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Electron tomography allows for the determination of the three-dimensional structures of cells and tissues at resolutions significantly higher than that which is possible with optical microscopy. Electron tomograms contain, in principle, vast amounts of information on the locations and architectures of large numbers of subcellular assemblies and organelles. The development of reliable quantitative approaches for the analysis of features in tomograms is an important problem, and a challenging prospect due to the low signal-to-noise ratios that are inherent to biological electron microscopic images. This is, in part, a consequence of the tremendous complexity of biological specimens. We report on a new method for the automated segmentation of HIV particles and selected cellular compartments in electron tomograms recorded from fixed, plastic-embedded sections derived from HIV-infected human macrophages. Individual features in the tomogram are segmented using a novel robust algorithm that finds their boundaries as global minimal surfaces in a metric space defined by image features. The optimization is carried out in a transformed spherical domain with the center an interior point of the particle of interest, providing a proper setting for the fast and accurate minimization of the segmentation energy. This method provides tools for the semi-automated detection and statistical evaluation of HIV particles at different stages of assembly in the cells and presents opportunities for correlation with biochemical markers of HIV infection. The segmentation algorithm developed here forms the basis of the automated analysis of electron tomograms and will be especially useful given the rapid increases in the rate of data acquisition. It could also enable studies of much larger data sets, such as those which might be obtained from the tomographic analysis of HIV-infected cells from studies of large populations.

Electron tomography allows determination of the three-dimensional structures of cells and tissues at resolutions significantly higher than is possible with optical microscopy. Electron tomograms contain, in principle, vast amounts of information on the locations and architectures of large numbers of subcellular assemblies and organelles. The development of reliable quantitative approaches for interpretation of features in tomograms, is an important problem, but is a challenging prospect because of the low signal-to-noise ratios that are inherent to biological electron microscopic images. As a first step in this direction, we report methods for the automated statistical analysis of HIV particles and selected cellular compartments in electron tomograms recorded from fixed, plastic-embedded sections derived from HIV-infected human macrophages. Individual features in the tomogram are segmented using a novel, robust algorithm that finds their boundaries as global minimal surfaces in a metric space defined by image features. Our expectation is that such methods will provide tools for semi-automated detection and statistical evaluation of HIV particles at different stages of assembly in the cells, and present opportunities for correlation with biochemical markers of HIV infection.

We report a method for semi-automated segmentation of extended features such as filamentous structures in electron tomograms. We present an application of this method for the automatic propagation of segmented neuro-filaments present along the axis of an axonal section of fixed, plastic-embedded mouse brain tissue imaged by three-dimensional electron microscopy. The algorithm uses the manually segmented features in a given slice to progressively extend the segmentation to successive slices based on knowledge of the centroids, shapes, and intensities of filaments in the previous slice. An iterative procedure allows for refinement of the most probable locations of the filaments in each slice. Evaluation of the performance of the propagation algorithm by visual inspection suggests that this approach can be used successfully for the rapid quantitative and statistical analysis of filamentous structures in cellular tomograms.

We have recently reported electron tomographic studies of sections obtained from chemically fixed E. coli cells overproducing the 60-kDa chemotaxis receptor Tsr. Membrane extracts from these cells prepared in the presence of Tween-80 display hexagonally close-packed microcrystalline assemblies of Tsr, with a repeating unit large enough to accommodate six Tsr molecules arranged as trimers of receptor dimers. Here, we report the direct visualization of the Tsr receptor clusters in (i) vitrified cell suspensions of cells overproducing Tsr, prepared by rapid plunge-freezing, and (ii) frozen-hydrated sections obtained from cells frozen under high pressure. The frozen-hydrated sections were generated by sectioning at -150 degrees C using a diamond knife with a 25 degrees knife angle, with nominal thicknesses ranging from 20 to 60 nm. There is excellent correspondence between the spatial arrangement of receptors in thin frozen-hydrated sections and the arrangements found in negatively stained membrane extracts and plunge-frozen cells, highlighting the potential of using frozen-hydrated sections for the study of macromolecular assemblies within cells under near-native conditions.

Membrane proteins that belong to the major facilitator superfamily (MFS) are found in organisms across the evolutionary spectrum and mediate the transport of a variety of substrates ranging from small metabolites to neurotransmitters. The oxalate transporter (OxlT) is a representative MFS protein, and exchanges formate for oxalate across the cytoplasmic membrane of the organism Oxalobacter formigenes. Here, we present a structural model for the protein conformational changes that occur during oxalate transport by combining a three-dimensional map of the oxalate-bound, “closed” state of OxlT at 6.5Å determined by cryo-electron microscopy with a model of the “open” state of OxlT based on the atomic structures of the related transporters, glycerol-3-phosphate transporter (GlpT) and lactose permease (LacY). We demonstrate that the principal structural change associated with substrate transport is a concerted rocking movement of the two structurally similar halves of the protein relative to each other. Our structural model places two positively charged residues, Arg-272 and Lys-355 in the central cavity, suggesting that electrostatic interactions between these residues and the oxalate anion is a key step in generating the conformational change between the open and closed states of the transporter.

Electron tomography is a powerful method for determining the three-dimensional structures of large macromolecular assemblies, such as cells, organelles, and multiprotein complexes, when crystallographic averaging methods are not applicable. Here we used electron tomographic imaging to determine the molecular architecture of Escherichia coli cells engineered to overproduce the bacterial chemotaxis receptor Tsr. Tomograms constructed from fixed, cryosectioned cells revealed that overproduction of Tsr led to formation of an extended internal membrane network composed of stacks and extended tubular structures. We present an interpretation of the tomogram in terms of the packing arrangement of Tsr using constraints derived from previous X-ray and electron-crystallographic studies of receptor clusters. Our results imply that the interaction between the cytoplasmic ends of Tsr is likely to stabilize the presence of the membrane networks in cells overproducing Tsr. We propose that membrane invaginations that are potentially capable of supporting axial interactions between receptor clusters in apposing membranes could also be present in wild-type E. coli and that such receptor aggregates could play an important role in signal transduction during bacterial chemotaxis.

▪ Abstract Emerging methods in cryo-electron microscopy allow determination of the three-dimensional architectures of objects ranging in size from small proteins to large eukaryotic cells, spanning a size range of more than 12 orders of magnitude. Advances in determining structures by “single particle” microscopy and by “electron tomography” provide exciting opportunities to describe the structures of subcellular assemblies that are either too large or too heterogeneous to be investigated by conventional crystallographic methods. Here, we review selected aspects of progress in structure determination by cryo-electron microscopy at molecular resolution, with a particular emphasis on topics at the interface of single particle and tomographic approaches. The rapid pace of development in this field suggests that comprehensive descriptions of the structures of whole cells and organelles in terms of the spatial arrangements of their molecular components may soon become routine.

Bacteriorhodopsin is a protein found in cell membranes of the organism H. salinarum, where it functions as an efficient light-driven proton pump. Because bacteriorhodopsin is one of the simplest ion pumps known in biology, it has been the subject of intensive investigations over the last three decades, using methods spanning the range from femtosecond spectroscopy and crystallography to biochemistry and molecular biology. Here, we focus on the structural basis for the function of this protein, with primary emphasis on the contributions of electron microscopy and crystallography towards unravelling the mechanism of vectorial proton pumping.

For over three decades, bacteriorhodopsin has served as a paradigm for the study of the mechanisms underlying ion pumping across biological membranes. It is perhaps among the simplest known ion pumps, which functions by converting light energy into an electrochemical gradient by pumping protons out of the cytoplasm. The combination of spectroscopic, biochemical and crystallographic studies on bacteriorhodopsin provides a unique opportunity to dissect the principal elements underlying the mechanism of transmembrane proton transport. Here, we provide a brief review of recent developments related to the determination of the structural changes during proton transport using crystallographic approaches. Taken together with previous spectroscopic and biochemical investigations, these studies allow the description of a detailed molecular mechanism of the main steps in vectorial proton transport by bacteriorhodopsin.

The major facilitator superfamily (MFS) represents one of the largest classes of evolutionarily related membrane transporter proteins. Here we present the three-dimensional structure at 6.5 A resolution of a bacterial member of this superfamily, OxlT. The structure, derived from an electron crystallographic analysis of two-dimensional crystals, reveals that the 12 helices in the OxlT molecule are arranged around a central cavity, which is widest at the center of the membrane. The helices divide naturally into three groups: a peripheral set comprising helices 3, 6, 9 and 12; a second set comprising helices 2, 5, 8 and 11 that faces the central substrate transport pathway across most of the length of the membrane; and a third set comprising helices 1, 4, 7 and 10 that participate in the pathway either on the cytoplasmic side (4 and 10) or on the periplasmic side (1 and 7). Overall, the architecture of the protein is remarkably symmetric, providing a compelling molecular explanation for the ability of such transporters to carry out bi-directional substrate transport.

Application of emerging methods in single particle cryo-electron microscopy (cryo-EM) has led to the determination of the structures of a variety of macromolecular complexes at sub-nanometer resolutions. The overwhelming majority of these are large complexes, mostly with high symmetry. Extending cryo-EM methods to routinely determine the structures of small macromolecular complexes with sizes of < 500 kDa to sub-nanometer resolution still remains an exciting frontier in modern structural biology. Working with small protein complexes has certain inherent challenges such as conformational flexibility and beam induced movement during exposure to electrons which can significantly reduce resolution. Further, during image processing, errors can be introduced from model-bias, over-refinement, and inaccuracy in orientation determination. Although these errors are problematic for all reconstructions, they are particularly detrimental for structural analysis of complexes that are small and have either low or no internal symmetry. We show a step-wise systematic workflow for structure determination by cryo-EM at ∼ 6 A resolution of s-galactosidase, a 464 kDa protein, whose structure has previously been determined at atomic resolution by X-ray crystallography. Our approach employs a number of recent developments in cryo-EM technology that include the use of direct electron detectors, the ability to fractionate the dose to use only those portions of the exposure that are recorded before there is significant radiation damage, and the use of image processing procedures that can track and correct for the movement of single molecules during the course of the exposure.

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

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

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

Extended abstract of a paper presented at Microscopy and Microanalysis 2008 in Albuquerque, New Mexico, USA, August 3 – August 7, 2008

Extended abstract of a paper presented at Microscopy and Microanalysis 2006 in Chicago, Illinois, USA, July 30 – August 3, 2005

Extended abstract of a paper presented at Microscopy and Microanalysis 2006 in Chicago, Illinois, USA, July 30 – August 3, 2006

A series of mini-antibodies (monovalent and bivalent Fabs) targeting the conserved internal trimeric coiled-coil of the N-heptad repeat (N-HR) of HIV-1 gp41 has been previously constructed and reported. Crystal structures of two closely related monovalent Fabs, one (Fab 8066) broadly neutralizing across a wide panel of HIV-1 subtype B and C viruses, and the other (Fab 8062) non-neutralizing, representing the extremes of this series, were previously solved as complexes with 5-Helix, a gp41 pre-hairpin intermediate mimetic. Binding of these Fabs to covalently stabilized chimeric trimers of N-peptides of HIV-1 gp41 (named (CCIZN36)3 or 3-H) has now been investigated using X-ray crystallography, cryo-electron microscopy, and a variety of biophysical methods. Crystal structures of the complexes between 3-H and Fab 8066 and Fab 8062 were determined at 2.8 and 3.0 Å resolution, respectively. Although the structures of the complexes with the neutralizing Fab 8066 and its non-neutralizing counterpart Fab 8062 were generally similar, small differences between them could be correlated with the biological properties of these antibodies. The conformations of the corresponding CDRs of each antibody in the complexes with 3-H and 5-Helix are very similar. The adaptation to a different target upon complex formation is predominantly achieved by changes in the structure of the trimer of N-HR helices, as well as by adjustment of the orientation of the Fab molecule relative to the N-HR in the complex, via rigid-body movement. The structural data presented here indicate that binding of three Fabs 8062 with high affinity requires more significant changes in the structure of the N-HR trimer compared to binding of Fab 8066. A comparative analysis of the structures of Fabs complexed to different gp41 intermediate mimetics allows further evaluation of biological relevance for generation of neutralizing antibodies, as well as provides novel structural insights into immunogen design.

Emerging methods in 3-D biological electron microscopy provide powerful tools and great promise to bridge a critical gap in imaging in the biomedical size spectrum. This comprises a size range of considerable interest that includes cellular protein machines, giant protein and nucleic acid assemblies, small subcellular organelles and bacteria. These objects are generally too large and/or too heterogeneous to be investigated by high resolution X-ray and NMR methods; yet the level of detail afforded by conventional light and electron microscopy is often not adequate to describe their structures at resolutions high enough to be useful in understanding the chemical basis of biological function. The long-term mission of our research program is to obtain an integrated molecular understanding of cellular architecture by combining novel technologies for 3-D biological imaging with advanced methods for image segmentation and computational analysis. I will review our recent progress in imaging and modeling dynamic biological systems, with particular emphasis on applications to signal transduction, HIV/AIDS and cancer. Lab website: http://electron.nci.nih.gov Citation Format: Sriram Subramaniam. 3-D electron microscopy in cellular and molecular imaging: Applications to cancer. [abstract]. In: Proceedings of the Twelfth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2013 Oct 27-30; National Harbor, MD. Philadelphia (PA): AACR; Can Prev Res 2013;6(11 Suppl): Abstract nr CN08-03.

Glutamate receptor ion channels are membrane proteins that mediate excitatory synaptic transmission in the central nervous system of vertebrates. Insight into the molecular mechanisms underlying their gating is limited by lack of structural information for receptors trapped in different conformational states. Here, we report the use of single particle cryo-electron tomography to determine the structures, at ∼ 25 A resolution, of full-length GluK2 kainate receptors trapped in resting and desensitized states. The resting state structure, stabilized by the competitive antagonist LY466195, closely resembles the crystal structure of the AMPA receptor GluA2, with well-resolved proximal and distal subunits exhibiting cross over between the amino terminal (ATD) and a 2-fold symmetric ligand binding domain (LBD) dimer of dimers assembly. In the desensitized state, the LBD regions undergo major rearrangements resulting in a remarkable separation of the four subunits by ∼ 30 A. From fits of the ATD and LBD domains into the density map of the desensitized state, we have derived a structural model for the differences in quaternary conformation between resting and desensitized states of glutamate receptor ion channels.View Large Image | View Hi-Res Image | Download PowerPoint Slide

The HIV envelope glycoprotein (Env) binds to CD4 and a chemokine co-receptor to mediate fusion of viral and target-cell membranes required for infection. We have previously shown that the unbound Env is a mushroom-shaped trimer with a stem composed of three membrane-anchored gp41 subunits capped by three gp120 subunits. We also showed that binding to CD4 and the co-receptor site binding Fab 17b leads to a significant outward movement of gp120 and to a rearrangement of density in gp41, resulting in an “open” conformation. Soluble constructs bearing the complete ectodomain of Env (gp140) display conformations that closely resemble the structure of native unliganded trimeric Env, and importantly, show identical ligand-induced transitions. Here we show that a key structural signature of the open Env conformation is a three-helix motif composed of α-helical segments derived from highly conserved, non-glycosylated N-terminal regions of the gp41 trimer. The three N-terminal helices in this novel, pre-fusion intermediate are much less compactly packed than in the post-fusion, six-helix bundle state. These findings suggest a new structural template for designing immunogens that can elicit antibodies targeting HIV at a vulnerable, pre-entry stage.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Extended abstract of a paper presented at Microscopy and Microanalysis 2004 in Savannah, Georgia, USA, August 1–5, 2004.

Structural changes are central to the mechanism of light-driven proton transport by bacteriorhodopsin, a seven-helix membrane protein. The main intermediate formed upon light absorption is M, which occurs between the proton release and uptake steps of the photocycle. To investigate the structure of the M intermediate, we have carried out electron diffraction studies with two-dimensional crystals of wild-type bacteriorhodopsin and the Asp96-->Gly mutant. The M intermediate was trapped by rapidly freezing the crystals in liquid ethane following illumination with a xenon flash lamp at 5 and 25 degrees C. Here, we present 3.5 A resolution Fourier projection maps of the differences between the M intermediate and the ground state of bacteriorhodopsin. The most prominent structural changes are observed in the vicinity of helices F and G and are localized to the cytoplasmic half of the membrane.