Cryo-electron microscopy of viruses

Adrian, Marc; Dubochet, Jacques; Lepault, Jean; McDowall, Alasdair W.

doi:10.1038/308032a0

Cited by 1,229 publications

(741 citation statements)

References 28 publications

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“…Adrian et al (1984) published the first cryoEM images of adenovirus single particles. Vogel et al (1986) used these images to calculate a map of the structure at 45 Å resolution and Stewart et al (1991) improved this to 35 Å.…”

Section: Field Emission Guns Column Stability and Automationmentioning

confidence: 99%

Single particle electron cryomicroscopy: trends, issues and future perspective

Vinothkumar

Henderson

2016

Quart. Rev. Biophys.

175

115

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Abstract. There has been enormous progress during the last few years in the determination of three-dimensional biological structures by single particle electron cryomicroscopy (cryoEM), allowing maps to be obtained with higher resolution and from fewer images than required previously. This is due principally to the introduction of a new type of direct electron detector that has 2-to 3-fold higher detective quantum efficiency than available previously, and to the improvement of the computational algorithms for image processing. In spite of the great strides that have been made, quantitative analysis shows that there are still significant gains to be made provided that the problems associated with image degradation can be solved, possibly by minimising beam-induced specimen movement and charge build up during imaging. If this can be achieved, it should be possible to obtain near atomic resolution structures of smaller single particles, using fewer images and resolving more conformational states than at present, thus realising the full potential of the method. The recent popularity of cryoEM for molecular structure determination also highlights the need for lower cost microscopes, so we encourage development of an inexpensive, 100 keV electron cryomicroscope with a high-brightness field emission gun to make the method accessible to individual groups or institutions that cannot afford the investment and running costs of a state-of-the-art 300 keV installation. A key requisite for successful high-resolution structure determination by cryoEM includes interpretation of images and optimising the biochemistry and grid preparation to obtain nicely distributed macromolecules of interest. We thus include in this review a gallery of cryoEM micrographs that shows illustrative examples of single particle images of large and small macromolecular complexes.

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Section: Field Emission Guns Column Stability and Automationmentioning

confidence: 99%

Single particle electron cryomicroscopy: trends, issues and future perspective

Vinothkumar

Henderson

2016

Quart. Rev. Biophys.

175

115

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“…Scale bar, 500 Å. Both samples were prepared and photographed with established cryomicroscopy procedures [14][15][16][17] . Roughly 4 ml each of a 2.0 mg ml −1 CPMV (b) or 0.5 mg ml −1 CPMV-Fab (c; stoichiometry of Fabs to virus in solution was 600:1) sample at pH 7.4 in PBS was applied to holey-carbon films, blotted with filter paper and rapidly plunged into liquid ethane at about −170 °C.…”

Section: Figmentioning

confidence: 99%

Identification of a Fab interaction footprint site on an icosahedral virus by cryoelectron microscopy and X-ray crystallography

Wang

Porta

Chen

et al. 1992

Nature

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Biological processes frequently require the formation of multi-protein or nucleoprotein complexes. Some of these complexes have been produced in homogeneous form, crystallized, and analysed at high resolution by X-ray crystallography (for example, see refs 1-3). Most, however, are too large or too unstable to crystallize. Individual components of such complexes can often be purified and analysed by crystallography. Here we report how the coordinated application of cryoelectron microscopy, three-dimensional image reconstruction, and X-ray crystallography provides a powerful approach to study large, unstable macromolecular complexes. Three-dimensional reconstructions of native cowpea mosaic virus (CMPV) and a complex of CPMV saturated with a Fab fragment of a monoclonal antibody against the virus have been determined at 23 Å resolution from low-irradiation images of unstained, frozen-hydrated samples. Despite the nominal resolution of the complex, the physical footprint of the Fab on the capsid surface and the orientation and position of the Fab have been determined to within a few angstroms by fitting atomic models of CPMV 4 and Fab (Kol) 5 to reconstructed density maps.Comoviruses are plant viruses whose structural and biological properties are strikingly similar to the animal picornaviruses 6,7 . For example, the protein capsids of CPMV, the type member of the comovirus group, and poliovirus, the most extensively studied picornavirus, are comparable (Fig. 1a). The CPMV coat contains 60 copies each of large (relative molecular mass 42,000 (M r 42K) and small (24K) protein subunits arranged with icosahedral symmetry. Each large subunit (L) is composed of two antiparallel β-barrel domains which are positioned close to the icosahedral 3-fold axes. The β-barrel domain at the amino-terminal end of L corresponds to the VP2 subunit in poliovirus and the β-barrel at the carboxy-terminal end of L corresponds to VP3 in poliovirus. The small subunits (S), packed near the icosahedral 5-fold axes, correspond to VP1 in poliovirus.CPMV is strongly immunogenic when injected into rabbits or mice. We prepared nine monoclonal antibodies against CPMV using standard methods 8 . The binding properties of these antibodies were analysed by enzyme-linked immunosorbent assay (ELISA) and all nine would only bind to virus particles presented in a double antibody sandwich-type ELISA © 1992 Nature Publishing Group Correspondence to: John E. Johnson. G.W. and C.P. are joint first authors. in which the virus is bound to a polyclonal antibody attached to the plastic plate. Denaturation of the virus by its direct adsorption onto a plastic surface destroys the antigenic sites for all nine monoclonal antibodies. Only monoclonal antibodies 5B2 and 10B7 reacted strongly with CPMV that was free in solution (antibody binding was assayed by electron microscopy analysis of negatively stained complexes marked with gold-labelled anti-mouse immunoglobulin). Fab fragments from monoclonal antibody 5B2 were prepared by digestion of IgGs with insoluble...

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“…After briefly blotting the excess liquid, the grid was rapidly freeze-plunged into liquid ethane and stored in liquid nitrogen until imaging. 38 Images were taken at liquid nitrogen conditions using a CM200 FEG electron microscope equipped with a Gatan cryoholder (Gatan GmbH, Munich, Germany). For electron microscopical analysis, a CCD camera (TVIPS) was used and particle dimensions were determined using the EMMENU software.…”

Section: Introductionmentioning

confidence: 99%

Characterization of Tailor-Made Copolymers of Oligo(ethylene glycol) Methyl Ether Methacrylate and N,N-Dimethylaminoethyl Methacrylate as Nonviral Gene Transfer Agents: Influence of Macromolecular Structure on Gene Vector Particle Properties and Transfection Efficiency

et al. 2009

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Oligo(ethylene glycol) methyl ether methacrylates (OEGMA) of various chain lengths (i.e., 9, 23, or 45 EG units) and N,N-dimethylaminoethyl methacrylate (DMAEMA) were copolymerized by atom transfer radical polymerization (ATRP), yielding well-defined P(DMAEMA-co-OEGMA) copolymers with increasing OEGMA molar fractions (F OEGMA) but a comparable degree of polymerization (DP ∼ 120). Increase of both F OEGMA and OEGMA chain lengths correlated inversely with gene vector size, morphology, and zeta potential. P(DMAEMA-co-OEGMA) copolymers prevented gene vector aggregation at high plasmid DNA (pDNA) concentrations in isotonic solution and did not induce cytotoxicity even at high concentrations. Transfection efficiency of the most efficient P(DMAEMA-co-OEGMA) copolymers was found to be >10-fold lower compared with branched polyethylenimine (PEI) 25 kDa. Although OEGMA copolymerization largely reduced gene vector binding with the cell surface, cellular internalization of the bound complexes was less affected. These observations suggest that inefficient endolysosomal escape limits transfection efficiency of P(DMAEMA-co-OEGMA) copolymer gene vectors. Despite this observation, optimized p(DMAEMA-co-OEGMA) gene vectors remained stable under conditions for in vivo application leading to 7-fold greater gene expression in the lungs compared with PEI. Tailor-made P(DMAEMA-co-OEGMA) copolymers are promising nonviral gene transfer agents that fulfill the requirements for successful in vivo gene delivery.

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Cryo-electron microscopy of viruses

Cited by 1,229 publications

References 28 publications

Single particle electron cryomicroscopy: trends, issues and future perspective

Single particle electron cryomicroscopy: trends, issues and future perspective

Identification of a Fab interaction footprint site on an icosahedral virus by cryoelectron microscopy and X-ray crystallography

Characterization of Tailor-Made Copolymers of Oligo(ethylene glycol) Methyl Ether Methacrylate and N,N-Dimethylaminoethyl Methacrylate as Nonviral Gene Transfer Agents: Influence of Macromolecular Structure on Gene Vector Particle Properties and Transfection Efficiency

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