Outcomes of the EMDataResource Cryo-EM Ligand Modeling Challenge (preprint)

The EMDataResource Ligand Model Challenge aimed to assess the reliability and reproducibility of modeling ligands bound to protein and protein/nucleic-acid complexes in cryogenic electron microscopy (cryo-EM) maps determined at near-atomic (1.9-2.5 Å) resolution. Three published maps were selected as targets: E. coli beta-galactosidase with inhibitor, SARS-CoV-2 RNA-dependent RNA polymerase with covalently bound nucleotide analog, and SARS-CoV-2 ion channel ORF3a with bound lipid. Sixty-one models were submitted from 17 independent research groups, each with supporting workflow details. We found that (1) the quality of submitted ligand models and surrounding atoms varied, as judged by visual inspection and quantification of local map quality, model-to-map fit, geometry, energetics, and contact scores, and (2) a composite rather than a single score was needed to assess macromolecule+ligand model quality. These observations lead us to recommend best practices for assessing cryo-EM structures of liganded macromolecules reported at near-atomic resolution.

Potent and Selective Covalent Inhibition of the Papain-like Protease from SARS-CoV-2 (preprint)

Direct-acting antivirals are needed to combat coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). The papain-like protease (PLpro) domain of Nsp3 from SARS-CoV-2 is essential for viral replication. In addition, PLpro dysregulates the host immune response by cleaving ubiquitin and interferon-stimulated gene 15 protein (ISG15) from host proteins. As a result, PLpro is a promising target for inhibition by small-molecule therapeutics. Here we have designed a series of covalent inhibitors by introducing a peptidomimetic linker and reactive electrophile onto analogs of the noncovalent PLpro inhibitor GRL0617. The most potent compound inhibited PLpro with kinact/KI = 10,000 M− 1 s− 1, achieved sub-µM EC50 values against three SARS-CoV-2 variants in mammalian cell lines, and did not inhibit a panel of human deubiquitinases at > 30 µM concentrations of inhibitor. An X-ray co-crystal structure of the compound bound to PLpro validated our design strategy and established the molecular basis for covalent inhibition and selectivity against structurally similar human DUBs. These findings present an opportunity for further development of covalent PLpro inhibitors.

Dual domain recognition determines SARS-CoV-2 PLpro selectivity for human ISG15 and K48-linked di-ubiquitin (preprint)

The Papain-like protease (PLpro) is a domain of a multi-functional, non-structural protein 3 of coronaviruses. PLpro cleaves viral polyproteins and posttranslational conjugates with poly-ubiquitin and protective ISG15, composed of two ubiquitin-like (UBL) domains. Across coronaviruses, PLpro showed divergent selectivity for recognition and cleavage of posttranslational conjugates despite sequence conservation. We show that SARS-CoV-2 PLpro binds human ISG15 and K48-linked di-ubiquitin (K48-Ub2) with nanomolar affinity and detect alternate weaker-binding modes. Crystal structures of untethered PLpro complexes with ISG15 and K48-Ub2 combined with solution NMR and cross-linking mass spectrometry revealed how the two domains of ISG15 or K48-Ub2 are differently utilized in interactions with PLpro. Analysis of protein interface energetics uncovered differential binding stabilities of the two UBL/Ub domains. We emphasize how substrate recognition can be tuned to cleave specifically ISG15 or K48-Ub2 modifications while retaining capacity to cleave mono-Ub conjugates. These results highlight alternative druggable surfaces that would inhibit PLpro function.

Dual domain recognition determines SARS-CoV-2 PLpro selectivity for human ISG15 and K48-linked di-ubiquitin (preprint)

The Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) genome is evolving as the viral pandemic continues its active phase around the world. The Papain-like protease (PLpro) is a domain of Nsp3 - a large multidomain protein that is an essential component of the replication-transcription complex, making it a good therapeutic target. PLpro is a multi-functional protein encoded in coronaviruses that can cleave viral polyproteins, poly-ubiquitin and protective Interferon Stimulated Gene 15 product, ISG15, which mimics a head-to-tail linked ubiquitin (Ub) dimer. PLpro across coronavirus families showed divergent selectivity for recognition and cleavage of these protein substrates despite sequence conservation. However, it is not clear how sequence changes in SARS-CoV-2 PLpro alter its selectivity for substrates and what outcome this has on the pathogenesis of the virus. We show that SARS-CoV-2 PLpro preferentially binds ISG15 over Ub and K48-linked Ub2. We determined crystal structures of PLpro in complex with human K48-Ub2 and ISG15 revealing that dual domain recognition of ISG15 drives substrate selectivity over Ub and Ub2. We also characterized the PLpro substrate interactions using solution NMR, cross-linking mass spectrometry to support that ISG15 is recognized via two domains while Ub2 binds primarily through one Ub domain. Finally, energetic analysis of the binding interfaces between PLpro from SARS-CoV-1 and SARS-CoV-2 with ISG15 and Ub2 define the sequence determinants for how PLpros from different coronaviruses recognize two topologically distinct substrates and how evolution of the protease altered its substrate selectivity. Our work reveals how PLpro substrate selectivity may evolve in PLpro coronaviruses variants enabling design of more effective therapeutics.

Potent and Selective Covalent Inhibitors of the Papain-like Protease from SARS-CoV-2 (preprint)

Direct-acting antivirals for the treatment of COVID-19, which is caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2), are needed to complement vaccination efforts. The papain-like protease (PLpro) of SARS-CoV-2 is essential for viral proliferation. In addition, PLpro dysregulates the host immune response by cleaving ubiquitin and interferon-stimulated gene 15 protein (ISG15) from host proteins. As a result, PLpro is a promising target for inhibition by small-molecule therapeutics. Here we have designed a series of covalent inhibitors by introducing a peptidomimetic linker and reactive electrophilic “warheads” onto analogs of the noncovalent PLpro inhibitor GRL0617. We show that the most promising PLpro inhibitor is potent and selective, with activity in cell-based antiviral assays rivaling that of the RNA-dependent RNA polymerase inhibitor remdesivir. An X-ray crystal structure of the most promising lead compound bound covalently to PLpro establishes the molecular basis for protease inhibition and selectivity against structurally similar human deubiquitinases. These findings present an opportunity for further development of potent and selective covalent PLpro inhibitors.

Integration of Transcriptome and Cell Surface Protein Expression by LinQ-View Identifies Unique Immune Subpopulations (preprint)

Single cell RNA sequencing is a powerful tool for characterizing the molecular and cellular heterogeneity of immune cells during their development and activation. Multimodal advances in single cell sequencing have enabled the simultaneous quantification of cell surface protein expression alongside unbiased transcriptional profiling. Here we present LinQ-View, a toolkit designed for multimodal single cell data visualization and analysis that links transcriptional and cell surface protein expression profiling data. Further, we propose a quantitative metric for cluster purity of CITE-seq data, enabling effective determination of clustering algorithms and their parameters, and finally demonstrate the utility of our toolkit through seamless integration with standard single cell analysis workflows on several public datasets. Through comparison to existing multimodal methods, we demonstrate that LinQ-View generates more accurate cell clusters and is highly efficient, even with massive datasets. LinQ-View is specialized in handling CITE-seq data with routine numbers of surface protein features (e.g. less than 50), by preventing variations in a single surface protein feature from affecting results. Finally, we utilized this method to integrate single cell transcriptional and protein expression data from COVID-19-infected patients and influenza-immunized subjects, revealing antigen-specific B cell subsets and previously unknown T cell subsets post-infection and vaccination.

COVID-19 Infection Induces Substantial Memory B Cell Maturation to Non-Neutralizing Viral Targets (preprint)

Dissecting the evolution of memory B cells (MBCs) against SARS-CoV-2 is critical for understanding antibody recall upon secondary exposure. Here, we utilized single-cell sequencing to profile SARS-CoV-2-reactive B cell subsets in 42 COVID-19 patients. We isolated thousands of B cells in multiple distinct subsets specific to the SARS-CoV-2 spike, endemic coronavirus (HCoV) spikes, nucleoprotein (NP), and open reading frame 8 (ORF8). Spike-specific cells were enriched in the memory compartment of acutely infected and convalescent patients 1.5–5 months post-infection. With severe acute infection, we identified substantial populations of endemic HCoV-reactive antibody-secreting cells with highly mutated variable genes, indicative of preexisting immunity. Finally, MBCs exhibited maturation to NP and ORF8 over time relative to spike, especially in older patients. Monoclonal antibodies against these targets were non-neutralizing and non-protective in vivo. These findings reveal considerable antibody adaptation to non-neutralizing antigens during infection, emphasizing the importance of vaccination for inducing neutralizing spike-specific MBCs.Trial Registration Number: This clinical trial was registered at ClinicalTrials.gov with identifier NCT04340050, and clinical information for patients included in the study is detailed in Table S1–S3.Funding: This project was funded in part by the National Institute of Allergy and Infectious Disease (NIAID); National Institutes of Health (NIH) grant numbers U19AI082724 (P.C.W.), U19AI109946 (P.C.W.), U19AI057266 (P.C.W.), the NIAID Centers of Excellence for Influenza Research and Surveillance (CEIRS) grant numbers HHSN272201400005C(P.C.W.). N.W.A. was supported by the Multi-disciplinary Training program in Cancer Research (MTCR) - NIH T32 CA009594. A.J. and R.P.J were supported by federal funds from the NIAID, NIH, and Department of Health and Human Services under Contract HHSN272201700060C. F.K and F.A. were funded by the NIAID CEIRS contractHHSN272201400008C, Collaborative Influenza Vaccine Innovation Centers (CIVIC) contract 75N93019C00051 and the generous support of the JPB foundation, the Open Philanthropy Project (#2020-215611) and other philanthropic donations. Y.K. and P.H.were funded by the Research Program on Emerging and Re-emerging Infectious Disease grant (JP19fk0108113) and the Japan Program for Infectious Diseases Research and Infrastructure (JP20fk0108272) from the Japan Agency for Medical Research and Development (AMED), NIAID CEIRS contract HHSN272201400008C, and CIVIC contract 75N93019C00051. D.F., C.N, Y.D., and P.D.H, were supported by NIAID contracts HHSN272201700060C and 75N93019C00062. M.S.D. and E.S.W. were supported by NIH grants R01 AI157155 and F30 AI152327, respectively.Conflict of Interest: Several antibodies generated from this work are being used by Now Diagnostics in Springdale, AR for the development of a diagnostic test. M.S.D. is a consultant for Inbios, Vir Biotechnology, NGM Biopharmaceuticals, and Carnival Corporation, and on the Scientific Advisory Boards of Moderna and Immunome. The Diamond laboratory has received funding support in sponsored research agreements from Moderna, Vir Biotechnology, and Emergent BioSolutions.Ethical Approval: All studies were performed with the approval of the University of Chicago institutional review board IRB20-0523 and University of Chicago, University of Wisconsin-Madison, and Washington University in St. Louis institutional biosafety committees. Informed consent was obtained after the research applications and possible consequences of the studies were disclosed to study subjects.

Distinct B cell subsets give rise to antigen-specific antibody responses against SARS-CoV-2 (preprint)

Discovery of durable memory B cell (MBC) subsets against neutralizing viral epitopes is critical for determining immune correlates of protection from SARS-CoV-2 infection. Here, we identified functionally distinct SARS-CoV-2-reactive B cell subsets by profiling the repertoire of convalescent COVID-19 patients using a high-throughput B cell sorting and sequencing platform. Utilizing barcoded SARS-CoV-2 antigen baits, we isolated thousands of B cells that segregated into discrete functional subsets specific for the spike, nucleocapsid protein (NP), and open reading frame (ORF) proteins 7a and 8. Spike-specific B cells were enriched in canonical MBC clusters, and monoclonal antibodies (mAbs) from these cells were potently neutralizing. By contrast, B cells specific to ORF8 and NP were enriched in naïve and innate-like clusters, and mAbs against these targets were exclusively non-neutralizing. Finally, we identified that B cell specificity, subset distribution, and affinity maturation were impacted by clinical features such as age, sex, and symptom duration. Together, our data provide a comprehensive tool for evaluating B cell immunity to SARS-CoV-2 infection or vaccination and highlight the complexity of the human B cell response to SARS-CoV-2.

nanoDoc: RNA modification detection using Nanopore raw reads with Deep One-Class Classification (preprint)

Advances in Nanopore single-molecule direct RNA sequencing (DRS) have presented the possibility of detecting comprehensive post-transcriptional modifications (PTMs) as an alternative to experimental approaches combined with high-throughput sequencing. It has been shown that the DRS method can detect the change in the raw electric current signal of a PTM; however, the accuracy and reliability still require improvement. Here, we presented a new software, called nanoDoc, for detecting PTMs from DRS data using a deep neural network. Current signal deviations caused by PTMs are analyzed via Deep One-Class Classification with a convolutional neural network. Using a ribosomal RNA dataset, the software archive displayed an area under the curve (AUC) accuracy of 0.96 for the detection of 23 different kinds of modifications in Escherichia coli and Saccharomyces cerevisiae. We also demonstrated a tentative classification of PTMs using unsupervised clustering. Finally, we applied this software to severe acute respiratory syndrome coronavirus 2 data and identified commonly modified sites among three groups. nanoDoc is open source (GPLv3) and available at https://github.com/uedaLabR/nanoDoc

SARS-CoV-2 infection severity is linked to superior humoral immunity against the spike (preprint)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is currently causing a global pandemic. The antigen specificity and kinetics of the antibody response mounted against this novel virus are not understood in detail. Here, we report that subjects with a more severe SARS-CoV-2 infection exhibit a larger antibody response against the spike and nucleocapsid protein and epitope spreading to subdominant viral antigens, such as open reading frame 8 and non-structural proteins. Subjects with a greater antibody response mounted a larger memory B cell response against the spike, but not the nucleocapsid protein. Additionally, we revealed that antibodies against the spike are still capable of binding the D614G spike mutant and cross-react with the SARS-CoV-1 receptor binding domain. Together, this study reveals that subjects with a more severe SARS-CoV-2 infection exhibit a greater overall antibody response to the spike and nucleocapsid protein and a larger memory B cell response against the spike.

Evaluation of stability and inactivation methods of SARS-CoV-2 in context of laboratory settings (preprint)

The novel coronavirus SARS-CoV-2 is the causative agent of the acute respiratory disease COVID-19, which has become a global concern due to its rapid spread. Laboratory work with SARS-CoV-2 in a laboratory setting was rated to biosafety level 3 (BSL-3) biocontainment level. However, certain research applications in particular in molecular biology require incomplete denaturation of the proteins, which might cause safety issues handling contaminated samples. In particular, it is critical to provide proof of inactivation before samples can be removed from the BSL-3. In this study, we evaluated common lysis buffers that are used in molecular biological laboratories for their ability to inactivate SARS-CoV-2. We have found that guanidine thiocyanate, SDS, and Triton-X containing lysis buffers were effective in inactivation of SARS-CoV-2, however, the M-PER lysis buffer containing a proprietary detergent failed to inactivate SARS-CoV-2. Furthermore, we compared chemical and non-chemical inactivation methods including ethanol, acetone-methanol mixture, PFA, UV-C light, and heat inactivation. In addition, the stability of the virus in cell culture media at 4{degrees}C and on surfaces used in laboratory environment was analyzed. In conclusion, careful evaluation of the used inactivation methods are required and additional inactivation steps are necessary before removal of lysed viral samples from BSL-3.

Age-dependent regulation of SARS-CoV-2 cell entry genes and cell death programs correlates with COVID-19 disease severity (preprint)

Angiotensin-converting enzyme 2 (ACE2) maintains cardiovascular and renal homeostasis but also serves as the entry receptor for the novel severe acute respiratory syndrome coronavirus (SARS-CoV-2), the causal agent of novel coronavirus disease 2019 (COVID-19)1. COVID-19 disease severity, while highly variable, is typically lower in pediatric patients than adults (particularly the elderly), but increased rates of hospitalizations requiring intensive care are observed in infants than in older children. The reasons for these differences are unknown. To detect potential age-based correlates of disease severity, we measured ACE2 protein expression at the single cell level in human lung tissue specimens from over 100 donors from [~]4 months to 75 years of age. We found that expression of ACE2 in distal lung epithelial cells generally increases with advancing age but exhibits extreme intra- and inter-individual heterogeneity. Notably, we also detected ACE2 expression on neonatal airway epithelial cells and within the lung parenchyma. Similar patterns were found at the transcript level: ACE2 mRNA is expressed in the lung and trachea shortly after birth, downregulated during childhood, and again expressed at high levels in late adulthood in alveolar epithelial cells. Furthermore, we find that apoptosis, which is a natural host defense system against viral infection, is also dynamically regulated during lung maturation, resulting in periods of heightened apoptotic priming and dependence on pro-survival BCL-2 family proteins including MCL-1. Infection of human lung cells with SARS-CoV-2 triggers an unfolded protein stress response and upregulation of the endogenous MCL-1 inhibitor Noxa; in juveniles, MCL-1 inhibition is sufficient to trigger apoptosis in lung epithelial cells - this may limit virion production and inflammatory signaling. Overall, we identify strong and distinct correlates of COVID-19 disease severity across lifespan and advance our understanding of the regulation of ACE2 and cell death programs in the mammalian lung. Furthermore, our work provides the framework for potential translation of apoptosis modulating drugs as novel treatments for COVID-19.

Methylation of RNA Cap in SARS-CoV-2 captured by serial crystallography (preprint)

The genome of the SARS-CoV-2 coronavirus contains 29 proteins, of which 15 are nonstructural. Nsp10 and Nsp16 form a complex responsible for the capping of mRNA at the 5' terminus. In the methylation reaction the S-adenosyl-L-methionine serves as the donor of the methyl group that is transferred to Cap-0 at the first transcribed nucleotide to create Cap-1. The presence of Cap-1 makes viral RNAs mimic the host transcripts and prevents their degradation. To investigate the 2'-O methyltransferase activity of SARS-CoV-2 Nsp10/16, we applied fixed-target serial synchrotron crystallography (SSX) which allows for physiological temperature data collection from thousands of crystals, significantly reducing the x-ray dose while maintaining a biologically relevant temperature. We determined crystal structures of Nsp10/16 that revealed the states before and after the methylation reaction, for the first time illustrating coronavirus Nsp10/16 complexes with the m7GpppAm2'-O Cap-1, where 2'OH of ribose is methylated. We compare these structures with structures of Nsp10/16 at 297 K and 100 K collected from a single crystal. This data provide important mechanistic insight and can be used to design small molecules that inhibit viral RNA maturation making SARS-CoV-2 sensitive to host innate response.

Structure of papain-like protease from SARS-CoV-2 and its complexes with non-covalent inhibitors (preprint)

The number of new cases world-wide for the COVID-19 disease is increasing dramatically, while efforts to contain Severe Acute Respiratory Syndrome Coronavirus 2 is producing varied results in different countries. There are three key SARS-CoV-2 enzymes potentially targetable with antivirals: papain-like protease (PLpro), main protease (Mpro), and RNA-dependent RNA polymerase. Of these, PLpro is an especially attractive target because it plays an essential role in several viral replication processes, including cleavage and maturation of viral polyproteins, assembly of the replicase-transcriptase complex (RTC), and disruption of host viral response machinery to facilitate viral proliferation and replication. Moreover, this enzyme is conserved across different coronaviruses and promising inhibitors have already been discovered for its SARS-CoV variant. Here we report a substantive body of structural, biochemical, and virus replication studies that identify several inhibitors of the enzyme from SARS-CoV-2 in both wild-type and mutant forms. These efforts include the first structures of wild-type PLpro, the active site C111S mutant, and their complexes with inhibitors, determined at 1.60-2.70 Angstroms. This collection of structures provides fundamental molecular and mechanistic insight to PLpro, and critically, illustrates details for inhibitors recognition and interactions. All presented compounds inhibit the peptidase activity of PLpro in vitro, and some molecules block SARS-CoV-2 replication in cell culture assays. These collated findings will accelerate further structure-based drug design efforts targeting PLpro, with the ultimate goal of identifying high-affinity inhibitors of clinical value for SARS-CoV-2.

Tipiracil binds to uridine site and inhibits Nsp15 endoribonuclease NendoU from SARS-CoV-2 (preprint)

ABSTRACTSARS-CoV-2 Nsp15 is a uridylate-specific endoribonuclease with C-terminal catalytic domain belonging to the EndoU family. It degrades the polyuridine extensions in (-) sense strand of viral RNA and some non-translated RNA on (+) sense strand. This activity seems to be responsible for the interference with the innate immune response and evasion of host pattern recognition. Nsp15 is highly conserved in coronaviruses suggesting that its activity is important for virus replication. Here we report first structures with bound nucleotides and show that SARS-CoV-2 Nsp15 specifically recognizes U in a pattern previously predicted for EndoU. In the presence of manganese ions, the enzyme cleaves unpaired RNAs. Inhibitors of Nsp15 have been reported but not actively pursued into therapeutics. The current COVID-19 pandemic brought to attention the repurposing of existing drugs and the rapid identification of new antiviral compounds. Tipiracil is an FDA approved drug that is used with trifluridine in the treatment of colorectal cancer. Here, we combine crystallography, biochemical and whole cell assays, and show that this compound inhibits SARS-CoV-2 Nsp15 and interacts with the uridine binding pocket of the enzyme’s active site, providing basis for the uracil scaffold-based drug development.Competing Interest StatementThe authors have declared no competing interest.View Full Text

Crystal structures of SARS-CoV-2 ADP-ribose phosphatase (ADRP): from the apo form to ligand complexes (preprint)

Among 15 nonstructural proteins (Nsps), the newly emerging SARS-CoV-2 encodes a large, multidomain Nsp3. One of its units is ADP-ribose phosphatase domain (ADRP, also known as macrodomain) which is believed to interfere with the host immune response. Such a function appears to be linked to the proteins ability to remove ADP-ribose from ADP-ribosylated proteins and RNA, yet the precise role and molecular targets of the enzyme remains unknown. Here, we have determined five, high resolution (1.07 - 2.01 [A]) crystal structures corresponding to the apo form of the protein and complexes with 2-(N-morpholino)ethanesulfonic acid (MES), AMP and ADPr. We show that the protein undergoes conformational changes to adapt to the ligand in a manner previously observed before for in close homologs from other viruses. We also identify a conserved water molecule that may participate in hydrolysis. This work builds foundations for future structure-based research of the ADRP, including search for potential antiviral therapeutics.

Structural Plasticity of the SARS-CoV-2 3CL Mpro Active Site Cavity Revealed by Room Temperature X-ray Crystallography (preprint)

The COVID-19 disease caused by the SARS-CoV-2 Coronavirus has become a pandemic health crisis. An attractive target for antiviral inhibitors is the main protease 3CL Mpro due to its essential role in processing the polyproteins translated from viral RNA. Here we report the room temperature X-ray structure of unliganded SARS-CoV-2 3CL Mpro, revealing the resting structure of the active site and the conformation of the catalytic site cavity. Comparison with previously reported low-temperature ligand-free and inhibitor-bound structures suggest that the room temperature structure may provide more relevant information at physiological temperatures for aiding in molecular docking studies.

The crystal structure of nsp10-nsp16 heterodimer from SARS CoV-2in complex with S-adenosylmethionine (preprint)

SARS-CoV-2 is a member of the coronaviridae family and is the etiological agent of the respiratory Coronavirus Disease 2019. The virus has spread rapidly around the world resulting in over two million cases and nearly 150,000 deaths as of April 17, 2020. Since no treatments or vaccines are available to treat COVID-19 and SARS-CoV-2, respiratory complications derived from the infections have overwhelmed healthcare systems around the world. This virus is related to SARS-CoV-1, the virus that caused the 2002-2004 outbreak of Severe Acute Respiratory Syndrome. In January 2020, the Center for Structural Genomics of Infectious Diseases implemented a structural genomics pipeline to solve the structures of proteins essential for coronavirus replication-transcription. Here we show the first structure of the SARS-CoV-2 nsp10-nsp16 2-O-methyltransferase complex with S-adenosylmethionine at a resolution of 1.80 [A]. This heterodimer complex is essential for capping viral mRNA transcripts for efficient translation and to evade immune surveillance.

Crystal structure of Nsp15 endoribonuclease NendoU from SARS-CoV-2 (preprint)

Severe Acute Respiratory Syndrome Coronavirus 2 is rapidly spreading around the world. There is no existing vaccine or proven drug to prevent infections and stop virus proliferation. Although this virus is similar to human and animal SARS- and MERS-CoVs the detailed information about SARS-CoV-2 proteins structures and functions is urgently needed to rapidly develop effective vaccines, antibodies and antivirals. We applied high-throughput protein production and structure determination pipeline at the Center for Structural Genomics of Infectious Diseases to produce SARS-CoV-2 proteins and structures. Here we report the high-resolution crystal structure of endoribonuclease Nsp15/NendoU from SARS-CoV-2 - a virus causing current world-wide epidemics. We compare this structure with previously reported models of Nsp15 from SARS and MERS coronaviruses.