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The ongoing global pandemic of coronavirus disease 2019 (COVID-19) has caused a huge number of human deaths. Currently, there are no specific drugs or vaccines available for this virus (SARS-CoV-2). The viral polymerase is a promising antiviral target. Here, we describe the near-atomic-resolution structure of the SARS-CoV-2 polymerase complex consisting of the nsp12 catalytic subunit and nsp7-nsp8 cofactors. This structure highly resembles the counterpart of SARS-CoV with conserved motifs for all viral RNA-dependent RNA polymerases and suggests a mechanism of activation by cofactors. Biochemical studies reveal reduced activity of the core polymerase complex and lower thermostability of individual subunits of SARS-CoV-2 compared with SARS-CoV. These findings provide important insights into RNA synthesis by coronavirus polymerase and indicate adaptation of SARS-CoV-2 toward humans with a relatively lower body temperature than the natural bat hosts.Cytokine-inducible SH2-containing protein (CIS; encoded by the gene CISH) is a key negative regulator of interleukin-15 (IL-15) signaling in natural killer (NK) cells. Here, we develop human CISH-knockout (CISH-/-) NK cells using an induced pluripotent stem cell-derived NK cell (iPSC-NK cell) platform. CISH-/- iPSC-NK cells demonstrate increased IL-15-mediated JAK-STAT signaling activity. Consequently, CISH-/- iPSC-NK cells exhibit improved expansion and increased cytotoxic activity against multiple tumor cell lines when maintained at low cytokine concentrations. CISH-/- iPSC-NK cells display significantly increased in vivo persistence and inhibition of tumor progression in a leukemia xenograft model. Mechanistically, CISH-/- iPSC-NK cells display improved metabolic fitness characterized by increased basal glycolysis, glycolytic capacity, maximal mitochondrial respiration, ATP-linked respiration, and spare respiration capacity mediated by mammalian target of rapamycin (mTOR) signaling that directly contributes to enhanced NK cell function. Together, these studies demonstrate that CIS plays a key role to regulate human NK cell metabolic activity and thereby modulate anti-tumor activity.The trinuclear copper center (TNC) of laccase reduces oxygen to water with very little overpotential. The arrangement of the coppers and ligands in the TNC is known to be from many crystal structures, yet information about possible dynamics of the ligands is absent. Here, we report dynamics at the TNC of small laccase from Streptomyces coelicolor using paramagnetic NMR and electron paramagnetic resonance spectroscopy. Fermi contact-shifted resonances tentatively assigned to histidine Hδ1 display a two-state chemical exchange with exchange rates in the order of 100 s-1. In the electron paramagnetic resonance spectra, at least two forms are observed with different gz-values. It is proposed that the exchange processes reflect the rotational motion of histidine imidazole rings that coordinate the coppers in the TNC.The F1 motor is a rotating molecular motor that ensures a tight chemomechanical coupling between ATP hydrolysis/synthesis reactions and rotation steps. However, the mechanism underlying this tight coupling remains to be elucidated. In this study, we used electrorotation in single-molecule experiments using an F1βE190D mutant to demonstrate that the stall torque was significantly smaller than the wild-type F1, indicating a loose coupling of this mutant, despite showing similar stepping torque as the wild-type. Experiments on the ATPase activity after heat treatment and gel filtration of the α3β3-subcomplex revealed the unstable structure of the βE190D mutant. Our results suggest that the tight chemomechanical coupling of the F1 motor relies on the structural stability of F1. [https://www.selleckchem.com/products/ly333531.html find more] We also discuss the difference between the stepping torque and the stall torque.Monolinks are produced in a chemical crosslinking mass spectrometry experiment and are more abundant than crosslinks. They convey residue exposure information, but so far have not been used in the modeling of protein structures. Here, we present the Monolink Depth Score (MoDS), for assessing structural models based on the depth of monolinked residues, corresponding to their distance to the nearest bulk water. Using simulated and reprocessed experimental data from the Proteomic Identification Database, we compare the performance of MoDS to MNXL, our previously developed score for assessing models based on crosslinking data. Our results show that MoDS can be used to effectively score models based on monolinks, and that a crosslink/monolink combined score (XLMO) leads to overall higher performance. The work strongly supports the use of monolink data in the context of integrative structure determination. We also present XLM-Tools, a program to assist in this effort, available at https//github.com/Topf-Lab/XLM-Tools.Predicting RNA three-dimensional structures from sequence could accelerate understanding of the growing number of RNA molecules being discovered across biology. Rosetta's Fragment Assembly of RNA with Full-Atom Refinement (FARFAR) has shown promise in community-wide blind RNA-Puzzle trials, but lack of a systematic and automated benchmark has left unclear what limits FARFAR performance. Here, we benchmark FARFAR2, an algorithm integrating RNA-Puzzle-inspired innovations with updated fragment libraries and helix modeling. In 16 of 21 RNA-Puzzles revisited without experimental data or expert intervention, FARFAR2 recovers native-like structures more accurate than models submitted during the RNA-Puzzles trials. Remaining bottlenecks include conformational sampling for &gt;80-nucleotide problems and scoring function limitations more generally. Supporting these conclusions, preregistered blind models for adenovirus VA-I RNA and five riboswitch complexes predicted native-like folds with 3- to 14 Å root-mean-square deviation accuracies. We present a FARFAR2 webserver and three large model archives (FARFAR2-Classics, FARFAR2-Motifs, and FARFAR2-Puzzles) to guide future applications and advances.It is widely assumed that decreasing transcription factor DNA-binding affinity reduces transcription initiation by diminishing occupancy of sequence-specific regulatory elements. However, in vivo transcription factors find their binding sites while confronted with a large excess of low-affinity degenerate motifs. Here, using the melanoma lineage survival oncogene MITF as a model, we show that low-affinity binding sites act as a competitive reservoir in vivo from which transcription factors are released by mitogen-activated protein kinase (MAPK)-stimulated acetylation to promote increased occupancy of their regulatory elements. Consequently, a low-DNA-binding-affinity acetylation-mimetic MITF mutation supports melanocyte development and drives tumorigenesis, whereas a high-affinity non-acetylatable mutant does not. The results reveal a paradoxical acetylation-mediated molecular clutch that tunes transcription factor availability via genome-wide redistribution and couples BRAF to tumorigenesis. Our results further suggest that p300/CREB-binding protein-mediated transcription factor acetylation may represent a common mechanism to control transcription factor availability.