We present a perspective of our view of the application of cryoelectron microscopy (cryo-EM) to structure-based drug design (SBDD). We discuss the basic needs and requirements for SBDD, the current ...state of cryo-EM, and the challenges that need to be overcome for this technique to reach its full potential in facilitating the process of drug discovery.
The ribosome is a complex macromolecular machine and serves as an ideal system for understanding biological macromolecular assembly. Direct observation of ribosome assembly in vivo is difficult, as ...few intermediates have been isolated and thoroughly characterized. Herein, we deploy a genetic system to starve cells of an essential ribosomal protein, which results in the accumulation of assembly intermediates that are competent for maturation. Quantitative mass spectrometry and single-particle cryo-electron microscopy reveal 13 distinct intermediates, which were each resolved to ∼4–5 Å resolution and could be placed in an assembly pathway. We find that ribosome biogenesis is a parallel process, that blocks of structured rRNA and proteins assemble cooperatively, and that the entire process is dynamic and can be “re-routed” through different pathways as needed. This work reveals the complex landscape of ribosome assembly in vivo and provides the requisite tools to characterize additional assembly pathways for ribosomes and other macromolecular machines.
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•Single-particle cryo-EM and quantitative mass spectrometry of assembling ribosomes•Ribosome assembly occurs through the coordinated folding of RNA and protein “blocks”•The order of ribosomal protein association is highly flexible and can be “re-routed”•Limitation of a single ribosomal protein has long-range structural effects
Assembly pathways for large macromolecular complexes can be determined using a combination of quantitative mass spectrometry and cryo-EM.
The organization of genomic DNA into nucleosomes profoundly affects all DNA-related processes in eukaryotes. The histone chaperone known as 'facilitates chromatin transcription' (FACT
) (consisting ...of subunits SPT16 and SSRP1) promotes both disassembly and reassembly of nucleosomes during gene transcription, DNA replication and DNA repair
. 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.
). 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.
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. Insulin receptor dysfunction has been associated with many diseases, including diabetes, cancer and Alzheimer's disease. The primary sequence of the receptor has been known since the 1980s, 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. The structures of insulin bound to an ECD fragment containing S1 and of the apo ectodomain have previously been reported, 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.
Lymphangioleiomyomatosis (LAM) is a metastatic neoplasm of reproductive-age women associated with mutations in tuberous sclerosis complex genes. LAM causes cystic remodeling of the lung and ...progressive respiratory failure. The sources and cellular characteristics of LAM cells underlying disease pathogenesis remain elusive.
Identification and characterization of LAM cells in human lung and uterus using a single-cell approach.
Single-cell and single-nuclei RNA sequencing on LAM (
= 4) and control (
= 7) lungs, immunofluorescence confocal microscopy, ELISA, and aptamer proteomics were used to identify and validate LAM
cells and secreted biomarkers, predict cellular origins, and define molecular and cellular networks in LAM.
A unique cell type termed LAM
was identified, which was distinct from, but closely related to, lung mesenchymal cells. LAM
cells expressing signature genes included known LAM markers such as
,
,
, and
and novel biomarkers validated by aptamer screening, ELISA, and immunofluorescence microscopy. LAM cells in lung and uterus are morphologically indistinguishable and share similar gene expression profiles and biallelic
mutations, supporting a potential uterine origin for the LAM
cell. Effects of LAM on resident pulmonary cell types indicated recruitment and activation of lymphatic endothelial cells.
A unique population of LAM
cells was identified in lung and uterus of patients with LAM, sharing close transcriptomic identity. LAM cell selective markers, secreted biomarkers, and the predicted cellular molecular features provide new insights into the signaling and transcriptional programs that may serve as diagnostic markers and therapeutic targets to influence the pathogenesis of LAM.
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 podocyte is a remarkable cell type, which encases the capillaries of the kidney glomerulus. Although mesodermal in origin it sends out axonal like projections that wrap around the capillaries. ...These extend yet finer projections, the foot processes, which interdigitate, leaving between them the slit diaphragms, through which the glomerular filtrate must pass. The podocytes are a subject of keen interest because of their key roles in kidney development and disease.
In this report we identified and characterized a novel transgenic mouse line, MafB-GFP, which specifically marked the kidney podocytes from a very early stage of development. These mice were then used to facilitate the fluorescent activated cell sorting based purification of podocytes from embryos at E13.5 and E15.5, as well as adults. Microarrays were then used to globally define the gene expression states of podocytes at these different developmental stages. A remarkable picture emerged, identifying the multiple sets of genes that establish the neuronal, muscle, and phagocytic properties of podocytes. The complete combinatorial code of transcription factors that create the podocyte was characterized, and the global lists of growth factors and receptors they express were defined.
The complete molecular character of the in vivo podocyte is established for the first time. The active molecular functions and biological processes further define their unique combination of features. The results provide a resource atlas of gene expression patterns of developing and adult podocytes that will help to guide further research of these incredible cells.
Following the discovery of the gravitational-wave source GW170817 by three Laser Interferometer Gravitational-wave Observatory (LIGO)/Virgo antennae (Abbott et al., 2017a), the MASTER Global Robotic ...Net telescopes obtained the first image of the NGC 4993 host galaxy. An optical transient, MASTER OTJ130948.10-232253.3/SSS17a was later found, which appears to be a kilonova resulting from the merger of two neutron stars (NSs). Here we describe this independent detection and photometry of the kilonova made in white light, and in B, V, and R filters. We note that the luminosity of this kilonova in NGC 4993 is very close to those measured for other kilonovae possibly associated with gamma-ray burst (GRB) 130603 and GRB 080503.
Low-dose images obtained by electron cryo-microscopy (cryo-EM) are often affected by blurring caused by sample motion during electron beam exposure, degrading signal especially at high resolution. We ...show here that we can align frames of movies, recorded with a direct electron detector during beam exposure of rotavirus double-layered particles, thereby greatly reducing image blurring caused by beam-induced motion and sample stage instabilities. This procedure increases the efficiency of cryo-EM imaging and enhances the resolution obtained in three-dimensional reconstructions of the particle. Using movies in this way is generally applicable to all cryo-EM samples and should also improve the performance of midrange electron microscopes that may have limited mechanical stability and beam coherence.
► Cryo-EM images are blurred by sample motion during electron beam exposure ► The sample motion degrades signal especially at high resolution ► Aligning frames of movies recorded with a direct electron detector reduces blurring ► This procedure enhances the resolution obtained in 3D reconstructions of the sample
In cryo-EM, low-dose images are often affected by blurring caused by sample motion during beam exposure. Melody et al. show that aligning frames of movies recorded with a direct electron detector during beam exposure greatly reduces blurring, increases the efficiency of cryo-EM imaging, and enhances resolution.
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