Retrospective on the early development of cryoelectron microscopy of macromolecules and a prospective on opportunities for the future

Taylor, Kenneth A.; Glaeser, Robert M.

doi:10.1016/j.jsb.2008.06.004

Cited by 151 publications

(129 citation statements)

References 66 publications

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“…Our observation of ∼100 structures per micrograph suggests that interactions of the support structure with the air-water interface, possibly both at the top and the bottom, may lead to a strong local enrichment in concentration. For proteins, a similar enrichment has been attributed to a sticky layer of denatured protein at the airwater interface (15). Because our samples without protein show a similar-or even stronger-enrichment in DNA support structures, unbound p53 tetramers are probably not required for the enrichment.…”

Section: Discussionsupporting

confidence: 69%

“…Second, during the short time between blotting and vitrification, the macromolecules are in a thin liquid film that extends for millimeters to the side, but is only a few hundred angstroms thick. Brownian motion will cause the macromolecules to collide with the air-water interface >1,000 times per second (15). Biological macromolecules may unfold when they hit the air-water interface (16), or they may adsorb to this interface in a nonrandom manner-for example, by presenting their most hydrophobic patch to it.…”

mentioning

confidence: 99%

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Design of a molecular support for cryo-EM structure determination

Martin

Bharat

Joerger

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Despite the recent rapid progress in cryo-electron microscopy (cryo-EM), there still exist ample opportunities for improvement in sample preparation. Macromolecular complexes may disassociate or adopt nonrandom orientations against the extended air-water interface that exists for a short time before the sample is frozen. We designed a hollow support structure using 3D DNA origami to protect complexes from the detrimental effects of cryo-EM sample preparation. For a first proof-of-principle, we concentrated on the transcription factor p53, which binds to specific DNA sequences on double-stranded DNA. The support structures spontaneously form monolayers of preoriented particles in a thin film of water, and offer advantages in particle picking and sorting. By controlling the position of the binding sequence on a single helix that spans the hollow support structure, we also sought to control the orientation of individual p53 complexes. Although the latter did not yet yield the desired results, the support structures did provide partial information about the relative orientations of individual p53 complexes. We used this information to calculate a tomographic 3D reconstruction, and refined this structure to a final resolution of ∼15 Å. This structure settles an ongoing debate about the symmetry of the p53 tetramer bound to DNA.cryo-EM | DNA-origami | single particle analysis | structural biology | p53 C ryo-electron microscopy (cryo-EM) structure determination of biological macromolecules is undergoing rapid progress. With the advent of efficient direct electron detectors and the development of powerful algorithms for image processing, numerous structures to near-atomic resolution have been reported in the past few years (1, 2). In cryo-EM single-particle analysis, solutions of purified protein and/or nucleic acid complexes are typically applied to a thin, amorphous carbon film with micrometer-sized holes in it that is held in place by a metal grid. Excess liquid is then blotted away with filter paper, and the sample is rapidly plunged in liquid ethane (3,4). This procedure ideally results in the formation of a film of vitreous ice that is only slightly thicker than the macromolecular complex of interest. Keeping the frozen sample at liquid nitrogen temperatures allows its insertion into the high vacuum of a transmission electron microscope and limits the effects of radiation damage by the electrons that are used for imaging (5). Images taken through the holes of the carbon film ideally contain 2D projections of many, assumedly identical copies of the macromolecular complex of interest, which are often called particles. Projections from different viewing directions can then be combined in a 3D reconstruction of the scattering potential of the molecule (6). If the resulting map approaches a resolution of 3 Å, it allows building an atomic model of the molecules, from which useful information about their function may be derived.A major hurdle in single-particle analysis is the need to recover the relative viewing angles...

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Section: Discussionsupporting

confidence: 69%

mentioning

confidence: 99%

Design of a molecular support for cryo-EM structure determination

Martin

Bharat

Joerger

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…Micron-sized holes engineered into carbon films provide a transparent background, ideal for imaging weak-phase objects such as protein machinery suspended in vitreous ice (reviewed by Taylor and Glaeser, 2008) [9]. One inherent limitation with the use of these support films is the beam-induced movement that is generated from illuminating the specimen suspended in the holes.…”

Section: Introductionmentioning

confidence: 99%

Molecular Surveillance of Viral Processes Using Silicon Nitride Membranes

et al. 2013

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Abstract:Here we present new applications for silicon nitride (SiN) membranes to evaluate biological processes. We determined that 50-nanometer thin films of SiN produced from silicon wafers were sufficiently durable to bind active rotavirus assemblies. A direct comparison of SiN microchips with conventional carbon support films indicated that SiN performs equivalent to the traditional substrate to prepare samples for Electron Microscopy (EM) imaging. Likewise, SiN films coated with Ni-NTA affinity layers concentrated rotavirus particles similarly to affinity-coated carbon films. However, affinity-coated SiN membranes outperformed glow-discharged conventional carbon films 5-fold as indicated by the number of viral particles quantified in EM images. In addition, we were able to recapitulate viral uncoating and transcription mechanisms directed onto the microchip surfaces. EM images of these processes revealed the production of RNA transcripts emerging from active rotavirus complexes. These results were confirmed by the functional incorporation of radiolabeled nucleotides into the nascent RNA transcripts. Collectively, we demonstrate new uses for SiN membranes to perform molecular surveillance on life processes in real-time. OPEN ACCESSMicromachines 2013, 4 91

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“…Biological samples present several technical challenges for TEM [7]: (i) the high vacuum is not compatible with hydrated samples; (ii) biological samples are mainly composed of light elements that are vulnerable to damage from high-energy electrons; and (iii) light elements interact with electrons weakly, lowering image contrast. In 1974, Robert M. Glaeser et al [8] discovered that freezing biological samples at liquid nitrogen temperatures dramatically reduced damage from the electrons.…”

Section: Basic Principles Of Cryo-emmentioning

confidence: 99%

Cryo-electron microscopy for structural biology: current status and future perspectives

Wang

2015

Sci. China Life Sci.

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Recently, significant technical breakthroughs in both hardware equipment and software algorithms have enabled cryo-electron microscopy (cryo-EM) to become one of the most important techniques in biological structural analysis. The technical aspects of cryo-EM define its unique advantages and the direction of development. As a rapidly emerging field, cryo-EM has benefitted from highly interdisciplinary research efforts. Here we review the current status of cryo-EM in the context of structural biology and discuss the technical challenges. It may eventually merge structural and cell biology at multiple scales.cryo-electron microscopy, structural biology, cell biology, three-dimensional reconstruction Citation:Wang HW. Cryo-electron microscopy for structural biology: current status and future perspectives. Sci China Life Sci, 2015Sci, , 58: 750 -756, doi: 10.1007 Since the 1980s, structural biology has become a booming area in life sciences. By determining the three-dimensional (3-D) structures of proteins, nucleic acids, and their complexes, structural biology has given us accurate insights into concerning the shapes of complicated bio-macromolecules, the arrangements of atoms and molecules, and physical properties such as electro-potential distributions and hydrophobicity. These structures provide a crucial understanding of how bio-macromolecules execute specific biological functions. As techniques of structural biology become more mature in the 21st century, they assume more significant roles in various sub-disciplines of life sciences. Of the more than 10 5 protein structures that have been deposited in the Worldwide Protein Data Bank, most were solved by X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy ( Figure 1A). Recent technical progress has stimulated the use of new structural biology tools. In December 2013, two landmark Nature articles revealed the atomic-resolution structure of the TRPV1 ion channel in membranes that was imaged by cryo-EM [1,2]. With cryo-EM, Yifan Cheng and David Julius et al. [1,2] at the University of California, San Francisco, imaged for the first time the 3-D structure of the membrane channel protein TRVP1 with a resolution of 3.4 Å, and constructed an atomic model. In addition, several nearly atomic models of viruses, proteasomes, and ribosomes have been resolved by cryo-EM in the past few years [36]. The significance of the work was to obtain high-resolution structural information from a very small amount of sample, without requiring protein crystallization, and to simultaneously obtain the structures of the protein complex in different states over a relatively short time. In only one year, cryo-EM has received extensive attention as a means to directly obtain atomic structures of bio-macromolecules.Electron microscopy has been used in structural biology for many years. Since the 1950s, it has revealed cellular, sub-cellular, and bio-macromolecular structures. Based on completely different principles from X-ray crystallography and NMR spectroscopy in so...

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Retrospective on the early development of cryoelectron microscopy of macromolecules and a prospective on opportunities for the future

Cited by 151 publications

References 66 publications

Design of a molecular support for cryo-EM structure determination

Design of a molecular support for cryo-EM structure determination

Molecular Surveillance of Viral Processes Using Silicon Nitride Membranes

Cryo-electron microscopy for structural biology: current status and future perspectives

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