QTL analysis for five yield-related traits, including spike yield, yield components and plant height (PH), in the Nongda3338/Jingdong6 doubled haploid (DH) population using a high-density SNP and SSR-based genetic map. A total of 12 major genomic regions with stable QTL controlling yield-related traits were detected on chromosomes 1B, 2A, 2B, 2D, 3A, 4A, 4B, 4D, 5A, 6A, and 7A across 12 different field trials with timely sown (normal) and late sown (heat stress) conditions. Co-location of yield components revealed significant tradeoffs between thousand grain weight (TGW) and grain number per spike (GNS) on chromosome 4A. Dissection of a "QTL-hotspot" region for grain weight on chromosome 4B was helpful in marker-assisted selection (MAS) breeding. Moreover, this study identified a novel QTL for heat susceptibility index of thousand grain weight (HSITGW) on chromosome 4BL that explains approximately 10% of phenotypic variation. QPh.cau-4B.2, QPh.cau-4D.1 and QPh.cau-2D.3 were coincident with the dwarfing genes Rht1, Rht2, and Rht8, and haplotype analysis revealed their pleiotropic architecture with yield components. Overall, our findings will be useful for elucidating the genetic architecture of yield-related traits and developing new wheat varieties with high and stable yield.

The potato transcription factor StbZIP61 regulates dynamic biosynthesis of salicylic acid in defense against Phytophthora infestans infection.

Salicylic acid (SA) signalling plays an essential role in plant innate immunity. In this study, we identified a component in the SA signaling pathway in potato (Solanum tuberosum), the transcription factor StbZIP61, and characterized its function in defence against Phytophthora infestans. Expression of StbZIP61 was induced upon P. infestans infection and following exposure to the defense signaling hormones SA, ethylene and jasmonic acid. Overexpression of StbZIP61 increased the tolerance of potato plants to P. infestans while RNA interference (RNAi) increased susceptibility. Yeast two-hybrid and pull down experiments revealed that StbZIP61 could interact with an NPR3-like protein (StNPR3L) that inhibited its DNA-binding and transcriptional activation activities. Moreover, StNPR3L interacted with StbZIP61 in an SA-dependent manner. Among candidate genes involved in SA-regulated defense responses, StbZIP61 had a significant impact on expression of StICS1, which encodes a key enzyme for SA biosynthesis. StICS1 transcription was induced upon P. infestans infection and this responsive expression to the pathogen was reduced in StbZIP61 RNAi plants. Accordingly, StICS1 expression was remarkably enhanced in StbZIP61-overexpressing plants. Together, our data demonstrate that StbZIP61 functions in concert with StNPR3L to regulate the temporal activation of SA biosynthesis, which contributes to SA-mediated immunity against P. infestans infection in potato.

Ectopic expression of SsPETE2, a plastocyanin from Suaeda salsa, improves plant tolerance to oxidative stress.

Accumulating evidence indicates that plant plastocyanin is involved in copper homeostasis, yet the physiological relevance remains elusive. In this study, we found that a plastocyanin gene (SsPETE2) from euhalophyte Suaeda salsa possessed a novel antioxidant function, which was associated with the copper-chelating activity of SsPETE2. In S. salsa, expression of SsPETE2 increased in response to oxidative stress and ectopic expression of SsPETE2 in Arabidopsis enhanced the antioxidant ability of the transgenic plants. SsPETE2 bound Cu ion and alleviated formation of hydroxyl radicals in vitro. Accordingly, SsPETE2 expression lowered the free Cu content that was associated with reduced H2O2 level under oxidative stress. Arabidopsis pete1 and pete2 mutants showed ROS-sensitive phenotypes that could be restored by expression of SsPETE2 or AtPETEs. In addition, SsPETE2-expressing plants exhibited more potent tolerance to oxidative stress than plants overexpressing AtPETEs, likely owing to the stronger copper-binding activity of SsPETE2 than AtPETEs. Taken together, these results demonstrated that plant PETEs play a novel role in oxidative stress tolerance by regulating Cu homeostasis under stress conditions, and SsPETE2, as an efficient copper-chelating PETE, potentially could be used in crop genetic engineering.

A Novel N-Methyltransferase in Arabidopsis Appears to Feed a Conserved Pathway for Nicotinate Detoxification among Land Plants and Is Associated with Lignin Biosynthesis.

The Preiss-Handler pathway, which salvages nicotinate (NA) for NAD synthesis, is an indispensable biochemical pathway in land plants. Various NA conjugations (mainly methylation and glycosylation) have been detected and have long been proposed for NA detoxification in plants. Previously, we demonstrated that NA O-glucosylation functions as a mobilizable storage form for NAD biosynthesis in the Brassicaceae. However, little is known about the functions of other NA conjugations in plants. In this study, we first found that N-methylnicotinate is a ubiquitous NA conjugation in land plants. Furthermore, we functionally identified a novel methyltransferase (At3g53140; NANMT), which is mainly responsible for N-methylnicotinate formation, from Arabidopsis (Arabidopsis thaliana). We also established that trigonelline is a detoxification form of endogenous NA in plants. Combined phylogenetic analysis and enzymatic assays revealed that NA N-methylation activity was likely derived from the duplication and subfunctionalization of an ancestral caffeic acid O-methyltransferase (COMT) gene in the course of land plant evolution. COMT enzymes, which function in S-lignin biosynthesis, also have weak NANMT activity. Our data suggest that NA detoxification conferred by NANMT and COMT might have facilitated the retention of the Preiss-Handler pathway in land plants.

Two Novel Vesicle-Inducing Proteins in Plastids 1 Genes Cloned and Characterized in Triticum urartu.

Vesicle-inducing protein in plastids 1 (Vipp1) is thought to play an important role both in thylakoid biogenesis and chloroplast envelope maintenance during stress. Vipp1 is conserved in photosynthetic organisms and forms a high homo-oligomer complex structure that may help sustain the membrane integrity of chloroplasts. This study cloned two novel VIPP1 genes from Triticum urartu and named them TuVipp1 and TuVipp2. Both proteins shared high identity with the homologous proteins AtVipp1 and CrVipp1. TuVipp1 and TuVipp2 were expressed in various organs of common wheat, and both genes were induced by light and various stress treatments. Purified TuVipp1 and TuVipp2 proteins showed secondary and advanced structures similar to those of the homologous proteins. Similar to AtVipp1, TuVipp1 is a chloroplast target protein. Additionally, TuVipp1 was able to rescue the phenotypes of pale leaves, lethality, and disordered chloroplast structures of AtVipp1 (-/-) mutant lines. Collectively, our data demonstrate that TuVipp1 and TuVipp2 are functional proteins in chloroplasts in wheat and may be critical for maintaining the chloroplast envelope under stress and membrane biogenesis upon photosynthesis.

Mapping QTLs of yield-related traits using RIL population derived from common wheat and Tibetan semi-wild wheat.

KEY MESSAGE: QTLs controlling yield-related traits were mapped using a population derived from common wheat and Tibetan semi-wild wheat and they provided valuable information for using Tibetan semi-wild wheat in future wheat molecular breeding. Tibetan semi-wild wheat (Triticum aestivum ssp tibetanum Shao) is a kind of primitive hexaploid wheat and harbors several beneficial traits, such as tolerance to biotic and abiotic stresses. And as a wild relative of common wheat, heterosis of yield of the progeny between them was significant. This study focused on mapping QTLs controlling yield-related traits using a recombined inbred lines (RILs) population derived from a hybrid between a common wheat line NongDa3331 (ND3331) and the Tibetan semi-wild wheat accession Zang 1817. In nine location-year environments, a total of 148 putative QTLs controlling nine traits were detected, distributed on 19 chromosomes except for 1A and 2D. Single QTL explained the phenotypic variation ranging from 3.12 to 49.95%. Of these QTLs, 56 were contributed by Zang 1817. Some stable QTLs contributed by Zang 1817 were also detected in more than four environments, such as QPh-3A1, QPh-4B1 and QPh-4D for plant height, QSl-7A1 for spike length, QEp-4B2 for ears per plant, QGws-4D for grain weight per spike, and QTgw-4D for thousand grain weight. Several QTL-rich Regions were also identified, especially on the homoeologous group 4. The TaANT gene involved in floral organ development was mapped on chromosome 4A between Xksm71 and Xcfd6 with 0.8 cM interval, and co-segregated with the QTLs controlling floret number per spikelet, explaining 4.96-11.84% of the phenotypic variation. The current study broadens our understanding of the genetic characterization of Tibetan semi-wild wheat, which will enlarge the genetic diversity of yield-related traits in modern wheat breeding program.