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The organization of genomic DNA into nucleosomes profoundly affects all DNA-related processes in eukaryotes. The histone chaperone known as ‘facilitates chromatin transcription’ (FACT 1 ) (consisting of subunits SPT16 and SSRP1) promotes both disassembly and reassembly of nucleosomes during gene transcription, DNA replication and DNA repair 2 . However, the mechanism by which FACT causes these opposing outcomes is unknown. Here we report two cryo-electron-microscopic structures of human FACT in complex with partially assembled subnucleosomes, with supporting biochemical and hydrogen–deuterium exchange data. We find that FACT is engaged in extensive interactions with nucleosomal DNA and all histone variants. The large DNA-binding surface on FACT appears to be protected by the carboxy-terminal domains of both of its subunits, and this inhibition is released by interaction with H2A–H2B, allowing FACT–H2A–H2B to dock onto a complex containing DNA and histones H3 and H4 (ref. 3 ). SPT16 binds nucleosomal DNA and tethers H2A–H2B through its carboxy-terminal domain by acting as a placeholder for DNA. SSRP1 also contributes to DNA binding, and can assume two conformations, depending on whether a second H2A–H2B dimer is present. Our data suggest a compelling mechanism for how FACT maintains chromatin integrity during polymerase passage, by facilitating removal of the H2A–H2B dimer, stabilizing intermediate subnucleosomal states and promoting nucleosome reassembly. Our findings reconcile discrepancies regarding the many roles of FACT and underscore the dynamic interactions between histone chaperones and nucleosomes. Two cryo-electron-microscopy images of the histone chaperone FACT interacting with components of nucleosomes shed light on how FACT manipulates nucleosomes to promote transcription, DNA repair and DNA replication.

Cryo-electron microscopy structures of insulin in a complex with the insulin receptor define the S2 binding site on the receptor and suggest a mechanism for downstream propagation of insulin signalling. Structure of insulin bound to its receptor Insulin is a peptide hormone with important roles in metabolism. Recent crystal structures of insulin bound to a truncated form of the insulin receptor have provided insights into the binding mode of this complex. Giovanna Scapin and colleagues now describe the three-dimensional structure of the ectodomain of the insulin receptor in complex with an insulin dimer, solved to a resolution of 4.6 Å using single-particle cryo-electron microscopy. The findings advance our structural and mechanistic knowledge about the formation of the insulin–receptor complex and receptor activation. The insulin receptor is a dimeric protein that has a crucial role in controlling glucose homeostasis, regulating lipid, protein and carbohydrate metabolism, and modulating brain neurotransmitter levels 1 , 2 . Insulin receptor dysfunction has been associated with many diseases, including diabetes, cancer and Alzheimer’s disease 1 , 3 , 4 . The primary sequence of the receptor has been known since the 1980s 5 , and is composed of an extracellular portion (the ectodomain, ECD), a single transmembrane helix and an intracellular tyrosine kinase domain. Binding of insulin to the dimeric ECD triggers auto-phosphorylation of the tyrosine kinase domain and subsequent activation of downstream signalling molecules. Biochemical and mutagenesis data have identified two putative insulin-binding sites, S1 and S2 6 . The structures of insulin bound to an ECD fragment containing S1 and of the apo ectodomain have previously been reported 7 , 8 , but details of insulin binding to the full receptor and the signal propagation mechanism are still not understood. Here we report single-particle cryo-electron microscopy reconstructions of the 1:2 (4.3 Å) and 1:1 (7.4 Å) complexes of the insulin receptor ECD dimer with insulin. The symmetrical 4.3 Å structure shows two insulin molecules per dimer, each bound between the leucine-rich subdomain L1 of one monomer and the first fibronectin-like domain (FnIII-1) of the other monomer, and making extensive interactions with the α-subunit C-terminal helix (α-CT helix). The 7.4 Å structure has only one similarly bound insulin per receptor dimer. The structures confirm the binding interactions at S1 and define the full S2 binding site. These insulin receptor states suggest that recruitment of the α-CT helix upon binding of the first insulin changes the relative subdomain orientations and triggers downstream signal propagation.

Cryo-FIB/SEM combined with cryo-ET has emerged from within the field of cryo-EM as the method for obtaining the highest resolution structural information of complex biological samples in-situ in native and non-native environments. However, challenges remain in conventional cryo-FIB/SEM workflows, including milling thick specimens with vitrification issues, specimens with preferred orientation, low-throughput when milling small and/or low concentration specimens, and specimens that distribute poorly across grid squares. Here we present a general approach called the ‘Waffle Method’ which leverages high-pressure freezing to address these challenges. We illustrate the mitigation of these challenges by applying the Waffle Method and cryo-ET to reveal the macrostructure of the polar tube in microsporidian spores in multiple complementary orientations, which was previously not possible due to preferred orientation. We demonstrate the broadness of the Waffle Method by applying it to three additional cellular samples and a single particle sample using a variety of cryo-FIB-milling hardware, with manual and automated approaches. We also present a unique and critical stress-relief gap designed specifically for waffled lamellae. We propose the Waffle Method as a way to achieve many advantages of cryo-liftout on the specimen grid while avoiding the long, challenging, and technically-demanding process required for cryo-liftout. Here the authors describe the Waffle Method, aimed at increasing the throughput of and solves several challenges present in cryo-FIB/SEM sample preparation for cryo-ET analysis — the highest-resolution method for obtaining 3D views of native biological specimens in-situ.

Most protein particles prepared in vitreous ice for single-particle cryo-electron microscopy (cryo-EM) are adsorbed to air–water or substrate–water interfaces, which can cause the particles to adopt preferred orientations. By using a rapid plunge-freezing robot and nanowire grids, we were able to reduce some of the deleterious effects of the air–water interface by decreasing the dwell time of particles in thin liquid films. We demonstrated this by using single-particle cryo-EM and cryo-electron tomography (cryo-ET) to examine hemagglutinin, insulin receptor complex, and apoferritin.

The HIV-1 envelope glycoprotein (Env) trimer contains the receptor binding sites and membrane fusion machinery that introduce the viral genome into the host cell. As the only target for broadly neutralizing antibodies (bnAbs), Env is a focus for rational vaccine design. We present a cryo-electron microscopy reconstruction and structural model of a cleaved, soluble Env trimer (termed BG505 SOSIP. 664 gp140) in complex with a CD4 binding site (CD4bs) bnAb, PGV04, at 5.8 angstrom resolution. The structure reveals the spatial arrangement of Env components, including the V1/V2, V3, HR1, and HR2 domains, as well as shielding glycans. The structure also provides insights into trimer assembly, gp120-gp41 interactions, and the CD4bs epitope cluster for bnAbs, which covers a more extensive area and defines a more complex site of vulnerability than previously described.

Influenza virus ribonucleoprotein complexes (RNPs) are central to the viral life cycle and in adaptation to new host species. RNPs are composed of the viral genome, viral polymerase, and many copies of the viral nucleoprotein. In vitro cell expression of all RNP protein components with four of the eight influenza virus gene segments enabled structural determination of native influenza virus RNPs by means of cryogenic electron microscopy (cryo-EM). The cryo-EM structure reveals the architecture and organization of the native RNP, defining the attributes of its largely helical structure and how polymerase interacts with nucleoprotein and the viral genome. Observations of branched-RNP structures in negative-stain electron microscopy and their putative identification as replication intermediates suggest a mechanism for viral replication by a second polymerase on the RNP template.

Neurite self-recognition and avoidance are fundamental properties of all nervous systems 1 . These processes facilitate dendritic arborization 2 , 3 , prevent formation of autapses 4 and allow free interaction among non-self neurons 1 , 2 , 4 , 5 . Avoidance among self neurites is mediated by stochastic cell-surface expression of combinations of about 60 isoforms of α-, β- and γ-clustered protocadherin that provide mammalian neurons with single-cell identities 1 , 2 , 4 – 13 . Avoidance is observed between neurons that express identical protocadherin repertoires 2 , 5 , and single-isoform differences are sufficient to prevent self-recognition 10 . Protocadherins form isoform-promiscuous cis dimers and isoform-specific homophilic trans dimers 10 , 14 – 20 . Although these interactions have previously been characterized in isolation 15 , 17 – 20 , structures of full-length protocadherin ectodomains have not been determined, and how these two interfaces engage in self-recognition between neuronal surfaces remains unknown. Here we determine the molecular arrangement of full-length clustered protocadherin ectodomains in single-isoform self-recognition complexes, using X-ray crystallography and cryo-electron tomography. We determine the crystal structure of the clustered protocadherin γB4 ectodomain, which reveals a zipper-like lattice that is formed by alternating cis and trans interactions. Using cryo-electron tomography, we show that clustered protocadherin γB6 ectodomains tethered to liposomes spontaneously assemble into linear arrays at membrane contact sites, in a configuration that is consistent with the assembly observed in the crystal structure. These linear assemblies pack against each other as parallel arrays to form larger two-dimensional structures between membranes. Our results suggest that the formation of ordered linear assemblies by clustered protocadherins represents the initial self-recognition step in neuronal avoidance, and thus provide support for the isoform-mismatch chain-termination model of protocadherin-mediated self-recognition, which depends on these linear chains 11 . Clustered protocadherin ectodomains spontaneously assemble to form a zipper-like lattice of alternating cis and trans interactions at membrane contact sites, which probably represents their mode of function in neuronal self-recognition.

Single particle cryo-electron microscopy (cryoEM) is often performed under the assumption that particles are not adsorbed to the air-water interfaces and in thin, vitreous ice. In this study, we performed fiducial-less tomography on over 50 different cryoEM grid/sample preparations to determine the particle distribution within the ice and the overall geometry of the ice in grid holes. Surprisingly, by studying particles in holes in 3D from over 1000 tomograms, we have determined that the vast majority of particles (approximately 90%) are adsorbed to an air-water interface. The implications of this observation are wide-ranging, with potential ramifications regarding protein denaturation, conformational change, and preferred orientation. We also show that fiducial-less cryo-electron tomography on single particle grids may be used to determine ice thickness, optimal single particle collection areas and strategies, particle heterogeneity, and de novo models for template picking and single particle alignment.

NOSs are homodimeric multidomain enzymes responsible for producing NO. In mammals, NO acts as an intercellular messenger in a variety of signaling reactions, as well as a cytotoxin in the innate immune response. Mammals possess three NOS isoforms— inducible, endothelial, and neuronal NOS—that are composed of an N-terminal oxidase domain and a C-terminal reductase domain. Calmodulin (CaM) activates NO synthesis by binding to the helical region connecting these two domains. Although crystal structures of isolated domains have been reported, no structure is available for full-length NOS. We used high-throughput single-particle EM to obtain the structures and higher-order domain organization of all three NOS holoenzymes. The structures of inducible, endothelial, and neuronal NOS with and without CaM bound are similar, consisting of a dimerized oxidase domain flanked by two separated reductase domains. NOS isoforms adopt many conformations enabled by three flexible linkers. These conformations represent snapshots of the continuous electron transfer pathway from the reductase domain to the oxidase domain, which reveal that only a single reductase domain participates in electron transfer at a time, and that CaM activates NOS by constraining rotational motions and by directly binding to the oxidase domain. Direct visualization of these large conformational changes induced during electron transfer provides significant insight into the molecular underpinnings governing NO formation.

Recent developments in detector hardware and image-processing software have revolutionized single particle cryo-electron microscopy (cryoEM) and led to a wave of near-atomic resolution (typically ∼3.3 Å) reconstructions. Reaching resolutions higher than 3 Å is a prerequisite for structure-based drug design and for cryoEM to become widely interesting to pharmaceutical industries. We report here the structure of the 700 kDa Thermoplasma acidophilum 20S proteasome (T20S), determined at 2.8 Å resolution by single-particle cryoEM. The quality of the reconstruction enables identifying the rotameric conformation adopted by some amino-acid side chains (rotamers) and resolving ordered water molecules, in agreement with the expectations for crystal structures at similar resolutions. The results described in this manuscript demonstrate that single particle cryoEM is capable of competing with X-ray crystallography for determination of protein structures of suitable quality for rational drug design. Proteins perform many critical tasks within cells, and to do so, they must first fold into specific shapes. Being able to visualize these shapes can help scientists to understand how proteins work, and help them create drugs that can interact with the proteins to treat diseases. The past few years have seen the rapid development of an imaging technique called single-particle cryo-electron microscopy (or cryoEM for short), and this technique is now increasingly used to investigate protein structures. First, proteins are embedded in a thin film of non-crystalline ice by rapidly cooling to around the temperature of liquid nitrogen (below −180°C). This traps the protein in the shape it has in solution. High-energy electrons are then transmitted through the protein sample and their interaction with the atoms in the protein is recorded by a direct electron camera. The analysis of a large series of images recorded in this way can be used to determine the approximate positions of the atoms in the protein. Previously, single-particle cryoEM techniques have not produced a detailed enough protein structure to be useful to scientists interested in drug development. By refining these techniques, Campbell, Veesler et al. have now obtained the most detailed cryoEM protein structure to date—a structure of an enzyme complex that helps get rid of proteins that are misfolded or that have become too abundant. The structure is so detailed that it reveals the shapes of some small groups of atoms that stick out from the sides of amino acids in the enzyme complex. (Amino acids are the building blocks of enzymes and all other proteins.) Moreover, the structure shows where individual water molecules are positioned around the protein. The level of detail in the structure produced by Campbell, Veesler et al. is high enough to be useful to drug researchers. Furthermore, because only 10% of the images Campbell, Veesler et al. collected were used to produce the structure, future work will investigate whether incorporating more of the images could reveal structures in even greater detail.