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The design of atomic-scale microstructural traps to limit the diffusion of hydrogen is one key strategy in the development of hydrogen-embrittlement-resistant materials. In the case of bearing steels, an effective trapping mechanism may be the incorporation of finely dispersed V-Mo-Nb carbides in a ferrite matrix. First, we charged a ferritic steel with deuterium by means of electrolytic loading to achieve a high hydrogen concentration. We then immobilized it in the microstructure with a cryogenic transfer protocol before atom probe tomography (APT) analysis. Using APT, we show trapping of hydrogen within the core of these carbides with quantitative composition profiles. Furthermore, with this method the experiment can be feasibly replicated in any APT-equipped laboratory by using a simple cold chain.

A cap-specific m6A writerN6,2′-O-dimethyladenosine (m6Am) is present at the transcription start nucleotide of capped mRNAs in vertebrates. Akichika et al. quantified the abundance of this modification in the transcriptome and identified the writer protein, cap-specific adenosine methyltransferase (CAPAM), needed for this modification. CAPAM contains a unique structure that recognizes cap-specific N6-methyladenosine (m6A) as the substrate. The protein interacts with RNA polymerase II, suggesting that the modification occurs cotranscriptionally. The m6Am promotes the translation of capped mRNAs in a eIF4E-independent fashion.Science, this issue p. eaav0080INTRODUCTIONN6-methyladenosine (m6A), an abundant modification in eukaryotic mRNAs and long-noncoding RNAs, has been recognized as a major epitranscriptome mark that plays critical roles in RNA metabolism and function. In addition to the internal m6A, N6, 2′-O-dimethyladenosine (m6Am) is present at the transcription start site of capped mRNAs in vertebrates. Previous studies reported that an eraser protein, FTO, demethylates N6-methyl group of m6Am and destabilizes a subset of mRNAs, suggesting a possible function of m6Am in stabilizing A-starting capped mRNAs. However, the biogenesis and functional role of m6Am remain elusive.RATIONALETo reveal the functional and physiological roles of m6Am, it is necessary to identify a writer protein for N6-methylation of m6Am. We first established a highly sensitive method to analyze 5′-terminal chemical structures of the capped mRNAs using mass spectrometry (RNA-MS), and then measured m6Am methylation status accurately. We employed RNA-MS to identify the writer gene by a reverse genetic approach. We chose several candidates of uncharacterized methyltransferases (MTases) that are conserved in vertebrates, but not in yeast, which does not have m6Am. Each of the candidates was knocked out in human cells. If the target gene is disrupted, RNA-MS can detect the absence of m6Am in mRNAs prepared from the knockout cells.RESULTSRNA-MS analysis revealed that m6Am modification in human mRNAs is more abundant (92%) than previously estimated. We identified human PCIF1 as cap-specific adenosine-N6-MTase (CAPAM) responsible for N6-methylation of m6Am. Indeed, m6Am disappeared completely and converted to Am modification in mRNAs prepared from the CAPAM knockout (KO) cells. The CAPAM KO cells were viable, but sensitive to oxidative stress, implying the physiological importance of m6Am. We showed that CAPAM catalyzes N6-methylation of m6Am in the capped mRNAs in an S-adenosylmethionine (SAM)–dependent manner. A series of biochemical studies revealed that CAPAM specifically recognizes the 7-methylguanosine (m7G) cap structure and preferentially N6-methylates m7GpppAm rather than m7GpppA, indicating the importance of the 2′-O-methyl group of the target site for efficient m6Am formation. CAPAM has a N-terminal WW domain that specifically interacts with the Ser5-phosphorylated C-terminal domain (CTD) of RNA polymerase II (RNAPII), suggesting that the CAPAM is recruited to the early elongation complex of RNAPII and introduces m6Am in a nascent mRNA chain cotranscriptionally. We also solved the crystal structure of CAPAM complexed with the cap analog and SAM analog. The core region of CAPAM is composed of MTase and helical domains. The m7G cap is bound to a pocket formed by these two domains. The SAM analog is recognized by an active site with the characteristic NPPF motif in the MTase domain. This structure reveals the molecular basis of cap-specific m6A formation. RNA-sequencing analysis of the CAPAM KO cells revealed that loss of m6Am does not affect transcriptome alteration. This result does not support the proposed function of m6Am in stabilizing A-starting capped mRNAs. Instead, ribosome profiling of the CAPAM KO cells showed that N6-methylation of m6Am promotes the translation of capped mRNAs.CONCLUSIONWe identified PCIF1/CAPAM as a cap-specific m6A writer for vertebrate mRNAs. Structural analysis revealed the molecular basis of cap-specific m6A formation by CAPAM. The ribosome profiling experiment revealed that CAPAM-mediated m6Am formation promotes translation of A-starting mRNAs, rather than stabilization of mRNAs.N6-methyladenosine (m6A), a major modification of messenger RNAs (mRNAs), plays critical roles in RNA metabolism and function. In addition to the internal m6A, N6, 2′-O-dimethyladenosine (m6Am) is present at the transcription start nucleotide of capped mRNAs in vertebrates. However, its biogenesis and functional role remain elusive. Using a reverse genetics approach, we identified PCIF1, a factor that interacts with the serine-5–phosphorylated carboxyl-terminal domain of RNA polymerase II, as a cap-specific adenosine methyltransferase (CAPAM) responsible for N6-methylation of m6Am. The crystal structure of CAPAM in complex with substrates revealed the molecular basis of cap-specific m6A formation. A transcriptome-wide analysis revealed that N6-methylation of m6Am promotes the translation of capped mRNAs. Thus, a cap-specific m6A writer promotes translation of mRNAs starting from m6Am.

Structures of voltage-gated sodium channelsIn “excitable” cells, like neurons and muscle cells, a difference in electrical potential is used to transmit signals across the cell membrane. This difference is regulated by opening or closing ion channels in the cell membrane. For example, mutations in human voltage-gated sodium (Nav) channels are associated with disorders such as chronic pain, epilepsy, and cardiac arrhythmia. Pan et al. report the high-resolution structure of a human Nav channel, and Shen et al. report the structures of an insect Nav channel bound to the toxins that cause pufferfish and shellfish poisoning in humans. Together, the structures give insight into the molecular basis of sodium ion permeation and provide a path toward structure-based drug discovery.Science, this issue p. eaau2486, p. eaau2596INTRODUCTIONThe nine subtypes of mammalian voltage-gated sodium (Nav) channels, Nav1.1 to Nav1.9, are responsible for the initiation and propagation of action potentials in specific excitable systems, among which Nav1.4 functions in skeletal muscle. Responding to membrane potential changes, Nav channels undergo sophisticated conformational shifts that lead to transitions between resting, activated, and inactivated states. Defects in Nav channels are associated with a variety of neurological, cardiovascular, muscular, and psychiatric disorders. In addition, Nav channels are targets for natural toxins and clinical therapeutics.Understanding the physiological and pathophysiological mechanisms of Nav channels requires knowing the structure of each conformational state. All eukaryotic Nav channels comprise a single polypeptide chain, the α subunit, that folds to four homologous repeats I to IV. Channel properties are modulated by one or two subtype-specific β subunits. Cryo–electron microscopy (cryo-EM) structures of two Nav channels, one from American cockroach and the other from electric eel, were resolved in two distinct conformations. However, the inability to record currents of either channel in heterologous systems prevented functional assignment of these structures. Structural elucidation of a functionally well-characterized Nav channel is required to establish a model for structure-function relationship studies.RATIONALEAfter extensive screening for expression systems, protein boundaries, chimeras, affinity tags, and combination with subtype-specific β subunits, we focused on human Nav1.4 in the presence of β1 subunit for cryo-EM analysis. The complex, which was transiently coexpressed in human embryonic kidney (HEK) 293F cells with BacMam viruses and purified through tandem affinity columns and size exclusion chromatography, was concentrated to ~0.5 mg/ml for cryo-EM sample preparation and data acquisition.RESULTSThe cryo-EM structure of human Nav1.4-β1 complex was determined to 3.2-Å resolution. The extracellular and transmembrane domains, including the complete pore domain, all four voltage-sensing domains (VSDs), and the β1 subunit, were clearly resolved, enabling accurate model building (see the figure).The well-resolved Asp/Glu/Lys/Ala (DEKA) residues, which are responsible for specific Na+ permeation through the selectivity filter, exhibit identical conformations to those seen in the other two Nav structures. A glyco-diosgenin (GDN) molecule, the primary detergent used for protein purification and cryo-EM sample preparation, penetrates the intracellular gate of the pore domain, holding it open to a diameter of ~5.6 Å. The central cavity of the pore domain is filled with lipid-like densities, which traverse the side wall fenestrations.Voltage sensing involves four to six Arg/Lys residues on helix S4 of the VSD. This helix moves “up” (away from the cytoplasm) in response to changes of the membrane potential, and this opens the channel finally. All four VSDs display up conformations. The movement of the gating charge residues is facilitated by coordination to acidic and polar residues on S1 to S3. The improved resolution allows detailed analysis of the coordination.The fast inactivation Ile/Phe/Met (IFM) motif on the short linker between repeats III and IV inserts into a hydrophobic cavity enclosed by the S6 and S4-S5 segments in repeats III and IV. Analysis of reported functional residues and disease mutations corroborates our recently proposed allosteric blocking mechanism for fast inactivation.CONCLUSIONThe structure provides important insight into the molecular basis for Na+ permeation, electromechanical coupling, asynchronous activation, and fast inactivation of the four repeats. It opens a new chapter for studying the structure-function relationships of Nav channels, affords an accurate template to map mutations associated with diseases such as myotonia and periodic paralysis hyperkalemic, and illuminates a path toward precise understanding and intervention with specific Nav channelopathies.Voltage-gated sodium (Nav) channels, which are responsible for action potential generation, are implicated in many human diseases. Despite decades of rigorous characterization, the lack of a structure of any human Nav channel has hampered mechanistic understanding. Here, we report the cryo–electron microscopy structure of the human Nav1.4-β1 complex at 3.2-Å resolution. Accurate model building was made for the pore domain, the voltage-sensing domains, and the β1 subunit, providing insight into the molecular basis for Na+ permeation and kinetic asymmetry of the four repeats. Structural analysis of reported functional residues and disease mutations corroborates an allosteric blocking mechanism for fast inactivation of Nav channels. The structure provides a path toward mechanistic investigation of Nav channels and drug discovery for Nav channelopathies.

When the DNA bases cytosine and guanine are next to each other, a methyl group is generally added to the pyrimidine, generating a mCpG dinucleotide. This modification alters DNA structure but can also affect function by inhibiting transcription factor (TF) binding. Yin et al. systematically analyzed the effect of CpG methylation on the binding of 542 human TFs (see the Perspective by Hughes and Lambert). In addition to inhibiting binding of some TFs, they found that mCpGs can promote binding of others, particularly TFs involved in development, such as homeodomain proteins. Science , this issue p. eaaj2239 ; see also p. 489 Genome-scale analysis reveals positive and negative binding of transcription factors to methylated CpG dinucleotides. The majority of CpG dinucleotides in the human genome are methylated at cytosine bases. However, active gene regulatory elements are generally hypomethylated relative to their flanking regions, and the binding of some transcription factors (TFs) is diminished by methylation of their target sequences. By analysis of 542 human TFs with methylation-sensitive SELEX (systematic evolution of ligands by exponential enrichment), we found that there are also many TFs that prefer CpG-methylated sequences. Most of these are in the extended homeodomain family. Structural analysis showed that homeodomain specificity for methylcytosine depends on direct hydrophobic interactions with the methylcytosine 5-methyl group. This study provides a systematic examination of the effect of an epigenetic DNA modification on human TF binding specificity and reveals that many developmentally important proteins display preference for mCpG-containing sequences.

Enzymatic conversion of molecular nitrogen to ammonia requires a dance of electrons and protons. The stage for that dance is the nitrogenase cofactor, a carefully constructed cluster of iron, sulfur, and carbon with homocitrate and, in some cases, bicarbonate appendages, as well as a secondary metal ion that defines the class of enzyme. The question of how this cofactor binds nitrogen has been vexingly difficult to answer. Sippel et al. report a high-resolution structure of the vanadium nitrogenase with a light atom, interpreted as nitrogen, bound to the FeV cofactor. A sulfur atom is displaced from the cofactor in this structure and is observed resting in a holding site formed by rearrangement of a glutamine residue. The putative bridging nitrogen atom suggests that diatomic nitrogen may bind to the cluster in a head-on manner, with the glutamine side chain stabilizing protonated intermediates as they are reduced. Science , this issue p. 1484 An atomic-resolution structure of a late-stage nitrogenase intermediate reveals nitrogen bound to iron in the active site. Reduction of N 2 by nitrogenases occurs at an organometallic iron cofactor that commonly also contains either molybdenum or vanadium. The well-characterized resting state of the cofactor does not bind substrate, so its mode of action remains enigmatic. Carbon monoxide was recently found to replace a bridging sulfide, but the mechanistic relevance was unclear. Here we report the structural analysis of vanadium nitrogenase with a bound intermediate, interpreted as a μ 2 -bridging, protonated nitrogen that implies the site and mode of substrate binding to the cofactor. Binding results in a flip of amino acid glutamine 176, which hydrogen-bonds the ligand and creates a holding position for the displaced sulfide. The intermediate likely represents state E 6 or E 7 of the Thorneley-Lowe model and provides clues to the remainder of the catalytic cycle.

Adaptive immunity had been long thought of as an exclusive feature of animals. However, the discovery of the CRISPR-Cas defense system, present in almost half of prokaryotic genomes, proves otherwise. Because of the everlasting parasite-host arms race, CRISPR-Cas has rapidly evolved through horizontal transfer of complete loci or individual modules, resulting in extreme structural and functional diversity. CRISPR-Cas systems are divided into two distinct classes that each consist of three types and multiple subtypes. We discuss recent advances in CRISPR-Cas research that reveal elaborate molecular mechanisms and provide for a plausible scenario of CRISPR-Cas evolution. We also briefly describe the latest developments of a wide range of CRISPR-based applications.

Induction of broadly neutralizing antibodies (bnAbs) is a major HIV vaccine goal. Germline-targeting immunogens aim to initiate bnAb induction by activating bnAb germline precursor B cells. Critical unmet challenges are to determine whether bnAb precursor naïve B cells bind germline-targeting immunogens and occur at sufficient frequency in humans for reliable vaccine responses. Using deep mutational scanning and multitarget optimization, we developed a germline-targeting immunogen (eOD-GT8) for diverse VRC01-class bnAbs. We then used the immunogen to isolate VRC01-class precursor naïve B cells from HIV-uninfected donors. Frequencies of true VRC01-class precursors, their structures, and their eOD-GT8 affinities support this immunogen as a candidate human vaccine prime. These methods could be applied to germline targeting for other classes of HIV bnAbs and for Abs to other pathogens.

The voltage-gated calcium channel Ca(v)1.1 is engaged in the excitation-contraction coupling of skeletal muscles. The Ca(v)1.1 complex consists of the pore-forming subunit α1 and auxiliary subunits α2δ, β, and γ. We report the structure of the rabbit Ca(v)1.1 complex determined by single-particle cryo-electron microscopy. The four homologous repeats of the α1 subunit are arranged clockwise in the extracellular view. The γ subunit, whose structure resembles claudins, interacts with the voltage-sensing domain of repeat IV (VSD(IV)), whereas the cytosolic β subunit is located adjacent to VSD(II) of α1. The α2 subunit interacts with the extracellular loops of repeats I to III through its VWA and Cache1 domains. The structure reveals the architecture of a prototypical eukaryotic Ca(v) channel and provides a framework for understanding the function and disease mechanisms of Ca(v) and Na(v) channels.

Nuclear pore complexes form a gateway between the cytoplasm and the nucleus (see the Perspective by Ullman and Powers). Stuwe et al. combined structural, biochemical, and functional analyses to elucidate the architecture of a six-protein complex that makes up the inner ring of the fungal nuclear pore. This includes a central trimeric complex homologous to the Nup62 complex found in metazoans that is incorporated into the nuclear pore inner-ring complex. Chug et al. report the structure of the metazoan trimeric Nup62 complex. Neither study supports a model in which the pore can dilate and constrict. Instead they suggest a rigid pore in which flexible domains called FG repeats fill the channel and form a barrier that can be traversed by receptors that carry cargos across. Science, this issue pp. 56 and 106; see also p. 33 Nuclear pore complexes (NPCs) conduct nucleocytoplasmic transport and gain transport selectivity through nucleoporin FG domains. Here, we report a structural analysis of the FG Nup62*58*54 complex, which is a crucial component of the transport system. It comprises a [approximate]13 nanometer-long trimerization interface with an unusual 2W3F coil, a canonical heterotrimeric coiled coil, and a kink that enforces a compact six-helix bundle. Nup54 also contains a ferredoxin-like domain. We further identified a heterotrimeric Nup93-binding module for NPC anchorage. The quaternary structure alternations in the Nup62 complex, which were previously proposed to trigger a general gating of the NPC, are incompatible with the trimer structure. We suggest that the highly elongated Nup62 complex projects barrier-forming FG repeats far into the central NPC channel, supporting a barrier that guards the entire cross section.

Motile cilia and flagella are hairlike cellular appendages that power the movement of individual cells or liquid across tissues, as exemplified by the cilia found in airways. The question of how they move in rhythmic oscillations has puzzled scientists for centuries. Lin and Nicastro used cryo–electron tomography (cryo-ET) to visualize the activity states of individual dynein motors with respect to their locations within beating flagella. They observed an asymmetric distribution of dynein activity and the switching of conformations of dyneins and their regulators between opposite sides of active flagella. The results confirm the switching aspect of the prevailing “switch-point” hypothesis but change the view with respect to how dynein activities are coordinated to drive flagellar motility. Science , this issue p. eaar1968 Ciliary motility is driven by the asymmetric distribution of dynein activity on alternating sides of the flagellum. Motile cilia and flagella are essential, highly conserved organelles, and their motility is driven by the coordinated activities of multiple dynein isoforms. The prevailing “switch-point” hypothesis posits that dyneins are asymmetrically activated to drive flagellar bending. To test this model, we applied cryo–electron tomography to visualize activity states of individual dyneins relative to their locations along beating flagella of sea urchin sperm cells. As predicted, bending was generated by the asymmetric distribution of dynein activity on opposite sides of the flagellum. However, contrary to predictions, most dyneins were in their active state, and the smaller population of conformationally inactive dyneins switched flagellar sides relative to the bending direction. Thus, our data suggest a “switch-inhibition” mechanism in which force imbalance is generated by inhibiting, rather than activating, dyneins on alternating sides of the flagellum.