Translation has a fundamental function in defining the fate of the transcribed genome. RNA-sequencing (RNA-seq) data enable the quantification of complex transcript mixtures, often detecting several transcript isoforms of unknown functions for one gene. Here, we describe ORFquant, a method to annotate and quantify translation at the level of single open reading frames (ORFs), using information from

The HIV-1 envelope glycoprotein (Env) trimer, composed of gp120 and gp41 subunits, mediates viral entry into cells. Recombinant Env trimers have been studied structurally, but characterization of Env embedded in intact virus membranes has been limited to low resolution. Here, we deploy cryo-electron tomography and subtomogram averaging to determine the structures of Env trimers on aldrithiol-2 (AT-2)-inactivated

Bivalent chromatin domains contain opposing histone modifications that assist cell lineage specification. Two studies report a role for Dppa2 and Dppa4 in the establishment of bivalency and the prevention of de novo DNA methylation at development-related genes in mouse embryonic stem cells.

How the epigenetic landscape is established in development is still being elucidated. Here, we uncover developmental pluripotency associated 2 and 4 (DPPA2/4) as epigenetic priming factors that establish a permissive epigenetic landscape at a subset of developmentally important bivalent promoters characterized by low expression and poised RNA-polymerase. Differentiation assays reveal that Dppa2/4 double

Transient receptor potential vanilloid subfamily member 3 (TRPV3) is a temperature-sensitive cation channel. Previous cryo-EM analyses of TRPV3 in detergent micelles or amphipol proposed that the lower gate opens by α-to-π helical transitions of the nearby S6 helix. However, it remains unclear how physiological lipids are involved in the TRPV3 activation. Here we determined the apo state structure

The transient receptor potential cation channel subfamily V member 3 (TRPV3) channel plays a critical role in skin physiology, and mutations in TRPV3 result in the development of a congenital skin disorder, Olmsted syndrome. Here we describe multiple cryo-electron microscopy structures of human TRPV3 reconstituted into lipid nanodiscs, representing distinct functional states during the gating cycle

Early mammalian development entails genome-wide epigenome remodeling, including DNA methylation erasure and reacquisition, which facilitates developmental competence. To uncover the mechanisms that orchestrate DNA methylation dynamics, we coupled a single-cell ratiometric DNA methylation reporter with unbiased CRISPR screening in murine embryonic stem cells (ESCs). We identify key genes and regulatory

Human islet amyloid polypeptide (hIAPP) functions as a glucose-regulating hormone but deposits as amyloid fibrils in more than 90% of patients with type II diabetes (T2D). Here we report the cryo-EM structure of recombinant full-length hIAPP fibrils. The fibril is composed of two symmetrically related protofilaments with ordered residues 14–37. Our hIAPP fibril structure (i) supports the previous hypothesis

Amyloid deposits consisting of fibrillar islet amyloid polypeptide (IAPP) in pancreatic islets are associated with beta-cell loss and have been implicated in type 2 diabetes (T2D). Here, we applied cryo-EM to reconstruct densities of three dominant IAPP fibril polymorphs, formed in vitro from synthetic human IAPP. An atomic model of the main polymorph, built from a density map of 4.2-Å resolution,

Transcription by RNA polymerase II (Pol II) is carried out by an elongation complex. We previously reported an activated porcine Pol II elongation complex, EC*, encompassing the human elongation factors DSIF, PAF1 complex (PAF) and SPT6. Here we report the cryo-EM structure of the complete EC* that contains RTF1, a dissociable PAF subunit critical for chromatin transcription. The RTF1 Plus3 domain

Controlled perturbation of protein activity is essential to study protein function in cells and living organisms. Small molecules that hijack the cellular protein ubiquitination machinery to selectively degrade proteins of interest, so-called degraders, have recently emerged as alternatives to selective chemical inhibitors, both as therapeutic modalities and as powerful research tools. These systems

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

RNA-binding sites (RBSs) can be identified by liquid chromatography and tandem mass spectrometry analyses of the protein–RNA conjugates created by crosslinking, but RBS mapping remains highly challenging due to the complexity of the formed RNA adducts. Here, we introduce RBS-ID, a method that uses hydrofluoride to fully cleave RNA into mono-nucleosides, thereby minimizing the search space to drastically

Noncoding RNAs (ncRNAs) direct a remarkable number of diverse functions in development and disease through their regulation of transcription, RNA processing and translation. Leading the charge in the RNA revolution is a class of ncRNAs that are synthesized at active enhancers, called enhancer RNAs (eRNAs). Here, we review recent insights into the biogenesis of eRNAs and the mechanisms underlying their

Prion diseases are caused by the misfolding of prion protein (PrP). Misfolded PrP forms protease-resistant aggregates in vivo (PrPSc) that are able to template the conversion of the native form of the protein (PrPC), a property shared by in vitro–produced PrP fibrils. Here we produced amyloid fibrils in vitro from recombinant, full-length human PrPC (residues 23–231) and determined their structure

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Human insulin and its current therapeutic analogs all show propensity, albeit varyingly, to self-associate into dimers and hexamers, which delays their onset of action and makes blood glucose management difficult for people with diabetes. Recently, we described a monomeric, insulin-like peptide in cone-snail venom with moderate human insulin-like bioactivity. Here, with insights from structural biology

Cyclic nucleotide–gated (CNG) channels convert cyclic nucleotide (CN) binding and unbinding into electrical signals in sensory receptors and neurons. The molecular conformational changes underpinning ligand activation are largely undefined. We report both closed- and open-state atomic cryo-EM structures of a full-length Caenorhabditis elegans cyclic GMP−activated channel TAX-4, reconstituted in lipid

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Cellular protein levels are finely tuned through microRNA-mediated gene regulation, triggered by RNA-induced silencing complexes (RISCs) that recognize, bind and silence mRNA targets. A recent study shows that, while mRNA target recognition is achieved with only about a third of the guide RNA sequence, formation of an efficient RISC conformation required for target silencing involves the coordination

The second Keystone Symposium on AAA+ proteins, “AAA+ Proteins: From Atomic Structures to Organisms”, was held in Tahoe City, USA in January 2020. The program highlighted recent advances from structural, biochemical and cellular approaches that have extended our understanding of these important ATP-driven molecular machines.

The Na+/I− symporter (NIS), the plasma membrane protein that actively transports I− (stoichiometry 2Na+:1I−) in thyroid physiology and radioiodide-based thyroid cancer treatment, also transports the environmental pollutant perchlorate (stoichiometry 1Na+:1ClO4−), which competes with I− for transport. Until now, the mechanism by which NIS transports different anion substrates with different stoichiometries

TENT4 enzymes generate ‘mixed tails’ of diverse nucleotides at 3′ ends of RNAs via nontemplated nucleotide addition to protect messenger RNAs from deadenylation. Here we discover extensive mixed tailing in transcripts of hepatitis B virus (HBV) and human cytomegalovirus (HCMV), generated via a similar mechanism exploiting the TENT4–ZCCHC14 complex. TAIL-seq on HBV and HCMV RNAs revealed that TENT4A

The ESCRT complexes drive membrane scission in HIV-1 release, autophagosome closure, multivesicular body biogenesis, cytokinesis, and other cell processes. ESCRT-I is the most upstream complex and bridges the system to HIV-1 Gag in virus release. The crystal structure of the headpiece of human ESCRT-I comprising TSG101-VPS28-VPS37B-MVB12A was determined, revealing an ESCRT-I helical assembly with a

The Vibrio parahaemolyticus T3SS effector VopQ targets host-cell V-ATPase, resulting in blockage of autophagic flux and neutralization of acidic compartments. Here, we report the cryo-EM structure of VopQ bound to the Vo subcomplex of the V-ATPase. VopQ inserts into membranes and forms an unconventional pore while binding directly to subunit c of the V-ATPase membrane-embedded subcomplex Vo. We show

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

How Gram-negative bacteria transport glycerophospholipids between membranes remains a major point of inquiry for cell envelope biologists. Two groups report cryo-EM structures and functional studies of LetB (YebT), providing support for its involvement in cell envelope maintenance.

Cryptochromes (CRYs) are a group of evolutionarily conserved flavoproteins found in many organisms. In plants, the well-studied CRY photoreceptor, activated by blue light, plays essential roles in plant growth and development. However, the mechanism of activation remains largely unknown. Here, we determined the oligomeric structures of the blue-light-perceiving PHR domain of Zea mays CRY1 and an Arabidopsis

The interplay between E2 and E3 enzymes regulates the polyubiquitination of substrates in eukaryotes. Among the several RING-domain E3 ligases in humans, many utilize two distinct E2s for polyubiquitination. For example, the cell cycle regulatory E3, human anaphase-promoting complex/cyclosome (APC/C), relies on UBE2C to prime substrates with ubiquitin (Ub) and on UBE2S to extend polyubiquitin chains

Oncogene activation during tumorigenesis generates DNA replication stress, a known driver of genome rearrangements. In response to replication stress, certain loci, such as common fragile sites and telomeres, remain under-replicated during interphase and subsequently complete locus duplication in mitosis in a process known as 'MiDAS'. Here, we demonstrate that RTEL1 (regulator of telomere elongation

The SLX4 tumor suppressor is a scaffold that plays a pivotal role in several aspects of genome protection, including homologous recombination, interstrand DNA crosslink repair and the maintenance of common fragile sites and telomeres. Here, we unravel an unexpected direct interaction between SLX4 and the DNA helicase RTEL1, which, until now, were viewed as having independent and antagonistic functions

Cryptochromes (CRYs) are blue-light receptors in plants that harbor FAD as a cofactor and regulate various physiological responses. Photoactivated CRYs undergo oligomerization, which increases the binding affinity to downstream signaling partners. Despite decades of research on the activation of CRYs, little is known about how they are inactivated. Binding of blue-light inhibitors of cryptochromes

Cryptochrome (CRY) photoreceptors undergo photoresponsive homo-oligomerization to become physiologically active, and BICs (blue-light inhibitors of CRYs) suppress homo-oligomerization. Structural elucidation of CRY–CRY homo-oligomers and a CRY–BIC heterodimer reveals how the activity of plant CRYs is regulated by alternative protein–protein interactions.

The antineoplastic drug carmofur is shown to inhibit the SARS-CoV-2 main protease (Mpro). Here, the X-ray crystal structure of Mpro in complex with carmofur reveals that the carbonyl reactive group of carmofur is covalently bound to catalytic Cys145, whereas its fatty acid tail occupies the hydrophobic S2 subsite. Carmofur inhibits viral replication in cells (EC50 = 24.30 μM) and is a promising lead

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Cas1 integrase associates with Cas2 to insert short DNA fragments into a CRISPR array, establishing nucleic acid memory in prokaryotes. Here we applied single-molecule FRET methods to the Enterococcus faecalis (Efa) Cas1-Cas2 system to establish a kinetic framework describing target-searching, integration, and post-synapsis events. EfaCas1-Cas2 on its own is not able to find the CRISPR repeat in the

Replication-dependent histones (RDH) are required for packaging of newly synthetized DNA into nucleosomes during the S phase when their expression is highly upregulated. However, the mechanisms of this upregulation in metazoan cells remain poorly understood. Using iCLIP and ChIP-seq, we found that human cyclin-dependent kinase 11 (CDK11) associates with RNA and chromatin of RDH genes primarily in the

Lipoteichoic acids (LTAs) are essential cell-wall components in Gram-positive bacteria, including the human pathogen Staphylococcus aureus, contributing to cell adhesion, cell division and antibiotic resistance. Genetic evidence has suggested that LtaA is the flippase that mediates the translocation of the lipid-linked disaccharide that anchors LTA to the cell membrane, a rate-limiting step in S. aureus

Amyloid appearance is a rare event that is promoted in the presence of other aggregated proteins. These aggregates were thought to act by templating the formation of an assembly-competent nucleation seed, but we find an unanticipated role for them in enhancing the persistence of amyloid after it arises. Specifically, Saccharomyces cerevisiae Rnq1 amyloid reduces chaperone-mediated disassembly of Sup35

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The coordination of DNA unwinding and synthesis at replication forks promotes efficient and faithful replication of chromosomal DNA. Disruption of the balance between helicase and polymerase activities during replication stress leads to fork progression defects and activation of the Rad53 checkpoint kinase, which is essential for the functional maintenance of stalled replication forks. The mechanism

Leading-strand template aberrations cause helicase-polymerase uncoupling and impede replication fork progression, but the details of how uncoupled forks are restarted remain uncertain. Using purified proteins from Saccharomyces cerevisiae, we have reconstituted translesion synthesis (TLS)-mediated restart of a eukaryotic replisome following collision with a cyclobutane pyrimidine dimer. We find that

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