bluuebunny/crossref_metadata_2025_split huggingface dataset:
Characterization of JEOL 3000f TEM Equipped for Electron Holography
Authors: Schofield M A , Zhu Y 
Published: July 2020

Abstract
Quantitative off-axis electron holography in a transmission electron microscope (TEM) requires careful design of experiment specific to instrumental characteristics. For example, the spatial resolution desired for a particular holography experiment imposes requirements on the spacing of the interference fringes to be recorded. This fringe spacing depends upon the geometric configuration of the TEM/electron biprism system, which is experimentally fixed, but also upon the voltage applied to the biprism wire of the holography unit, which is experimentally adjustable. Hence, knowledge of the holographic interference fringe spacing as a function of applied voltage to the electron biprism is essential to the design of a specific holography experiment. Furthermore, additional instrumental parameters, such as the coherence and virtual size of the electron source, for example, affect the quality of recorded holograms through their effect on the contrast of the holographic fringes.
Structure of Membrane Proteins Revealed by Electron Crystallography
Authors: Mitsuoka K 
Published: July 2020

Abstract
Because membrane proteins are localized in a continuous lipid bilayer in the native environment, the situation of membrane proteins in the two-dimensional (2D) crystals is quite similar to the environment in vivo. Thus, electron crystallography using 2D crystals is one of the suitable techniques for structure determination of membrane proteins at atomic or near-atomic resolution. Here we describe the structures of the two membrane proteins, bacteriorhodopsin and aquaporin-1, which were solved by electron crystallography at 2.5 and 4.0 Å resolution, respectively.
Bacteriorhodopsin (bR) is a light-driven proton pump found in Halobacterium salinarium. The atomic model of the protein was first proposed by electron crystallography and we improved the resolution of the structure determination up to 3.0 Å by collecting 366 electron diffraction patterns and 129 images. The resulted map showed not only a bR molecule but also eight surrounding lipids in the asymmetric unit.
Isolation and Characterization of Connexin26 Gap Junctions from Transfected Hela Cells
Authors: Hand G , Nguyen V , Downing K , Mueller D , Engel A , Nicholson Bruce , Sosinsky G 
Published: July 2020

Abstract
Gap junctions are composed of a multigene family of proteins called the connexins with a similar sequence and folding topology. Connexons, half-channels made from a particular connexin, will dock and form functional channels with some, but not all, connexons made of other isoforms. This is surprising considering that the primary sequences of the docking domains are conserved and in some cases there are limited amino acid changes in the extracellular loops between compatible connexins. Therefore, some tertiary or quaternary interactions between the extracellular loops of the two docking hemichannels must contribute to the compatibility of a connexon assembled from one connexin for a connexon made from a different connexin. The 3D structures of gap junctions composed of only three isoforms have been investigated with only one case of a gap junction structure at better than 1 nm resolution [1]. The rat liver hemichannel structure is the only one that contains an exposed extracellular surface [2].
Cryo-EM 3D Reconstruction of Skeletal Muscle α-Actinin
Authors: Tang Jinghua , Taylor Dianne W , Taylor Kenneth A 
Published: July 2020

Abstract
α-Actinin is a member of the spectrin superfamily of actin crosslinking proteins. The molecule is an antiparallel homodimer with a polypeptide chain weight of 94-103 kDa. Each chain can be divided into three domains: the N-terminal 250 amino acids forms an actin binding domain, the central domain consisting of four spectrin-like, triple-helical repeats and the Cterminal which contains two EF hand motifs (Baron et al, 1987).
Two models have been proposed for the alignment of the triple helical repeats in the α-actinin structure, an aligned model (Baron et al., 1987) and a staggered model (Taylor and Taylor, 1993). In order to resolve the controversy, we proceeded with the cryo-EM 3D reconstruction from 2D crystal grown on a positively charged lipid monolayer. From rabbit erector spinae aactinin, we obtain better ordered 2D crystals from which we have calculated a 3D reconstruction to ∼15 Å resolution.
Vive La Différence! Mapping Macromolecular Complexes by Generalized Difference Imaging
Authors: Steven Alasdair C , Conway James F , Cheng Naiqian , Watts Norman R , Wingfield Paul T 
Published: July 2020

Abstract
Overview. Three-dimensional cryo-electron microscopy holds great potential for the investigation of macromolecular complexes, including viruses, the cytoskeleton, and the nanomachines that carry out many vital cellular functions. Such complexes often contain protein subunits of different kinds, with cumulative molecular weights running well into the megadalton range. To rationalize their functional assignments in structural terms, the first order of business is to map the locations of the component subunits of a given complex. A more detailed objective then becomes to understand the organization of their domains and the overall layout of the polypeptide chains. With current methodology, resolutions below 20Å are often achievable in cryo-EM, extending below 10 Å in favorable cases. Although resolutions of this order yield valuable morphological information, they do not usually suffice to identify individual subunits within a complex. Generalized difference imaging offers a solution to this problem of molecular mapping.
Three-Dimensional Structure of Isometric Capsids of Bacteriophage T4
Authors: Baker T S , Olson N H , Gingery M , Eiserling F A 
Published: July 2020

Abstract
Coliphage T4 is a large, double-stranded DNA virus (family Myoviridae) which infects Escherichia coli. It is the classic example of a contractile-tailed phage and a wealth of information on viral morphogenesis processes have been obtained on it. Extensive studies of T4 by negative-stain and metal-shadow electron microscopy have shown that mature virions, which contain over 40 different polypeptides, have a prolate capsid that encapsidates the genome (∼175 × 106Da), and several other structural components, including the collar with whiskers, neck, a contractile sheath, and a baseplate with fibers. A complete understanding of processes such as assembly of the viral capsids will require atomic-scale knowledge of the various protein interactions. Details of the process can be extrapolated from knowledge of the initial, transitional, and final states of a structure.
We have initiated an examination of T4 head morphogenesis by studying mutant, isometric capsids ('isometrics’) using cryo-electron microscopy and three-dimensional (3D) image reconstruction methods as applied to particles with icosahedral symmetry.
Conformational Changes In GROEL Induced by a Protein Substrate
Authors: Falke S , Fisher M , Gogol E 
Published: July 2020

Abstract
The GroEL/GroES chaperonin system of E. coli facilitates nucleotide dependent folding of select proteins. The structure of GroEL has been described by three-dimensional electron microscopy and at higher resolution by X-ray crystallography. The GroEL oligomer is a cylindrical tetradecamer composed of two heptameric rings of 57 kDa protein subunits, stacked back to back. Each subunit is comprised of two large domains, equatorial and apical, connected by a smaller intermediate domain. The intermediate “hinge” domain links the apical and equatorial domain and provides flexibility for en bloc rearrangement associated with nucleotide and GroES binding. Equatorial domains are responsible for interactions between the two heptamers and contain the ATPase activity of GroEL. Each ring of GroEL has a central cavity that is the binding site for denatured protein substrate. GroES and denatured substrate binding sites are located on the apical domains facing the central cavity.
An Integrated System for Transmission Electron Microscopy.
Authors: Carragher B , Kisseberth N , Kriegman D , Milligan RA , Potter CS , Pulokas J , Reilein A 
Published: July 2020

Abstract
Macromolecular microscopy is becoming an increasingly important tool for structural biology. The development of improved capabilities for three-dimensional electron microscopy is critical for optimal progress in emerging integrative research in molecular cell biology. These techniques currently suffer from several severe disadvantages related to the tremendous time and effort required to acquire and analyze the data.
For several years we have been developing software for automated and intelligent acquisition of transmission electron micrographs [1,2,3]. Our overall goal is to develop a system for rapid and routine structure determination of macromolecular assemblies from specimens preserved in vitreous ice. Ultimately we plan to develop an integrated system that can produce an electron density map of a structure within a few hours of inserting a specimen into the electron microscope. With this goal in mind it is essential that the images be acquired using a digital camera rather than film.
Cryo-Em Imaging of a Pseudotyped Adenovhtus with Ocular Cell Tropism
Authors: Stewart PL , Chiu CY , Von Seggern D , Nemerow GR 
Published: July 2020

Abstract
Human adenovirus (Ad) provides unique model system for studying the multistage events in viral entry. We are applying cryo-electron microscopy and single particle reconstruction methods to study the cell entry pathway of Ad as well as to examine the structure of reengineered Ad vectors. Ad cell entry is mediated by interactions with two different cellular receptors: the fiber receptor, CAR for most Ad serotypes or a 50 kDa receptor for a subset of serotypes including Ad37, for attachment; and the αvβ3 and αvβ5 integrins for internalization. We have previously reconstructed both Ad2 and Adl2 virus particles complexed with a soluble form of αvβ5 integrin. We learned during our comparative cryo-EM study of the Ad2/αvβ5 and Adl2/αvβ5 complexes that the length of the variable RGD loop in the penton base affects the relative height of the integrin density over the penton base.
An Interactive User Interface for Automated Acquisition of Transmission Electron Micrographs
Authors: Pulokas J , Kisseberth N , Potter CS , Carragher B 
Published: July 2020

Abstract
For several years we have been developing a software application, called Leginon, for the automated control and acquisition of images from a transmission electron microscope. The system has been developed around a Philips CM200 and a Gatan MSC CCD camera. One of the primary motivations in developing this software is to provide for a system that can acquire many hundreds of images under low dose conditions from a specimen embedded in vitreous ice. In order to set up and manage this system we have developed a number of simple interactive graphical tools that enable the user to design, oversee and manage protocols for controlling the microscope and collecting the images. Many of these simple tools have also proved generally useful as stand alone applications.
An Automated System for Maintaining Liquid Nitrogen Levels in the Gatan Cryostage.
Authors: Robinson SJ , Fried G , Pulokas J 
Published: July 2020

Abstract
For several years we have been developing a system for the automated control and acquisition of images from a transmission electron microscope [1,2,3]. The system has been developed around a Philips CM200 equipped with a Gatan cryostage and a Gatan MSC CCD camera. One of the primary motivations in developing this software is to provide for a system that can acquire many hundreds of images over several days of completely unattended operation.
The current implementation of our automated system allows data to be automatically collected from the TEM for more than 36 hours. There is no intervention required from the operator except for one critical exception, refilling the Gatan cryostage. The cryostage, which maintains the temperature of the vitreous ice specimens, must be refilled with liquid nitrogen (LN2) approximately every two hours.
Three-Dimensional Structure Of C3D From Low Temperature Scanning Transmission Electron Microscopy and X-Ray Crystallography
Authors: Martin DJ , Ottensmeyer FP 
Published: July 2020

Abstract
Macromolecular structure can be solved by x-ray crystallography to atomic resolution provided that the molecule can be crystallized, that the crystals diffract x-rays to high resolution, and that the phases of the diffracted x-rays can be determined. Though the resolution of single particle imaging by electron microscopy is lower than that of x-ray diffraction by crystals, electron microscopy can directly image a large molecular weight range of macromolecules in a non-crystalline environment, and provide the basis for the three-dimensional reconstruction of these structures. To investigate combining structural information from x-ray crystallography and electron microscopy for unknown structures, we have imaged a small protein of known structure (1), the 35 kDa human complement protein fragment C3d, in a scanning transmission electron microscope (STEM). The intention is to eventually combine the knowledge of electron densities and molecular boundaries from electron microscopy to assist in phase determination in x-ray crystallography.
Ultrastructural Localization of Antigens in the Mitotic Spindle of Budding Yeast Using High Presure Freezing and Freeze Substitution
Authors: Müller-Reichert Thomas , Ashford Anthony , Antony Claude , Hyman Anthony 
Published: July 2020

Abstract
The preservation of S. cerevisiae ultrastructure is not a trivial task, because the cell wall serves as a diffusion barrier during all steps of preparation. In addition, a high density of proteins in the cytoplasm and in the nucleus renders the visualization of cellular details difficult.
High pressure freezing in combination with freeze substitution has proven to be an advantageous technique for the preservation of the yeast mitotic spindle (1). However, the composition of the freeze substitution “cocktail” is crucial, when immunolabeling of antigens in the sample is intended. A high concentration of crosslinking agents might improve ultrastructural preservation of the sample, but will possibly destroy the antigenicity of the target proteins. In contrast, under conditions of high labeling efficiency, the ultrastructural detail may be lost. We were interested in a method, which gives a good ultrastructural preservation of the mitotic spindle and maintains the antigenicity of proteins associated with the spindle.
Cryosections for Immunocytochemistry
Authors: Webster Paul 
Published: July 2020

Abstract
Immunocytochemistry describes methods for visualization of specific intracellular molecules within cells. These methods use high affinity markers to specifically bind to target molecules in tissues or cells. For electron microscopy, the sites where these markers are bound are visualized using electron opaque probes. Successful immunocytochemistry depends upon immobilization of target molecules, retention of subcellular morphology, and upon total accessibility of affinity marker to the target molecules.
Accessibility to extracellular molecules is not usually a problem, but for intracellular molecules, labeling protocols must be able to preserve subcellular morphology and allow the affinity markers to gain access to their target molecules. Currently, the best way to gain access to the inside of cells for electron microscopy (TEM) is to cut thin sections through them. In this way, morphology is retained and affinity markers can be applied to the cut face of the cells.
The Utilization of Cryopreservation in Small Gold Surface Labeling
Authors: Ackerley CA , Tilups A , Kanazawa T , Becker LE 
Published: July 2020

Abstract
Cryopreservation has many advantages over routine chemical fixation. Two of these are the almost spontaneous arrest of biological activity and the retention of material that normally would be lost with chemical fixation and dehydration. In the current study 2 systems were evaluated. Hyaluronic acid (HA) was covalently bound to 1.4nm nanogold® and applied to astrocytoma cell cultures and the uptake monitored over time using both cryopreparative methods and conventional chemical fixation. The second system evaluated was the detection of liposomes containing CFTR covalently bound to 1.4nm gold in the airways of mice from a phenotype of an inbred congenic strain of CFTR-knockout mice that spontaneously develop lung disease.
In the HA uptake experiment, astrocytoma cell cultures were treated with HA gold for either 5, 10 or 20 minutes. At the appropriate time point, cells were either plunge frozen in liquid ethane or fixed with 2.5% glutaraldehyde in phosphate buffer.
Combining Cryo and Conventional Specimen Preparation Methods to Improve the Preservation of Cell Ultrastructure
Authors: McDonald Kent L 
Published: July 2020

Abstract
Poor specimen preparation techniques affect all subsequent EM imaging operations, whether simple descriptions or sophisticated computer reconstruction and analysis. Therefore, EM researchers continue to put effort into developing methods that will preserve the fine structure of the cell as close to the native state as possible. Much attention has been paid to the primary or initial fixation because this is probably the singlemost important step in EM processing. Evidence from a variety of sources. shows that cryofixation is more likely to give good preservation than conventional chemical fixation for most cell types. Among the choices for cryofixation, high pressure freezing is probably the best for most samples, even those that are small enough to be frozen by other means.
Dehydration is another key point in processing where high resolution structure can be lost or distorted, though it has received relatively little attention compared to primary fixation.
Electron Tomography of Frozen-Hydrated Samples
Authors: Marko M , Hsieh C-E , Rath BK , Mannella CA , McEwen BF 
Published: July 2020

Abstract
Cryo-electron tomography offers a means of obtaining the 3-D ultrastructure of specimens that have not been chemically fixed or stained. The technique is still under development, but it has already been applied to several biological specimens including prokaryotic cells, centrioles, sperm axonemes, mitochondria, spindle-pole bodies, lipid vesicles, isolated skeletal-muscle triad junctions, chromatin fragments, actin bundles, and macromolecules.
Electron tomography itself is a nearly mature technology. It is most often used with conventional plastic sections 100- 1000nm thick. The specimen is imaged in the TEM, using a tilt stage to record a series of projection images over a large angular range. Using image-processing techniques, the tilt images are back-projected to form a reconstructed volume of the specimen. The reconstruction is a 3-D array of pixels or volume elements (voxels), and can be sliced in any direction to form a series of 2-D images.
The Potential for Correlative and Multiple Labeling With Colloidal Particles of Differing Shapes
Authors: Albrecht RM , Meyer DA 
Published: July 2020

Abstract
Labeling specific tissues, cells, cell components, or individual molecular species is often critical to correlative microscopic studies. In the case of larger structures such as tissues or cells it is often possible to use fluoresecent labels or cytologic/cytochemical based stains which are observable via photon based imaging systems. Specific locations on cells and tissues identified by the labeling or staining procedure can be delineated and then can be viewed via electron or force based instrumentation. This is provided preparative methodologies can be designed which: 1. are compatible with observation of the particular sample by the various desired imaging modes and 2. do not compromise structure viewed at the higher levels of resolution provided by electron and force based imaging technologies. This approach is particularly valuable where simultaneous multiple labeling is required since color differences permit discrimination of differing fluorescent and/or chromatic stains.
Gold Cluster Crystals
Authors: Hainfeld JF , Powell RD , Furuya FR , Wall JS 
Published: July 2020

Abstract
Gold clusters are gold compounds with a core of gold atoms and organic groups covalently bound to the surface gold atoms. An example is undecagold, Au11(P(C6H5)3)7, whose structure was solved by x-ray crystallography using 3-dimensional crystals. These differ from colloidal gold, which are suspensions of metal particles, usually formed by metal ion reduction; although the particles may be approximately the same size, they vary due to the statistical process of formation. Gold clusters are compounds with a definite formula, and should all be perfectly identical. However, it is known that there is a family of stable gold cluster compounds, such as Au6, Au11, Au13, AU55, Au67, etc. In a given preparation of gold clusters, there is usually some mixture of these, thus leading to some size variation. Methods such as gel filtration column chromatography and ultrafiltration can be used to separate most of these species, so that relatively pure preparations may be achieved.
Sodium Dodecyl Sulfate Enhances Immunolabeling for Certain Antigens
Authors: Vandré D D , Robinson J M 
Published: July 2020

Abstract
Immunocytochemistry provides unique information concerning the spatial and temporal distribution of biomolecules in cells and tissues. While techniques associated with immunocytochemical labeling are well developed, there are always improvements that can be achieved. In this abstract, a modification to immunocytochemical methodology that increases the labeling efficiency for certain antigens is described. This modification includes incubation of the sample with sodium dodecyl sulfate (SDS) prior to application of the primary antibody.
Tubulin in microtubules of human neutrophils has been extremely difficult to analyze with antibodies to α-tubulin or β-tubulin using immunocytochemical methods. Using an immunofluorescence assay, we have analyzed several preparative procedures in order to improve the labeling efficiency of these microtubules using anti-tubulin antibodies. We find that treatment of chemically fixed neutrophils with the chaotropic agent SDS improves labeling efficiency for neutrophil microtubules in the immunofluorescence assay.
Free-Floating Cryostat Sections for Immunoelectron Microscopy: Bridging the Gap from Light to Electron Microscopy
Authors: Kan RK , Pleva CM , Backof D , Hamilton T , Petrali JP 
Published: July 2020

Abstract
Since skin basement membrane protein components are labile to conventional chemical fixation and since skin is not amenable to vibratome sectioning, frozen skin sections are routinely used for light microscopic immunohistochemical study of the skin basement membrane zone. However, inherent limitations of conventional frozen sections, including compromised morphology and a requirement for glass slidemounting, usually limit study to the light microscopic level. In the present study, we introduce the use of unfixed, free-floating cryostat sections to characterize immunolocalizations of selected basement membrane protein components at both the light and electron microscopy level. The new procedure employs free-floating cryostat sections that can be processed as routine tissue specimens and can be subjected to a variety of special staining procedures including immunohistochemistry. Especially useful is the ease of progressive processing of the same tissue specimen from light microscopy to electron microscopy.