Cryo-electron microscopy analysis of small membrane proteins.
Ontology highlight
ABSTRACT:
SUBMITTER: Nygaard R
PROVIDER: S-EPMC7665978 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
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:The bacteriophage T4 capsid contains two accessory surface proteins, the small outer capsid protein (Soc, 870 copies) and the highly antigenic outer capsid protein (Hoc, 155 copies). As these are dispensable for capsid formation, they can be used for displaying proteins and macromolecular complexes on the T4 capsid surface. Anthrax toxin components were attached to the T4 capsid as a fusion protein of the N-terminal domain of the anthrax lethal factor (LFn) with Soc. The LFn-Soc fusion protein was complexed in vitro with Hoc(-)Soc(-)T4 phage. Subsequently, cleaved anthrax protective antigen heptamers (PA63)(7) were attached to the exposed LFn domains. A cryo-electron microscopy study of the decorated T4 particles shows the complex of PA63 heptamers with LFn-Soc on the phage surface. Although the cryo-electron microscopy reconstruction is unable to differentiate on its own between different proposed models of the anthrax toxin, the density is consistent with a model that had predicted the orientation and position of three LFn molecules bound to one PA63 heptamer.
| S-EPMC2062529 | biostudies-literature
Project description:Improved technology for reconstructing cryo-electron microscopy (cryo-EM) images has now made it possible to determine secondary structural features of membrane proteins in enveloped viruses. The structure of mature dengue virus particles was determined to a resolution of 9.5 A by cryo-EM and image reconstruction techniques, establishing the secondary structural disposition of the 180 envelope (E) and 180 membrane (M) proteins in the lipid envelope. The alpha-helical 'stem' regions of the E molecules, as well as part of the N-terminal section of the M proteins, are buried in the outer leaflet of the viral membrane. The 'anchor' regions of E and the M proteins each form antiparallel E-E and M-M transmembrane alpha-helices, leaving their C termini on the exterior of the viral membrane, consistent with the predicted topology of the unprocessed polyprotein. This is one of only a few determinations of the disposition of transmembrane proteins in situ and shows that the nucleocapsid core and envelope proteins do not have a direct interaction in the mature virus.
| S-EPMC4148076 | biostudies-literature
Project description:Correct reconstruction of macromolecular structure by cryo-electron microscopy (cryo-EM) relies on accurate determination of the orientation of single-particle images. For small (<100 kDa) DNA-binding proteins, obtaining particle images with sufficiently asymmetric features to correctly guide alignment is challenging. We apply DNA origami to construct molecular goniometers-instruments that precisely orient objects-and use them to dock a DNA-binding protein on a double-helix stage that has user-programmable tilt and rotation angles. We construct goniometers with 14 different stage configurations to orient and visualize the protein just above the cryo-EM grid surface. Each goniometer has a distinct barcode pattern that we use during particle classification to assign angle priors to the bound protein. We use goniometers to obtain a 6.5-Å structure of BurrH, an 82-kDa DNA-binding protein whose helical pseudosymmetry prevents accurate image orientation using traditional cryo-EM. Our approach should be adaptable to other DNA-binding proteins as well as small proteins fused to DNA-binding domains.
| S-EPMC7956247 | biostudies-literature
Project description:Recent advances in three-dimensional electron microscopy (3D EM) have enabled the quantitative visualization of the structural building blocks of proteins at improved resolutions. We provide algorithms to detect the secondary structures (α-helices and β-sheets) from proteins for which the volumetric maps are reconstructed at 6-10Å resolution. Additionally, we show that when the resolution is coarser than 10Å, some of the supersecondary structures can be detected from 3D EM maps. For both these algorithms, we employ tools from computational geometry and differential topology, specifically the computation of stable/unstable manifolds of certain critical points of the distance function induced by the molecular surface. Our results connect mathematically well-defined constructions with bio-chemically induced structures observed in proteins.
| S-EPMC3312805 | 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:Cationic coatings can enhance the stability of synthetic DNA objects in low ionic strength environments such as physiological fluids. Here, we used single-particle cryo-electron microscopy (cryo-EM), pseudoatomic model fitting, and single-molecule mass photometry to study oligolysine and polyethylene glycol (PEG)-oligolysine-coated multilayer DNA origami objects. The coatings preserve coarse structural features well on a resolution of multiple nanometers but can also induce deformations such as twisting and bending. Higher-density coatings also led to internal structural deformations in the DNA origami test objects, in which a designed honeycomb-type helical lattice was deformed into a more square-lattice-like pattern. Under physiological ionic strength, where the uncoated objects disassembled, the coated objects remained intact but they shrunk in the helical direction and expanded in the direction perpendicular to the helical axis. Helical details like major/minor grooves and crossover locations were not discernible in cryo-EM maps that we determined of DNA origami coated with oligolysine and PEG-oligolysine, whereas these features were visible in cryo-EM maps determined from the uncoated reference objects. Blunt-ended double-helical interfaces remained accessible underneath the coating and may be used for the formation of multimeric DNA origami assemblies that rely on stacking interactions between blunt-ended helices. The ionic strength requirements for forming multimers from coated DNA origami differed from those needed for uncoated objects. Using single-molecule mass photometry, we found that the mass of coated DNA origami objects prior to and after incubation in low ionic strength physiological conditions remained unchanged. This finding indicated that the coating effectively prevented strand dissociation but also that the coating itself remained stable in place. Our results validate oligolysine coatings as a powerful stabilization method for DNA origami but also reveal several potential points of failure that experimenters should watch to avoid working with false premises.
| S-EPMC8223477 | biostudies-literature
Project description:Huntingtin (HTT) is a large (348?kDa) protein that is essential for embryonic development and is involved in diverse cellular activities such as vesicular transport, endocytosis, autophagy and the regulation of transcription. Although an integrative understanding of the biological functions of HTT is lacking, the large number of identified HTT interactors suggests that it serves as a protein-protein interaction hub. Furthermore, Huntington's disease is caused by a mutation in the HTT gene, resulting in a pathogenic expansion of a polyglutamine repeat at the amino terminus of HTT. However, only limited structural information regarding HTT is currently available. Here we use cryo-electron microscopy to determine the structure of full-length human HTT in a complex with HTT-associated protein 40 (HAP40; encoded by three F8A genes in humans) to an overall resolution of 4 Å. HTT is largely ?-helical and consists of three major domains. The amino- and carboxy-terminal domains contain multiple HEAT (huntingtin, elongation factor 3, protein phosphatase 2A and lipid kinase TOR) repeats arranged in a solenoid fashion. These domains are connected by a smaller bridge domain containing different types of tandem repeats. HAP40 is also largely ?-helical and has a tetratricopeptide repeat-like organization. HAP40 binds in a cleft and contacts the three HTT domains by hydrophobic and electrostatic interactions, thereby stabilizing the conformation of HTT. These data rationalize previous biochemical results and pave the way for improved understanding of the diverse cellular functions of HTT.
| S-EPMC5837020 | biostudies-literature
Project description:Sample fixation by vitrification is critical for the optimal structural preservation of biomolecules and subsequent high-resolution imaging by cryo-correlative light and electron microscopy (cryoCLEM). There is a large resolution gap between cryo fluorescence microscopy (cryoFLM), ~400-nm, and the sub-nanometre resolution achievable with cryo-electron microscopy (cryoEM), which hinders interpretation of cryoCLEM data. Here, we present a general approach to increase the resolution of cryoFLM using cryo-super-resolution (cryoSR) microscopy that is compatible with successive cryoEM investigation in the same region. We determined imaging parameters to avoid devitrification of the cryosamples without the necessity for cryoprotectants. Next, we examined the applicability of various fluorescent proteins (FPs) for single-molecule localisation cryoSR microscopy and found that all investigated FPs display reversible photoswitchable behaviour, and demonstrated cryoSR on lipid nanotubes labelled with rsEGFP2 and rsFastLime. Finally, we performed SR-cryoCLEM on mammalian cells expressing microtubule-associated protein-2 fused to rsEGFP2 and performed 3D cryo-electron tomography on the localised areas. The method we describe exclusively uses commercially available equipment to achieve a localisation precision of 30-nm. Furthermore, all investigated FPs displayed behaviour compatible with cryoSR microscopy, making this technique broadly available without requiring specialised equipment and will improve the applicability of this emerging technique for cellular and structural biology.
| S-EPMC6362030 | biostudies-literature