Cryo-electron microscopy of the liposome-embedded type 1 ryanodine receptor
Ontology highlight
ABSTRACT:
SUBMITTER: Andrew R Marks
PROVIDER: EMPIAR-10793 | biostudies-other |
REPOSITORIES: biostudies-other
Similar Datasets
Project description: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.
| S-EPMC3510009 | biostudies-literature
Project description:The strychnine-sensitive glycine receptor (GlyR) mediates inhibitory synaptic transmission in the spinal cord and brainstem and is linked to neurological disorders, including autism and hyperekplexia. Understanding of molecular mechanisms and pharmacology of glycine receptors has been hindered by a lack of high-resolution structures. Here we report electron cryo-microscopy structures of the zebrafish α1 GlyR with strychnine, glycine, or glycine and ivermectin (glycine/ivermectin). Strychnine arrests the receptor in an antagonist-bound closed ion channel state, glycine stabilizes the receptor in an agonist-bound open channel state, and the glycine/ivermectin complex adopts a potentially desensitized or partially open state. Relative to the glycine-bound state, strychnine expands the agonist-binding pocket via outward movement of the C loop, promotes rearrangement of the extracellular and transmembrane domain 'wrist' interface, and leads to rotation of the transmembrane domain towards the pore axis, occluding the ion conduction pathway. These structures illuminate the GlyR mechanism and define a rubric to interpret structures of Cys-loop receptors.
| S-EPMC4659708 | biostudies-literature
Project description:To study non-enveloped virus cell entry, a versatile in vitro model system was developed in which liposomes containing nickel-chelating lipids were decorated with His-tagged poliovirus receptors and bound to virus. This system provides an exciting opportunity for structural characterization of the early steps in cell entry in the context of a membrane. Here we report the three-dimensional structure of a poliovirus-receptor-membrane complex solved by cryo-electron microscopy (cryo-EM) at a resolution of 32 A. Methods were developed to establish the symmetry of the complex objectively. This reconstruction demonstrates that receptor binding brings a viral five-fold axis close to the membrane. Density is clearly defined for the icosahedral virus, for receptors (including known glycosylation sites) and for the membrane bilayer. Apparent perturbations of the bilayer close to the viral five-fold axis may function in subsequent steps of cell entry.
| S-EPMC1500892 | biostudies-literature
Project description:AMPA-subtype ionotropic glutamate receptors (AMPARs) mediate fast excitatory neurotransmission and contribute to high cognitive processes such as learning and memory. In the brain, AMPAR trafficking, gating, and pharmacology is tightly controlled by transmembrane AMPAR regulatory proteins (TARPs). Here, we used cryo-electron microscopy to elucidate the structural basis of AMPAR regulation by one of these auxiliary proteins, TARP ?2, or stargazin (STZ). Our structures illuminate the variable interaction stoichiometry of the AMPAR-TARP complex, with one or two TARP molecules binding one tetrameric AMPAR. Analysis of the AMPAR-STZ binding interfaces suggests that electrostatic interactions between the extracellular domains of AMPAR and STZ play an important role in modulating AMPAR function through contact surfaces that are conserved across AMPARs and TARPs. We propose a model explaining how TARPs stabilize the activated state of AMPARs and how the interactions between AMPARs and their auxiliary proteins control fast excitatory synaptic transmission.
| S-EPMC5125255 | biostudies-literature
Project description:Glycine receptors (GlyRs) are pentameric ion channels that mediate fast inhibitory neurotransmission. GlyRs are found in the central nervous system including the spinal cord, brain stem, and cerebellum, as well as in the retina, sperm, macrophages, hippocampus, cochlea, and liver. Due to their crucial roles in counter-balancing excitatory signals and pain signal transmission, GlyR dysfunction can lead to severe diseases, and as a result, compounds that modify GlyR activity may have tremendous therapeutic potential. Despite this potential, the development of GlyR-specific small-molecule ligands is lacking. Over the past few years, high-resolution structures of both homomeric and heteromeric GlyRs structures in various conformations have provided unprecedented details defining the pharmacology of ligand binding, subunit composition, and mechanisms of channel gating. These high-quality structures will undoubtedly help with the development of GlyR-targeted therapies.
| S-EPMC9395720 | biostudies-literature
Project description:Membrane proteins (MPs) used to be the most difficult targets for structural biology when X-ray crystallography was the mainstream approach. With the resolution revolution of single-particle electron cryo-microscopy (cryo-EM), rapid progress has been made for structural elucidation of isolated MPs. The next challenge is to preserve the electrochemical gradients and membrane curvature for a comprehensive structural elucidation of MPs that rely on these chemical and physical properties for their biological functions. Toward this goal, here we present a convenient workflow for cryo-EM structural analysis of MPs embedded in liposomes, using the well-characterized AcrB as a prototype. Combining optimized proteoliposome isolation, cryo-sample preparation on graphene grids, and an efficient particle selection strategy, the three-dimensional (3D) reconstruction of AcrB embedded in liposomes was obtained at 3.9 Å resolution. The conformation of the homotrimeric AcrB remains the same when the surrounding membranes display different curvatures. Our approach, which can be widely applied to cryo-EM analysis of MPs with distinctive soluble domains, lays out the foundation for cryo-EM analysis of integral or peripheral MPs whose functions are affected by transmembrane electrochemical gradients or/and membrane curvatures.
| S-EPMC7414195 | biostudies-literature
Project description:The basic unit of chromatin, the nucleosome core particle (NCP), controls how DNA in eukaryotic cells is compacted, replicated and read. Since its discovery, biochemists have sought to understand how this protein-DNA complex can help to control so many diverse tasks. Recent electron-microscopy (EM) studies on NCP-containing assemblies have helped to describe important chromatin transactions at a molecular level. With the implementation of recent technical advances in single-particle EM, our understanding of how nucleosomes are recognized and read looks to take a leap forward. In this review, the authors highlight recent advances in the architectural understanding of chromatin biology elucidated by EM.
| S-EPMC5458496 | biostudies-literature
Project description:Image restoration techniques are used to obtain, given experimental measurements, the best possible approximation of the original object within the limits imposed by instrumental conditions and noise level in the data. In molecular electron microscopy (EM), we are mainly interested in linear methods that preserve the respective relationships between mass densities within the restored map. Here, we describe the methodology of image restoration in structural EM, and more specifically, we will focus on the problem of the optimum recovery of Fourier amplitudes given electron microscope data collected under various defocus settings. We discuss in detail two classes of commonly used linear methods, the first of which consists of methods based on pseudoinverse restoration, and which is further subdivided into mean-square error, chi-square error, and constrained based restorations, where the methods in the latter two subclasses explicitly incorporates non-white distribution of noise in the data. The second class of methods is based on the Wiener filtration approach. We show that the Wiener filter-based methodology can be used to obtain a solution to the problem of amplitude correction (or "sharpening") of the EM map that makes it visually comparable to maps determined by X-ray crystallography, and thus amenable to comparative interpretation. Finally, we present a semiheuristic Wiener filter-based solution to the problem of image restoration given sets of heterogeneous solutions. We conclude the chapter with a discussion of image restoration protocols implemented in commonly used single particle software packages.
| S-EPMC3166661 | biostudies-literature
Project description:Structure-based drug design (SBDD) has gained popularity owing to its ability to develop more potent drugs compared to conventional drug-discovery methods. The success of SBDD relies heavily on obtaining the three-dimensional structures of drug targets. X-ray crystallography is the primary method used for solving structures and aiding the SBDD workflow; however, it is not suitable for all targets. With the resolution revolution, enabling routine high-resolution reconstruction of structures, cryogenic electron microscopy (cryo-EM) has emerged as a promising alternative and has attracted increasing attention in SBDD. Cryo-EM offers various advantages over X-ray crystallography and can potentially replace X-ray crystallography in SBDD. To fully utilize cryo-EM in drug discovery, understanding the strengths and weaknesses of this technique and noting the key advancements in the field are crucial. This review provides an overview of the general workflow of cryo-EM in SBDD and highlights technical innovations that enable its application in drug design. Furthermore, the most recent achievements in the cryo-EM methodology for drug discovery are discussed, demonstrating the potential of this technique for advancing drug development. By understanding the capabilities and advancements of cryo-EM, researchers can leverage the benefits of designing more effective drugs. This review concludes with a discussion of the future perspectives of cryo-EM-based SBDD, emphasizing the role of this technique in driving innovations in drug discovery and development. The integration of cryo-EM into the drug design process holds great promise for accelerating the discovery of new and improved therapeutic agents to combat various diseases.
| S-EPMC10945328 | biostudies-literature
Project description:Signal transduction in bacterial chemotaxis is initiated by the binding of extracellular ligands to a specialized family of methyl-accepting chemoreceptor proteins. Chemoreceptors cluster at distinct regions of the cell and form stable ternary complexes with the histidine autokinase CheA and the adapter protein CheW. Here we report the direct visualization and spatial organization of chemoreceptor arrays in intact Escherichia coli cells by using cryo-electron tomography and biochemical techniques. In wild-type cells, ternary complexes are arranged as an extended lattice, which may or may not be ordered, with significant variations in the size and specific location among cells in the same population. In the absence of CheA and CheW, chemoreceptors do not form observable clusters and are diffusely localized to the cell pole. At disproportionately high receptor levels, membrane invaginations containing nonfunctional, axially interacting receptor assemblies are formed. However, functional chemoreceptor arrays can be reestablished by increasing cellular levels of CheA and CheW. Our results demonstrate that chemotaxis in E. coli requires the presence of chemoreceptor arrays and that the formation of these arrays requires the scaffolding interactions of the signaling molecules CheA and CheW.
| S-EPMC1820660 | biostudies-literature