Visualizing the translation landscape in human cells at high resolution.

Obtaining comprehensive structural descriptions of macromolecules within their natural cellular context holds immense potential for understanding fundamental biology and improving health. Here, we present the landscape of protein synthesis inside human cells in unprecedented detail obtained using an approach which combines automated cryo-focused ion beam (FIB) milling and in situ single-particle cryo-electron microscopy (cryo-EM). With this in situ cryo-EM approach we resolved a 2.19 Å consensus structure of the human 80S ribosome and unveiled its 21 distinct functional states, nearly all higher than 3 Å resolution. In contrast to in vitro studies, we identified protein factors, including SERBP1, EDF1 and NAC/3, not enriched on purified ribosomes. Most strikingly, we observed that SERBP1 binds to the ribosome in almost all translating and non-translating states to bridge the 60S and 40S ribosomal subunits. These newly observed binding sites suggest that SERBP1 may serve an important regulatory role in translation. We also uncovered a detailed interface between adjacent translating ribosomes which can form the helical polysome structure. Finally, we resolved high-resolution structures from cells treated with homoharringtonine and cycloheximide, and identified numerous polyamines bound to the ribosome, including a spermidine that interacts with cycloheximide bound at the E site of the ribosome, underscoring the importance of high-resolution in situ studies in the complex native environment. Collectively, our work represents a significant advancement in detailed structural studies within cellular contexts.

Unfolding the downloads of datasets: A multifaceted exploration of influencing factors.

Scientific data are essential to advancing scientific knowledge and are increasingly valued as scholarly output. Understanding what drives dataset downloads is crucial for their effective dissemination and reuse. Our study, analysing 55,473 datasets from 69 data repositories, identifies key factors driving dataset downloads, focusing on interpretability, reliability, and accessibility. We find that while lengthy descriptive texts can deter users due to complexity and time requirements, readability boosts a dataset's appeal. Reliability, evidenced by factors like institutional reputation and citation counts of related papers, also significantly increases a dataset's attractiveness and usage. Additionally, our research shows that open access to datasets increases their downloads and amplifies the importance of interpretability and reliability. This indicates that easy access enhances the overall attractiveness and usage of datasets in the scholarly community. By emphasizing interpretability, reliability, and accessibility, this study offers a comprehensive framework for future research and guides data management practices toward ensuring clarity, credibility, and open access to maximize the impact of scientific datasets.

Mutation-induced shift of the photosystem II active site reveals insight into conserved water channels.

Photosystem II (PSII) is the water-plastoquinone photo-oxidoreductase central to oxygenic photosynthesis. PSII has been extensively studied for its ability to catalyze light-driven water oxidation at a Mn4CaO5 cluster called the oxygen-evolving complex (OEC). Despite these efforts, the complete reaction mechanism for water oxidation by PSII is still heavily debated. Previous mutagenesis studies have investigated the roles of conserved amino acids, but these studies have lacked a direct structural basis that would allow for a more meaningful interpretation. Here, we report a 2.14-Å resolution cryo-EM structure of a PSII complex containing the substitution Asp170Glu on the D1 subunit. This mutation directly perturbs a bridging carboxylate ligand of the OEC, which alters the spectroscopic properties of the OEC without fully abolishing water oxidation. The structure reveals that the mutation shifts the position of the OEC within the active site without markedly distorting the Mn4CaO5 cluster metal-metal geometry, instead shifting the OEC as a rigid body. This shift disturbs the hydrogen-bonding network of structured waters near the OEC, causing disorder in the conserved water channels. This mutation-induced disorder appears consistent with previous FTIR spectroscopic data. We further show using quantum mechanics/molecular mechanics methods that the mutation-induced structural changes can affect the magnetic properties of the OEC by altering the axes of the Jahn-Teller distortion of the Mn(III) ion coordinated to D1-170. These results offer new perspectives on the conserved water channels, the rigid body property of the OEC, and the role of D1-Asp170 in the enzymatic water oxidation mechanism.

Choice of refractive surgery types for myopia assisted by machine learning based on doctors' surgical selection data.

In recent years, corneal refractive surgery has been widely used in clinics as an effective means to restore vision and improve the quality of life. When choosing myopia-refractive surgery, it is necessary to comprehensively consider the differences in equipment and technology as well as the specificity of individual patients, which heavily depend on the experience of ophthalmologists. In our study, we took advantage of machine learning to learn about the experience of ophthalmologists in decision-making and assist them in the choice of corneal refractive surgery in a new case. Our study was based on the clinical data of 7,081 patients who underwent corneal refractive surgery between 2000 and 2017 at the Department of Ophthalmology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences. Due to the long data period, there were data losses and errors in this dataset. First, we cleaned the data and deleted the samples of key data loss. Then, patients were divided into three groups according to the type of surgery, after which we used SMOTE technology to eliminate imbalance between groups. Six statistical machine learning models, including NBM, RF, AdaBoost, XGBoost, BP neural network, and DBN were selected, and a ten-fold cross-validation and grid search were used to determine the optimal hyperparameters for better performance. When tested on the dataset, the multi-class RF model showed the best performance, with agreement with ophthalmologist decisions as high as 0.8775 and Macro F1 as high as 0.8019. Furthermore, the results of the feature importance analysis based on the SHAP technique were consistent with an ophthalmologist's practical experience. Our research will assist ophthalmologists in choosing appropriate types of refractive surgery and will have beneficial clinical effects.

Occupancy Analysis of Water Molecules inside Channels within 25 Å Radius of the Oxygen-Evolving Center of Photosystem II in Molecular Dynamics Simulations.

At room temperature and neutral pH, the oxygen-evolving center (OEC) of photosystem II (PSII) catalyzes water oxidation. During this process, oxygen is released from the OEC, while substrate waters are delivered to the OEC and protons are passed from the OEC to the lumen through water channels known as the narrow or the O4 channel, broad or the Cl1 channel, and large or the O1 channel. Protein residues lining the surfaces of these channels play a critical role in stabilizing the hydrogen-bonding networks that assist in the process. We carried out an occupancy analysis to better understand the structural and possible substrate water dynamics in full PSII monomer molecular dynamics (MD) trajectories in both the S1 and S2 states. We find that the equilibrated positions of water molecules derived from MD-derived electron density maps largely match the experimentally observed positions in crystallography. Furthermore, the occupancy reduction in MD simulations of some water molecules inside the single-filed narrow channel also correlates well with the crystallographic data during a structural transition when the S1 state of the OEC advances to the S2 state. The overall reduced occupancies of water molecules are the source of their "vacancy-hopping" dynamic nature inside these channels, unlike water molecules inside an ice lattice where all water molecules have a fixed unit occupancy. We propose on the basis of findings in our structural and molecular dynamics analysis that the water molecule occupying a pocket formed by D1-D61, D1-S169, and O4 of the OEC could be the last steppingstone to enter into the OEC and that the broad channel may be favored for proton transfer.

Urine biomarkers individually and as a consensus model show high sensitivity and specificity for detecting UTIs.

BACKGROUND: Current diagnoses of urinary tract infection (UTI) by standard urine culture (SUC) has significant limitations in sensitivity, especially for fastidious organisms, and the ability to identify organisms in polymicrobial infections. The significant rate of both SUC "negative" or "mixed flora/contamination" results in UTI cases and the high prevalence of asymptomatic bacteriuria indicate the need for an accurate diagnostic test to help identify true UTI cases. This study aimed to determine if infection-associated urinary biomarkers can differentiate definitive UTI cases from non-UTI controls. METHODS: Midstream clean-catch voided urine samples were collected from asymptomatic volunteers and symptomatic subjects ≥ 60 years old diagnosed with a UTI in a urology specialty setting. Microbial identification and density were assessed using a multiplex PCR/pooled antibiotic susceptibility test (M-PCR/P-AST) and SUC. Three biomarkers [neutrophil gelatinase-associated lipocalin (NGAL), and Interleukins 8 and 1ß (IL-8, and IL-1ß)] were also measured via enzyme-linked immunosorbent assay (ELISA). Definitive UTI cases were defined as symptomatic subjects with a UTI diagnosis and positive microorganism detection by SUC and M-PCR, while definitive non-UTI cases were defined as asymptomatic volunteers. RESULTS: We observed a strong positive correlation (R2 > 0.90; p < 0.0001) between microbial density and the biomarkers NGAL, IL-8, and IL-1ß for symptomatic subjects. Biomarker consensus criteria of two or more positive biomarkers had sensitivity 84.0%, specificity 91.2%, positive predictive value 93.7%, negative predictive value 78.8%, accuracy 86.9%, positive likelihood ratio of 9.58, and negative likelihood ratio of 0.17 in differentiating definitive UTI from non-UTI cases, regardless of non-zero microbial density. NGAL, IL-8, and IL-1ß showed a significant elevation in symptomatic cases with positive microbe identification compared to asymptomatic cases with or without microbe identification. Biomarker consensus exhibited high accuracy in distinguishing UTI from non-UTI cases. CONCLUSION: We demonstrated that positive infection-associated urinary biomarkers NGAL, IL-8, and IL-1ß, in symptomatic subjects with positive SUC and/or M-PCR results was associated with definitive UTI cases. A consensus criterion with ≥ 2 of the biomarkers meeting the positivity thresholds showed a good balance of sensitivity (84.0%), specificity (91.2%), and accuracy (86.9%). Therefore, this biomarker consensus is an excellent supportive diagnostic tool for resolving the presence of active UTI, particularly if SUC and M-PCR results disagree.

The Prevalence and Association of Different Uropathogens Detected by M-PCR with Infection-Associated Urine Biomarkers in Urinary Tract Infections.

Background: Many emerging uropathogens are currently identified by multiplex polymerase chain reaction (M-PCR) in suspected UTI cases. Standard urine culture (SUC) has significantly lower detection rates, raising questions about whether these organisms are associated with UTIs and truly cause inflammation. Objective: To determine if microbes detected by M-PCR were likely causative of UTI by measuring inflammatory biomarkers in the urine of symptomatic patients. Design Setting and Participants: Midstream voided urine was collected from subjects ≥60 years presenting to urology clinics with symptoms of UTI (n = 1132) between 01/2023 and 05/2023. Microbe detection was by M-PCR and inflammation-associated biomarker (neutrophil gelatinase-associated lipocalin, interleukin 8, and interleukin 1ß) was by enzyme-linked immunosorbent assay. Biomarker positivity was measured against individual and groups of organisms, E. coli and non-E. coli cases, emerging uropathogens, monomicrobial and polymicrobial cases. Outcome Measurements and Statistical Analysis: Distributions were compared using 2-sample Wilcoxon Rank Sum test with 2-tailed p-values < 0.05 considered statistically significant. Results and Limitations: M-PCR was positive in 823 (72.7%) specimens with 28 of 30 (93%) microorganisms/groups detected. Twenty-six of twenty-eight detected microorganisms/groups (93%) had ≥2 biomarkers positive in >66% of cases. Both non-E. coli cases and E. coli cases had significant biomarker positivity (p < 0.05). Limitations were that a few organisms had low prevalence making inferences about their individual significance difficult. Conclusion: The majority of microorganisms identified by M-PCR were associated with active inflammation measured by biomarker positivity, indicating they are likely causative of UTIs in symptomatic patients. This includes emerging uropathogens frequently not detected by standard urine culture.

Bridging the Coordination Chemistry of Small Compounds and Metalloproteins Using Machine Learning.

Metalloproteins require metal ions as cofactors to catalyze specific reactions with remarkable efficiency and specificity. In various electron transfer reactions, metals in the active sites change their oxidation states to facilitate the biochemical reactions. Cryogenic electron microscopy, X-ray, and X-ray free electron laser (XFEL) crystallography are used to image metalloproteins to understand the reaction mechanisms. However, radiation damage in cryoEM and X-ray crystallography, and the challenge of generating homogeneous crystals and keeping the appropriate experimental conditions for all the crystals in XFEL crystallography, may alter the oxidation states. Here, we build machine learning models trained on a large data set from the Cambridge Crystallographic Data Center to evaluate the metal oxidation states. The models yield high accuracy scores (from 82% to 94%) for all metals in the small molecules. Then, they were used to predict the oxidation states of more than 30 000 metal clusters in metalloproteins with Fe, Mn, Co, and Cu in their active sites. We found that most of the metals exist in the lower oxidation states (Fe2+ 77%, Mn2+ 85%, Co2+ 65%, and Cu+ 64%), and these populations correlate with the standard reduction potentials of the metal ions. Furthermore, we found no clear correlation between these populations and the resolution of the structures, which suggests no significant dependence of these predictions on the resolution. Our models represent a valuable tool for evaluating the oxidation states of the metals in metalloproteins imaged with different techniques. The data files and the machine learning code are available in a public GitHub repository: https://github.com/mamin03/OxitationStatesMetalloprotein.git.

Computing the Relative Affinity of Chlorophylls a and b to Light-Harvesting Complex II.

In plants and algae, the primary antenna protein bound to photosystem II is light-harvesting complex II (LHCII), a pigment-protein complex that binds eight chlorophyll (Chl) a molecules and six Chl b molecules. Chl a and Chl b differ only in that Chl a has a methyl group (-CH3) on one of its pyrrole rings, while Chl b has a formyl group (-CHO) at that position. This blue-shifts the Chl b absorbance relative to Chl a. It is not known how the protein selectively binds the right Chl type at each site. Knowing the selection criteria would allow the design of light-harvesting complexes that bind different Chl types, modifying an organism to utilize the light of different wavelengths. The difference in the binding affinity of Chl a and Chl b in pea and spinach LHCII was calculated using multiconformation continuum electrostatics and free energy perturbation. Both methods have identified some Chl sites where the bound Chl type (a or b) has a significantly higher affinity, especially when the protein provides a hydrogen bond for the Chl b formyl group. However, the Chl a sites often have little calculated preference for one Chl type, so they are predicted to bind a mixture of Chl a and b. The electron density of the spinach LHCII was reanalyzed, which, however, confirmed that there is negligible Chl b in the Chl a-binding sites. It is suggested that the protein chooses the correct Chl type during folding, segregating the preferred Chl to the correct binding site.

Comparison Shows that Multiplex Polymerase Chain Reaction Identifies Infection-associated Urinary Biomarker-positive Urinary Tract Infections That Are Missed by Standard Urine Culture.

Background: Multiplex polymerase chain reaction (M-PCR) has increased sensitivity for microbial detection compared with standard urine culture (SUC) in cases diagnosed as urinary tract infections (UTIs), leading to questions whether detected microbes are likely causative of UTIs or are incidental findings. Objective: To compare infection-associated biomarker levels against M-PCR and SUC results in symptomatic cases with a presumptive diagnosis of a UTI by a urologist. Design setting and participants: Participants were ≥60 yr old and presented to urology clinics between January and April 2023 with symptoms of UTIs (n = 583). Urine microbial detection was by M-PCR and SUC. Three infection-associated biomarkers (neutrophil gelatinase-associated lipocalin, interleukin-8, and interleukin-1ß) were measured by enzyme-linked immunosorbent assay. Symptomatic cases with elevated biomarkers, detection of uropathogens, and a specialist clinical diagnosis of a UTI were considered definitive UTI cases. Outcome measurements and statistical analysis: Distributions were compared using two-sample Wilcoxon rank sum test, with two-tailed p values of <0.05 considered statistically significant. Results and limitations: In cases with M-PCR-positive/SUC-negative results (n = 80), all median biomarker levels were significantly higher (p < 0.0001) than in cases with M-PCR-negative/SUC-negative results (n = 107). Two or more biomarkers were positive in 76% of M-PCR-positive/SUC-negative specimens. Limitation was an inability to examine associations between each individual organism and inflammation. Conclusions: A significant number of M-PCR-positive/SUC-negative cases had elevated levels of infection-related urinary biomarkers, especially when infection was caused by organisms other than Escherichia coli. This is a strong indication that microbes detected by M-PCR, which would be missed by SUC, are associated with UTIs. Patient summary: We compared infection-associated biomarkers in patients diagnosed with urinary tract infections (UTIs) against the detection of microorganisms by standard urine culture (SUC) and multiplex polymerase chain reaction (M-PCR). We found that most patients with microorganisms detected by M-PCR, which were missed by SUC, had elevated markers of inflammation, indicating that these organisms were likely causative of UTIs.

Substrate-independent activation pathways of the CRISPR-Cas9 HNH nuclease.

A hallmark of tightly regulated high-fidelity enzymes is that they become activated only after encountering cognate substrates, often by an induced-fit mechanism rather than conformational selection. Upon analysis of molecular dynamics trajectories, we recently discovered that the Cas9 HNH domain exists in three conformations: 1) Y836 (which is two residues away from the catalytic D839 and H840 residues) is hydrogen bonded to the D829 backbone amide, 2) Y836 is hydrogen bonded to the backbone amide of D861 (which is one residue away from the third catalytic residue N863), and 3) Y836 is not hydrogen bonded to either residue. Each of the three conformers differs from the active state of HNH. The conversion between the inactive and active states involves a local unfolding-refolding process that displaces the Cα and side chain of the catalytic N863 residue by ∼5 Å and ∼10 Å, respectively. In this study, we report the two largest principal components of coordinate variance of the HNH domain throughout molecular dynamics trajectories to establish the interconversion pathways of these conformations. We show that conformation 2 is an obligate step between conformations 1 and 3, which are not directly interconvertible without conformation 2. The loss of hydrogen bonding of the Y836 side chain in conformation 3 likely plays an essential role in activation during local unfolding-refolding of an α-helix containing the catalytic N863. Three single Lys-to-Ala mutants appear to eliminate this substrate-independent activation pathway of the wild-type HNH nuclease, thereby enhancing the fidelity of HNH cleavage.

Triple-action inhibitory mechanism of allosteric TYK2-specific inhibitors.

Deucravacitinib, 6-(cyclopropanecarbonylamido)-4-[2-methoxy-3-(1-methyl-1,2,4-triazol-3-yl)anilino]-N-(trideuteriomethyl)pyridazine-3-carboxamide, is a highly selective inhibitor of protein tyrosine kinase 2 (TYK2) that targets the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway. The structural basis for its selectivity and allosteric inhibition remains poorly understood. Here, we investigate the inhibition mechanism through analysis of available structures relevant to the STAT pathway, including crystal structures of the truncated TYK2 FERM-SH2 domain bound to the IFNα type I receptor (IFNαR1) and the truncated TYK2 JH2-JH1 domain. Our computational analysis provides a mechanistic hypothesis for the relatively rapid interferon-induced gene expression mediated by TYK2 relative to other cytokines. We find that deucravacitinib inhibits TYK2 kinase in three distinct states: the autoinhibited state and two activated states for autophosphorylation and phosphorylation of downstream protein substrates. Its binding to the TYK2 pseudokinase domain in the autoinhibited state restricts the essential dynamics of the TYK2 kinase domain required for kinase activity. Furthermore, it binds competitively with ATP in the pseudokinase domain, and also directly prevents formation of the active state of TYK2 through steric clashes.

Enhanced production of glutaric acid by biocatalyst-recycled bioconversion process integrated with in situ product recovery by adsorption.

Product inhibition caused by organic acids is a serious issue in establishing economical biochemical production systems. Herein, for enhanced production of glutaric acid by overcoming product inhibition triggered by glutaric acid, a whole-cell bioconversion system equipped with biocatalyst recycling process and in situ product recovery by adsorption was developed successfully. From the whole-cell bioconversion reaction, we found that both dissociated and undissociated forms of glutaric acid acted as an inhibitor in the whole-cell bioconversion reaction, wherein bioconversion was hindered beyond 200 mM glutaric acid regardless of reaction pH. Therefore, as the promising solution for the inhibition issue by glutaric acid, the biocatalyst-recycled bioconversion process integrated with in situ product recovery by adsorption was introduced in the whole-cell bioconversion. As a result, 592 mM glutaric acid was produced from 1000 mM 5-aminovaleric acid with 59.2% conversion. We believe that our system will be a promising candidate for economically producing organic acids with high titer.

A quantitative assessment of (bacterio)chlorophyll assignments in the cryo-EM structure of the Chloracidobacterium thermophilum reaction center.

Chlorophylls and bacteriochlorophylls are the primary pigments used by photosynthetic organisms for light harvesting, energy transfer, and electron transfer. Many molecular structures of (bacterio)chlorophyll-containing protein complexes are available, some of which contain mixtures of different (bacterio)chlorophyll types. Differentiating these, which sometimes are structurally similar, is challenging but is required for leveraging structural data to gain functional insight. The reaction center complex from Chloroacidobacterium thermophilum has a hybrid (bacterio)chlorophyll antenna system containing both chlorophyll a and bacteriochlorophyll a molecules. The recent availability of its cryogenic electron microscopy (cryo-EM) structure provides an opportunity for a quantitative analysis of their identities and chemical environments. Here, we describe a theoretical basis for differentiating chlorophyll a and bacteriochlorophyll a in a cryo-EM map, and apply the approach to the experimental cryo-EM maps of the (bacterio)chlorophyll sites of the chloroacidobacterial reaction center. The comparison reveals that at ~ 2.2-Å resolution, chlorophyll a and bacteriochlorophyll a are easily distinguishable, but the orientation of the bacteriochlorophyll a acetyl moiety is not; however, the latter can confidently be assigned by identifying a hydrogen bond donor from the protein environment. This study reveals the opportunities and challenges in assigning (bacterio)chlorophyll types in structural biology, the accuracy of which is vital for downstream investigations.

Elevated UTI Biomarkers in Symptomatic Patients with Urine Microbial Densities of 10,000 CFU/mL Indicate a Lower Threshold for Diagnosing UTIs.

The literature lacks consensus on the minimum microbial density required for diagnosing urinary tract infections (UTIs). This study categorized the microbial densities of urine specimens from symptomatic UTI patients aged ≥ 60 years and correlated them with detected levels of the immune response biomarkers neutrophil gelatinase-associated lipocalin (NGAL), interleukin-8 (IL-8), and interleukin-1-beta (IL-1ß). The objective was to identify the microbial densities associated with significant elevation of these biomarkers in order to determine an optimal threshold for diagnosing symptomatic UTIs. Biobanked midstream voided urine samples were analyzed for microbial identification and quantification using standard urine culture (SUC) and multiplex-polymerase chain reaction (M-PCR) testing, while NGAL, IL-8, and IL-1ß levels were measured via enzyme-linked immunosorbent assay (ELISA). NGAL, IL-8, and IL-1ß levels were all significantly elevated at microbial densities ≥ 10,000 cells/mL when measured via M-PCR (p < 0.0069) or equivalent colony-forming units (CFUs)/mL via SUC (p < 0.0104) compared to samples with no detectable microbes. With both PCR and SUC, a consensus of two or more elevated biomarkers correlated well with microbial densities > 10,000 cells/mL or CFU/mL, respectively. The association between ≥10,000 cells and CFU per mL with elevated biomarkers in symptomatic patients suggests that this lower threshold may be more suitable than 100,000 CFU/mL for diagnosing UTIs.

Valproate-coenzyme A conjugate blocks opening of receptor binding domains in the spike trimer of SARS-CoV-2 through an allosteric mechanism.

The receptor-binding domains (RBDs) of the SARS-CoV-2 spike trimer exhibit "up" and "down" conformations often targeted by neutralizing antibodies. Only in the "up" configuration can RBDs bind to the ACE2 receptor of the host cell and initiate the process of viral multiplication. Here, we identify a lead compound (3-oxo-valproate-coenzyme A conjugate or Val-CoA) that stabilizes the spike trimer with RBDs in the down conformation. Val-CoA interacts with three R408 residues, one from each RBD, which significantly reduces the inter-subunit R408-R408 distance by ∼ 13 Å and closes the central pore formed by the three RBDs. Experimental evidence is presented that R408 is part of a triggering mechanism that controls the prefusion to postfusion state transition of the spike trimer. By stabilizing the RBDs in the down configuration, this and other related compounds can likely attenuate viral transmission. The reported findings for binding of Val-CoA to the spike trimer suggest a new approach for the design of allosteric antiviral drugs that do not have to compete for specific virus-receptor interactions but instead hinder the conformational motion of viral membrane proteins essential for interaction with the host cell. Here, we introduce an approach to target the spike protein by identifying lead compounds that stabilize the RBDs in the trimeric "down" configuration. When these compounds trimerize monomeric RBD immunogens as co-immunogens, they could also induce new types of non-ACE2 blocking antibodies that prevent local cell-to-cell transmission of the virus, providing a novel approach for inhibition of SARS-CoV-2.

Translocation pause of remdesivir-containing primer/template RNA duplex within SARS-CoV-2's RNA polymerase complexes.

The mechanism of remdesivir incorporation into the RNA primer by the RNA-dependent RNA polymerase (RdRp) of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) remains to be fully established at the molecular level. Here, we compare molecular dynamics (MD) simulations after incorporation of either remdesivir monophosphate (RMP) or adenosine monophosphate (AMP). We find that the Mg2+-pyrophosphate (PPi) binds more tightly to the polymerase when the added RMP is at the third primer position than in the AMP added complex. The increased affinity of Mg2+-PPi to the RMP-added primer/template (P/T) RNA duplex complex introduces a new hydrogen bond of a substituted cyano group in RMP with the K593 sidechain. The new interactions disrupt a switching mechanism of a hydrogen bond network that is essential for translocation of the P/T duplex product and for opening of a vacant NTP-binding site necessary for next primer extension. Furthermore, steric interactions between the sidechain of S861 and the 1'-cyano group of RMP at position i+3 hinders translocation of RMP to the i + 4 position, where i labels the insertion site. These findings are particularly valuable to guide the design of more effective inhibitors of SARS-CoV-2 RNA polymerase.

How to correct relative voxel scale factors for calculations of vector-difference Fourier maps in cryo-EM.

The atomic coordinates derived from cryo-electron microscopy (cryo-EM) maps can be inaccurate when the voxel scaling factors are not properly calibrated. Here, we describe a method for correcting relative voxel scaling factors between pairs of cryo-EM maps for the same or similar structures that are expanded or contracted relative to each other. We find that the correction of scaling factors reduces the amplitude differences of Fourier-inverted structure factors from voxel-rescaled maps by up to 20-30%, as shown by two cryo-EM maps of the SARS-CoV-2 spike protein measured at pH 4.0 and pH 8.0. This allows for the calculation of the difference map after properly scaling, revealing differences between the two structures for individual amino acid residues. Unexpectedly, the analysis uncovers two previously overlooked differences of amino acid residues in structures and their local structural changes. Furthermore, we demonstrate the method as applied to two cryo-EM maps of monomeric apo-photosystem II from the cyanobacteria Synechocystis sp. PCC 6803 and Thermosynechococcus elongatus. The resulting difference maps reveal many changes in the peripheral transmembrane PsbX subunit between the two species.

Structural Insights into Binding of Remdesivir Triphosphate within the Replication-Transcription Complex of SARS-CoV-2.

Remdesivir is an adenosine analogue that has a cyano substitution in the C1' position of the ribosyl moiety and a modified base structure to stabilize the linkage of the base to the C1' atom with its strong electron-withdrawing cyano group. Within the replication-transcription complex (RTC) of SARS-CoV-2, the RNA-dependent RNA polymerase nsp12 selects remdesivir monophosphate (RMP) over adenosine monophosphate (AMP) for nucleotide incorporation but noticeably slows primer extension after the added RMP of the RNA duplex product is translocated by three base pairs. Cryo-EM structures have been determined for the RTC with RMP at the nucleotide-insertion (i) site or at the i + 1, i + 2, or i + 3 sites after product translocation to provide a structural basis for a delayed-inhibition mechanism by remdesivir. In this study, we applied molecular dynamics (MD) simulations to extend the resolution of structures to the measurable maximum that is intrinsically limited by MD properties of these complexes. Our MD simulations provide (i) a structural basis for nucleotide selectivity of the incoming substrates of remdesivir triphosphate over adenosine triphosphate and of ribonucleotide over deoxyribonucleotide, (ii) new detailed information on hydrogen atoms involved in H-bonding interactions between the enzyme and remdesivir, and (iii) direct information on the catalytically active complex that is not easily captured by experimental methods. Our improved resolution of interatomic interactions at the nucleotide-binding pocket between remedesivir and the polymerase could help to design a new class of anti-SARS-CoV-2 inhibitors.

Ethnolinguistic diversity and the spread of communicable diseases: a cross-country study on the COVID-19 pandemic.

Motivated by the varying effectiveness of government intervention policies to contain the COVID-19 pandemic, and the potential positive relationship between ethnolinguistic diversity and social distance, this paper aims to provide empirical evidence on the relationship between ethnolinguistic diversity and the spread of COVID-19. In particular, using global data from 113 developed and developing countries during the early stages of the pandemic (from 31 December 2019 to 8 July 2020), we have found a significant negative effect of ethnolinguistic diversity on the spread of the virus. The result is robust to alternative measures of ethnolinguistic diversity and estimator that addresses endogeneity. Moreover, we also show that the impact of ethnolinguistic diversity on the spread of COVID-19 differs in economies characterized by different levels of democracy, policy stringency on addressing COVID-19 and health expenditure.