Extended abstract of a paper presented at Microscopy and Microanalysis 2012 in Phoenix, Arizona, USA, July 29 – August 2, 2012.

Extended abstract of a paper presented at Microscopy and Microanalysis 2010 in Portland, Oregon, USA, August 1 – August 5, 2010.

Cryo-electron tomography of frozen-hydrated specimens holds considerable promise for high-resolution three-dimensional imaging of organelles and macromolecular complexes in their native cellular environment. While the technique has been successfully used with small, plunge-frozen cells and organelles, application to bulk mammalian tissue has proven to be difficult. We report progress with cryo-electron tomography of frozen-hydrated sections of rat liver prepared by high-pressure freezing and cryo-ultramicrotomy. Improvements include identification of suitable grids for mounting sections for tomography, reduction of surface artifacts on the sections, improved image quality by the use of energy filtering, and more rapid tissue excision using a biopsy needle. Tomographic reconstructions of frozen-hydrated liver sections reveal the native structure of such cellular components as mitochondria, endoplasmic reticulum, and ribosomes, without the selective attenuation or enhancement of ultrastructural details associated with the osmication and post-staining used with freeze-substitution.

Three-dimensional motif search is becoming increasingly important both in the search for molecular signatures within a tomographic reconstruction, at low resolution, and in the search for atomic structures within high-resolution cryo-EM maps of macromolecular complexes. The present work describes the implementation of a fast local correlation algorithm suitable for template matching in the SPIDER environment. Two examples are given, one in each of the areas of application: (i) within a 7.8Å single-particle reconstruction of the Escherichia coli ribosome, four proteins and one RNA structure were located with high accuracy; (ii) within a cryo-tomogram of sarcoplasmic reticulum vesicles, ryanodine receptors were located in positions that agreed with expert knowledge.

Journal Article Electron Tomographic Comparison of Frozen-Hydrated and Freeze-Substituted Sections of High-Pressure Frozen Rat-Liver Tissue Get access C-E Hsieh, C-E Hsieh Resource for Visualization of Biological Complexity, Wadsworth Center, Albany, NY 12201 Search for other works by this author on: Oxford Academic Google Scholar M Marko, M Marko Resource for Visualization of Biological Complexity, Wadsworth Center, Albany, NY 12201 Search for other works by this author on: Oxford Academic Google Scholar A Leith, A Leith Resource for Visualization of Biological Complexity, Wadsworth Center, Albany, NY 12201 Search for other works by this author on: Oxford Academic Google Scholar J Frank, J Frank Resource for Visualization of Biological Complexity, Wadsworth Center, Albany, NY 12201Howard Hughes Medical Institute, Wadsworth Center, Empire State Plaza, Albany, NY 12201 Search for other works by this author on: Oxford Academic Google Scholar CA Mannella CA Mannella Resource for Visualization of Biological Complexity, Wadsworth Center, Albany, NY 12201 Search for other works by this author on: Oxford Academic Google Scholar Microscopy and Microanalysis, Volume 9, Issue S02, 1 August 2003, Pages 1178–1179, https://doi.org/10.1017/S1431927603445893 Published: 24 July 2003

Cryo-electron microscopy (Cryo-EM) of single particles has developed into a widely used technique for determining the 3-dimensional structure of large molecules and molecular assemblies. The low signal-to-noise ratio of cryo-EM requires thousands of images of single molecules to be averaged together. The field has matured to the point where realization of high-resolution structures is limited primarily by computational constraints. These constraints are at the algorithmic level, as well as the control level, where dozens of complex procedures and thousands of mathematical operations are applied to the raw data. SPIDER is a highly modular and flexible software package for single particle reconstruction. A typical reconstruction project involves dozens of procedure files, which in turn draw on hundreds of available low-level operations. In the present system, it is virtually impossible to rerun the system from selected branching points in the process flow to determine the effects of various parameters values. A Reconstruction Engine (RE) is being developed as a high-level "shell" for controlling processes in the SPIDER software system. The RE allows backtracking, optimization of parameters, and automation of processing flow. The RE is implemented in a scripting language, Python, which provides an overall management capability at the global level of the project.

The aim of this study was to develop a model for the detection of individual cell adhesion molecules (CAMs) in the glycocalyx of spread human platelets using high-resolution cryo-field emission scanning electron microscopy (cryoFESEM). Three surface glycoprotein CAMs, P-selectin (CD62P), GPIba in the GPI-IX complex (CD42a/CD42b alpha,b beta), and the integrin GPIIbIIIa (CD41/CD61) in the human platelet were selected on the basis of their unique topographic shape. Spread human platelets were indirectly immunolabeled with 10-nm colloidal gold and then cryoimmobilized. After sublimation of water from the cryoimmobilized sample, partially freeze-dried platelets were coated unidirectionally with Pt, stabilized with carbon, and examined in an in-lens cryoFESEM using high-resolution backscattered electron imaging. CAMs were detected by indirect immunogold labeling and the length of each type of CAM was determined using analysis of differences in parallax as measured in the software program Sterecon. Our results demonstrate the efficacy of using high-resolution cryoFESEM to recognize and detect individual CAMs in the glycocalyx. Further advances in production of metal coatings with finer granularity, together with improvements in imaging (tilting and angle of stereo images), may provide better definition of the topography associated with glycosylation and formation of multimeric CAM complexes. (J Histochem Cytochem 49:809-819, 2001)

Background: This study addresses the general problem of dividing a density map of a nucleic-acid-protein complex obtained by cryo-electron microscopy (cryo-EM) or X-ray crystallography into its two components. When the resolution of the density map approaches similar to 3 Angstrom it is generally possible to interpret its shape (i.e., the envelope obtained for a standard choice of threshold) in terms of molecular structure, and assign protein and nucleic acid elements on the basis of their known sequences. The interpretation of low-resolution maps in terms of proteins and nucleic acid elements of known structure is of increasing importance in the study of large macromolecular complexes, but such analyses are difficult.Results: Here we show that it is possible to separate proteins from nucleic acids in a cryo-EM density map, even at 11.5 Angstrom resolution. This is achieved by analysing the (continuous-valued) densities using the difference in scattering density between protein and nucleic acids, the contiguity constraints that the image of any nucleic acid molecule must obey, and the knowledge of the molecular volumes of all proteins.Conclusions: The new method, when applied to an 11.5 Angstrom cryo-EM map of the Escherichia coli 70S ribosome, reproduces boundary assignments between rRNA and proteins made from higher-resolution X-ray maps of the ribosomal subunits with a high degree of accuracy. Plausible predictions for the positions of as yet unassigned proteins and RNA components are also possible. One of the conclusions derived from this separation is that 23S rRNA is solely responsible for the catalysis of peptide bond formation. Application of the separation method to any nucleoprotein complex appears feasible.

Cubic membranes occur in a variety of membrane-bound organelles in many cell types. By transmission electron microscopy (TEM) these membrane systems appear to consist of highly curved periodic surfaces that fit mathematical models analogous to those used to describe lipidic cubic phases. For the first time, a naturally occurring cubic membrane system has been reconstructed in three dimensions by electron microscopic tomography, and its periodicity directly characterized. Double-tilt tomographic reconstruction of mitochondria in the amoeba, Chaos carolinensis, confirms that their cristae (inner membrane infoldings) have the cubic structure suggested by modeling studies based on thin-section TEM images. Analysis of the membrane surfaces in the reconstruction reveals the connectivity of the internal compartments within the mitochondria. In the cubic regions, the matrix is highly condensed and confined to a continuous, small space between adjacent cristal membranes. The cristae form large, undulating cisternae that communicate with the peripheral (inner membrane) compartment through narrow tubular segments as seen in other types of mitochondria. The cubic periodicity of these mitochondrial membranes provides an ideal specimen for measuring geometrical distortions in biological electron tomography. It may also prove to be a useful model system for studies of the correlation of cristae–matrix organization with mitochondrial activity.

A software system for interactive manipulation of three-dimensional data has been developed, based on the Open Inventor tool kit. The primary use of this software system is in the segmentation of tomographic reconstructions of subcellular structures. To this end, the reconstruction is represented by volume rendering and displayed in stereo. A three-dimensional cursor with adjustable shape and size is used to define and isolate regions of interest inside the volume, based on the user's expert knowledge. Once isolated, the region of interest can be conveniently analyzed and displayed.

The SPIDER system has evolved into a comprehensive tool set for image processing, making use of modern graphics interfacing in the VMS and UNIX environment. SPIDER and WEB handle the complementary tasks of batch processing and visualization of the results. The emphasis of the SPIDER system remains in the area of single particle averaging and reconstruction, although a variety of other application areas have been added. Novel features are a suite of operations relating to the determination, modeling, and correction of the contrast transfer function and the availability of the entire documentation in hypertext format.

Transfer RNA (tRNA) molecules play a crucial role in protein biosynthesis in all organisms. Their interactions with ribosomes mediate the translation of genetic messages into polypeptides. Three tRNAs bound to the Escherichia coli 70S ribosome were visualized directly with cryoelectron microscopy and three-dimensional reconstruction. The detailed arrangement of A- and P-site tRNAs inferred from this study allows localization of the sites for anticodon interaction and peptide bond formation on the ribosome.