Development of a superfluid helium stage for high-resolution electron microscopy

Fujiyoshi, Yoshinori; Mizusaki, T.; Morikawa, Kosuke; Yamagishi, Hideo; Aoki, Yasumichi; Kihara, H.; Harada, Yoshiyasu

doi:10.1016/0304-3991(91)90159-4

Cited by 192 publications

(116 citation statements)

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“…It was preliminarily found that crystals of LPS are easily destroyed during observation in a conventional electron microscope. Therefore, for observation and analysis of crystals, a high-resolution cryo-electron microscope (12,13) (JEOL, Ltd., Akishima, Japan) operating at 400 kV and equipped with a cryo-stage which was cooled with superfluid helium (superfluid cryo-stage) and a cryo-transfer system was used unless indicated otherwise. For the preparation of specimens to be tested, a drop of crystals was placed on a supermicrogrid which was coated with carbon and then gold, and the liquid was removed from the reverse side of the grid with filter paper.…”

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Crystallization of R-form lipopolysaccharides from Salmonella minnesota and Escherichia coli

et al. 1990

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SalmoneUa minnesota Re and Ra lipopolysaccharides (LPSs) and Escherichia coli K-12 LPS formed three-dimensional crystals, either hexagonal plates (preferential growth along the a axis) or solid columns (preferential growth along the c axis), when they were precipitated by the addition of 2 volumes of 95% ethanol containing 375 mM MgCl2 and incubated in 70% ethanol containing 250 mM MgCl2 at 4TC for 10 days.Analyses of crystals suggested that they consist of hexagonal lattices with the a axis (a side of the lozenge as a unit cell on the basal plane) of 0.462 nm for all these three kinds of LPSs and the c axes (perpendicular to the basal plane) of 5. 85, 8.47, and 8.75 nm for S. minnesota Re and Ra LPSs and E. coli K-12 LPS, respectively, and that hydrocarbon chains of the lipid A portion play the leading part in crystallization, whereas the hydrophilic part of the lipid A (the disaccharide backbone) and R core exhibit a disordered structure or are in a random orientation. The phenomenon of doubling of the a axis to 0.924 nm was observed with crystals of S.minnesota Re LPS when they were incubated in 70% ethanol for an additional 180 days, but not with crystals of S. minnesota Ra LPS or E. coli K-12 LPS. S. minnesota S-form LPS possessing the 0-antigen-specific polysaccharide and S. minnesota free lipid A obtained by acid hydrolysis of Re LPS did not crystallize under the same experimental conditions. Bacterial lipopolysaccharide (LPS) is the constituent of the outer membrane of gram-negative bacteria and consists of the polysaccharide (O antigen) which is linked to the R core consisting of oligosaccharide, which in turn is linked to the lipid portion termed lipid A (49). LPS strongly elicits a variety of host reactivities through interactions with humoral and cellular factors of the host. It has been widely accepted that the lipid A portion is mostly responsible for the biological activities of LPS (14,17,47), although the polysaccharide or the R core can modify the strength of action in some biological activities. Recently, studies on the relationship between chemical structure and biological activity of lipid A components have progressed greatly, since synthetic preparations of lipid A components and related compounds have been available (16,18,20,22,23,26,32,33,46). Although several studies have been done on the three-dimensional structure of LPS (4,10,11,34,35,39,40,56) and a schematic model of LPS has been proposed (34), the conformation of LPS has not been determined conclusively. We have found (29-31) that certain of the R-form LPSs form ordered two-dimensional hexagonal structures in the presence of MgCl2. During the experiments, we noticed that R-form LPSs from Salmonella minnesota and Escherichia coli K-12 form three-dimensional crystals when they are precipitated by the addition of 2 volumes of 95% ethanol containing 375 mM MgCl2 and suspended in 70% ethanol containing 250 mM MgCl2 and incubated at 4°C. We present here analyses of crystals of these R-form LPSs. This report is the first one that has de...

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“…2A). The crystals were labile to the electron beam, even at a specimen temperature lower than 8 K, which could be reached by high-resolution cryo-electron microscopy (12,13). The electron diffraction spots taken at the second exposure were very weak (Fig.…”

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Crystallization of R-form lipopolysaccharides from Salmonella minnesota and Escherichia coli

et al. 1990

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“…We have therefore developed a super fluid helium stage that achieved a resolution of 2.6 Å in 1991. 19 Thermal shield by liquid Nitrogen and Helium tank is gold plated to minimize radial heat (Upper right in Fig. 5).…”

Section: Development Of the Cryo-electron Microscopementioning

confidence: 99%

Structural physiology based on electron crystallography

Fujiyoshi

2011

Protein Science

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There are many questions in brain science, which are extremely interesting but very difficult to answer. For example, how do education and other experiences during human development influence the ability and personality of the adult? The molecular mechanisms underlying such phenomena are still totally unclear. However, technological and instrumental advancements of electron microscopy have facilitated comprehension of the structures of biological components, cells, and organelles. Electron crystallography is especially good for studying the structure and function of membrane proteins, which are key molecules of signal transduction in neural and other cells. Electron crystallography is now an established technique to analyze the structures of membrane proteins in lipid bilayers, which are close to their natural biological environment. By utilizing cryo-electron microscopes with helium cooled specimen stages, which were developed through a personal motivation to understand functions of neural systems from a structural point of view, structures of membrane proteins were analyzed at a resolution higher than 3 Å . This review has four objectives. First, it is intended to introduce the new research field of structural physiology. Second, it introduces some of the personal struggles, which were involved in developing the cryo-electron microscope. Third, it discusses some of the technology for the structural analysis of membrane proteins based on cryo-electron microscopy. Finally, it reviews structural and functional analyses of membrane proteins.

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“…Specimen stages cooled with both liquid nitrogen and liquid helium were developed. (Although cooling all the way to 4°K turned out to be detrimental for single-particle techniques, because of an unexpected change in the physical properties of ice below 30°K under electron bombardment, the development of liquid helium stages [22] had a significant impetus on modern instrument design.) Another driving force was the implementation of protocols for low-dose electron microscopy, going back to the ground-breaking protocols of Unwin and Henderson for visualizing glucose-embedded 2D crystals of bacteriorhodopsin [5] and on early radiation damage studies by Glaeser [12].…”

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Generalized single-particle cryo-EM – a historical perspective

Frank

2015

Microscopy (Tokyo)

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This is a brief account of the earlier history of single-particle cryo-EM of biological molecules lacking internal symmetry, which goes back to the mid-seventies. The emphasis of this review is on the mathematical concepts and computational approaches. It is written as the field experiences a turning point in the wake of the introduction of digital cameras capable of single electron counting, and near-atomic resolution can be reached even for smaller molecules.Key words: 3D reconstruction, image processing, ribosome, molecular structureThe spectacular, fast-paced advances in single-particle cryogenic electron microscopy (cryo-EM) in the past 3 years, following the introduction of novel direct detection device (DDD) cameras with superior signal-to-noise ratio and resolution [1,2], are currently the subject of many commentaries and news and views articles conveying the general excitement of the structural biology community and the scientific community at large. As someone who has been in the field since the early days, I would like to contribute by a recollection of how the field developed.Single-particle EM, as a novel approach to structural biology, required a heretical concept going against the grain of wisdom which held that quantitative structure determination by 3D reconstruction from EM projections [3] is not feasible unless molecules are arranged in crystalline order. These ordered structures include those with helical symmetry, as in DeRosier and Klug's reconstruction of a bacteriophage tail [3], arranged in a two-dimensional crystal [4,5], or with high symmetry as in viruses [6]. Viruses, although classifiable as single particles, contain structural information in a highly redundant form. For instance, the projection of a virus with icosahedral symmetry contains 60 projections of its asymmetric unit. Not only is the spatial arrangement of these units fixed and recoverable from the Fourier transform, but the signal is also retrieved with an instant bonus of nearly 8-fold reduction in the power of noise. Crowther and coworkers, in developing the common lines approach for recovering the structure of an icosahedral virus from its projection, did state that 'there is in principle no reason why the method should not be extended to systems with lower symmetry' [6], but it was not apparent how this could be practically accomplished for objects lacking symmetry altogether.At the time, the idea of single-particle averaging of entirely asymmetric molecules [7] therefore created some excitement: If such, [i.e. asymmetric single-particle] methods were to be perfected, then, in the words of one scientist, the sky would be the limit [8]. Thus, Robinson's Research News article started with an appreciation of Unwin and Henderson's seminal work, the 3D reconstruction of bacteriorhodopsin

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Development of a superfluid helium stage for high-resolution electron microscopy

Cited by 192 publications

References 7 publications

Crystallization of R-form lipopolysaccharides from Salmonella minnesota and Escherichia coli

Crystallization of R-form lipopolysaccharides from Salmonella minnesota and Escherichia coli

Structural physiology based on electron crystallography

Generalized single-particle cryo-EM – a historical perspective

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