A charge coupled device camera with electron decelerator for intermediate voltage electron microscopy

Downing, Kenneth H.; Mooney, P.E.

doi:10.1063/1.2902853

Cited by 17 publications

(12 citation statements)

References 27 publications

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“…Cryo-TEMimages were acquired on a JEOL-3100 electron microscope equipped with a field emission gun electron source operating at 300 kV, an Omega energy filter, a Gatan 795 4 K × 4 K CCD camera mounted at the exit of an electron decelerator (24), and a cryo-transfer stage. The decelerator was operated at 200 kV resulting in images formed by electron beam accelerated with approximately 100 kV at the CCD.…”

Section: Methodsmentioning

confidence: 99%

Direct observation of kinetic traps associated with structural transformations leading to multiple pathways of S-layer assembly

Shin

Chung

Sanii

et al. 2012

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

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The concept of a folding funnel with kinetic traps describes folding of individual proteins. Using in situ Atomic Force Microscopy to investigate S-layer assembly on mica, we show this concept is equally valid during self-assembly of proteins into extended matrices. We find the S-layer-on-mica system possesses a kinetic trap associated with conformational differences between a long-lived transient state and the final stable state. Both ordered tetrameric states emerge from clusters of the monomer phase, however, they then track along two different pathways. One leads directly to the final low-energy state and the other to the kinetic trap. Over time, the trapped state transforms into the stable state. By analyzing the time and temperature dependencies of formation and transformation we find that the energy barriers to formation of the two states differ by only 0.7 kT, but once the high-energy state forms, the barrier to transformation to the low-energy state is 25 kT. Thus the transient state exhibits the characteristics of a kinetic trap in a folding funnel.biological assembly | protein crystal growth | two-step crystallization | protein folding funnel P roteins that naturally self-assemble into extended ordered structures often adopt conformations that are distinct from those of the individual monomeric proteins (1-4). For example, collagen matrices, which constitute the organic scaffolds of bones and teeth in all higher organisms, are constructed from triple helices of the individual collagen monomers (4). These helices further assemble into highly organized twisted fibrils exhibiting a pseudohexagonal symmetry. In some cases, assembly is inexorably linked to folding transformations, as in the case of prion or amyloid fibrils, where misfolding of the monomers triggers assembly, which in turn drives misfolding of new monomers (1).When individual proteins transform from an unstructured state to the final equilibrium state, the concept of a folding funnel characterized by a large number of potential initial states higher in energy than the final state is often invoked (5, 6). The walls of the funnel are presumed not to be smooth and the resulting bumps and valleys define kinetic traps where the protein exhibits nonequilibrium structures for extended periods of time. These transient structures can be disordered "molten globules" or partially ordered intermediates (7). This physical picture of folding in which one fraction of a protein population promptly reaches the folded state while the remaining fraction gets trapped in metastable states has been referred as the kinetic partitioning mechanism (8). The energy landscape that defines the funnel as well as the pathways through it have been explored in some detail at the single molecule level for a number of small proteins and protein subdomains through both simulations (8-10) and experiments (11-13). However, despite the fact that conformational transformations are part and parcel of protein self-assembly into extended architectures, this concept of the folding fu...

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Section: Methodsmentioning

confidence: 99%

Direct observation of kinetic traps associated with structural transformations leading to multiple pathways of S-layer assembly

Shin

Chung

Sanii

et al. 2012

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

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“…Cryo-TEM images were acquired on a Jeol 3100-FFC electron microscope equipped with a field emission gun (FEG) electron source operating at 300 kV, an Omega energy filter, a cryotransfer stage, and a Gatan 795 4,000 (4K) ϫ 4K charge-coupled-device (CCD) camera mounted at the exit of an electron decelerator held at a constant voltage of 200 kV (28). The stage was cooled with liquid nitrogen to 80 K during acquisition of all data sets.…”

Section: Methodsmentioning

confidence: 99%

U(VI) Reduction by Diverse Outer Surface c -Type Cytochromes of Geobacter sulfurreducens

Orellana

Leavitt

Comolli

et al. 2013

Appl Environ Microbiol

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Early studies with Geobacter sulfurreducens suggested that outer-surface c-type cytochromes might play a role in U(VI) reduction, but it has recently been suggested that there is substantial U(VI) reduction at the surface of the electrically conductive pili known as microbial nanowires. This phenomenon was further investigated. A strain of G. sulfurreducens, known as Aro-5, which produces pili with substantially reduced conductivity reduced U(VI) nearly as well as the wild type, as did a strain in which the gene for PilA, the structural pilin protein, was deleted. In order to reduce rates of U(VI) reduction to levels less than 20% of the wild-type rates, it was necessary to delete the genes for the five most abundant outer surface c-type cytochromes of G. sulfurreducens. X-ray absorption near-edge structure spectroscopy demonstrated that whereas 83% ؎ 10% of the uranium associated with wild-type cells correspond to U(IV) after 4 h of incubation, with the quintuple mutant, 89% ؎ 10% of uranium was U(VI). Transmission electron microscopy and X-ray energy dispersion spectroscopy revealed that wild-type cells did not precipitate uranium along pili as previously reported, but U(IV) was precipitated at the outer cell surface. These findings are consistent with those of previous studies, which have suggested that G. sulfurreducens requires outer-surface c-type cytochromes but not pili for the reduction of soluble extracellular electron acceptors.

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“…The stage was cooled to 80 K using liquid nitrogen. Approximately 10% of the data were acquired using a modified Gatan 795 4Kx4K CCD camera with 15-m pixels mounted at the exit of an electron decelerator (15). The decelerator was operated at 248 kV, resulting in images formed by a 52-kV electron beam at the CCD.…”

Section: Methodsmentioning

confidence: 99%

Analysis of the Intact Surface Layer of Caulobacter crescentus by Cryo-Electron Tomography

et al. 2010

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The surface layers (S layers) of those bacteria and archaea that elaborate these crystalline structures have been studied for 40 years. However, most structural analysis has been based on electron microscopy of negatively stained S-layer fragments separated from cells, which can introduce staining artifacts and allow rearrangement of structures prone to self-assemble. We present a quantitative analysis of the structure and organization of the S layer on intact growing cells of the Gram-negative bacterium Caulobacter crescentus using cryo-electron tomography (CET) and statistical image processing. Instead of the expected long-range order, we observed different regions with hexagonally organized subunits exhibiting short-range order and a broad distribution of periodicities. Also, areas of stacked double layers were found, and these increased in extent when the S-layer protein (RsaA) expression level was elevated by addition of multiple rsaA copies. Finally, we combined high-resolution amino acid residue-specific Nanogold labeling and subtomogram averaging of CET volumes to improve our understanding of the correlation between the linear protein sequence and the structure at the 2-nm level of resolution that is presently available. The results support the view that the U-shaped RsaA monomer predicted from negative-stain tomography proceeds from the N terminus at one vertex, corresponding to the axis of 3-fold symmetry, to the C terminus at the opposite vertex, which forms the prominent 6-fold symmetry axis. Such information will help future efforts to analyze subunit interactions and guide selection of internal sites for display of heterologous protein segments.Surface layers (S layers) are the outermost cell wall component in many archaea and bacteria (6, 44). Most S layers are composed of a single protein or glycoprotein species that selforganizes into two-dimensional (2D) lattices of various sizes, usually with square or hexagonal symmetry (7,14,43). This geometrical arrangement is almost the only commonality among species, since sequence homology between S-layer proteins is low and functionality differs in many cases. In many archaea, the S layer is the only cell wall component, so it may have a role in shape determination. However, in bacteria such as Caulobacter crescentus, the role is more likely related to protection against a variety of predatorial assaults (8).One interest in understanding S layers comes from their potential applications in nanotechnology (46) and therapeutic applications, such as anti-HIV microbicide development (37) and cancer therapy (9). The concept is to display heterologous proteins from within the S-layer structure in order to create dense arrays of foreign insertions. Resolving the S-layer organization and structure at high resolution in cells as close to a native state as possible is crucial to understand or predict where proteins are displayed in the array, particularly when more than one foreign peptide is being displayed simultaneously.A significant limitation has been the dif...

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A charge coupled device camera with electron decelerator for intermediate voltage electron microscopy

Cited by 17 publications

References 27 publications

Direct observation of kinetic traps associated with structural transformations leading to multiple pathways of S-layer assembly

Direct observation of kinetic traps associated with structural transformations leading to multiple pathways of S-layer assembly

U(VI) Reduction by Diverse Outer Surface c -Type Cytochromes of Geobacter sulfurreducens

Analysis of the Intact Surface Layer of Caulobacter crescentus by Cryo-Electron Tomography

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