Electron spectroscopic imaging: parallel energy filtering and microanalysis in the fixed-beam electron microscope

A new imaging modality in electron microscopy uses energy filtration to produce micrographs with elastically scattered electrons or with electrons that have lost a specific, often characteristic amount of energy in interacting with the specimen. No deleterious effects on microscope performance are encountered. Instead, microanalysis of specimens is made possible with a spatial resolution of 3 to 5 A and a sensitivity of detection of 2 X 10(-21) g corresponding to about 50 atoms of phosphorus. Elements detected range from hydrogen (Z = 1) to uranium (Z = 92). Examples of elemental mapping show membrane structure, DNA within nucleosomes, and RNA within ribosomal particles.     

Methods for reconstructing density maps of "single" particles from cryoelectron micrographs to subnanometer resolution

Recently the resolution attainable in density maps calculated from cryo-electron micrographs of free-standing virus capsids has advanced to resolutions below 1 nm. This represents a significant milestone in that resolutions of this order potentially allow direct visualization of individual elements of protein secondary structure (i.e., alpha-helices), in addition to the shapes and connectivity of subdomains. We describe here a computational strategy for structural analyses at this level of detail: its principal innovation is a procedure for correcting the contrast transfer function of the electron microscope. Also important is the practice of combining data from pairs of differently defocused micrographs to improve the signal-to-noise ratio of the images, thereby allowing more precise determinations of the particles' orientations and origins and contributing to higher resolution reconstructions. These procedures proved instrumental in our analysis of the capsid of hepatitis B virus at 9-A resolution (Conway et al., 1997, Nature 386, 91-94). Finally, we discuss the prospects for achieving comparable resolutions for isolated macromolecular complexes with lower symmetry or no symmetry and for further extension of the resolution.     

Electron microscopic studies of negatively stained liposomes and membrane intrinsic polypeptide

High resolution electron microscopy has been applied to the study of the permeability properties of liposomes reconstituted with lens fibre protein. The experiments showed that liposomes formed in the presence of main intrinsic polypeptide (MIP) of bovine lens fibres appeared to become modified by changes in shape; also the bilayers seen in the electron micrographs of negatively stained specimens showed evidence of some structural changes or granularity. The morphological appearance of liposomes formed in the presence of MIP were interpreted as being consistent with the results of experiments performed by other methods.     

Applications of a bilateral denoising filter in biological electron microscopy

Due to the sensitivity of biological sample to the radiation damage, the low dose imaging conditions used for electron microscopy result in extremely noisy images. The processes of digitization, image alignment, and 3D reconstruction also introduce additional sources of noise in the final 3D structure. In this paper, we investigate the effectiveness of a bilateral denoising filter in various biological electron microscopy applications. In contrast to the conventional low pass filters, which inevitably smooth out both noise and structural features simultaneously, we found that bilateral filter holds a distinct advantage in being capable of effectively suppressing noise without blurring the high resolution details. In as much, we have applied this technique to individual micrographs, entire 3D reconstructions, segmented proteins, and tomographic reconstructions.     

Molecular structure determination by electron microscopy of unstained crystalline specimens.

The projected structures of two unstained periodic biological specimens, the purple membrane and catalase, have been determined by electron microscopy to resolutions of 7 Å and 9 Å, respectively. Glucose was used to facilitate their in vacuo preservation and extremely low electron doses were applied to avoid their destruction.The information on which the projections are based was extracted from defocussed bright-field micrographs and electron diffraction patterns. Fourier analysis of the micrograph data provided the phases of the Fourier components of the structures; measurement of the electron diffraction patterns provided the amplitudes.Large regions of the micrographs (3000 to 10,000 unit cells) were required for each analysis because of the inherently low image contrast (<1%) and the statistical noise due to the low electron dose.Our methods appear to be limited in resolution only by the performance of the microscope at the unusually low magnifications which were necessary. Resolutions close to 3 Å should ultimately be possible.     

The reproducible observation of unstained embedded cellular material in thin sections: visualisation of an integral membrane protein by a new mode of imaging for STEM

The contrast on micrographs obtained by conventional imaging in the conventional transmission electron microscope and in the scanning transmission electron microscope (STEM) (brightfield and darkfield) reflects mainly the variations of the mass-density and of the thickness of the specimen. The density differences in resin-embedded, unstained materials are too small to give enough contrast when compared to that produced by the surface perturbations introduced by sectioning. By darkfield imaging, therefore, this variable surface relief does not lead reproducibly to interpretable micrographs of high quality. Imaging by the ratio of elastically over inelastically scattered electrons in the STEM (Z-contrast) depends primarily on the atomic composition of the material. We present here the first experimental tests of theoretical predictions with thin sections; Z-contrast micrographs of septate junctions reveal the transmembrane proteins which are not visible in uranyl acetate stained sections viewed by conventional brightfield imaging.     

Sharpening high resolution information in single particle electron cryomicroscopy

Advances in single particle electron cryomicroscopy have made possible to elucidate routinely the structure of biological specimens at subnanometer resolution. At this resolution, secondary structure elements are discernable by their signature. However, identification and interpretation of high resolution structural features are hindered by the contrast loss caused by experimental and computational factors. This contrast loss is traditionally modeled by a Gaussian decay of structure factors with a temperature factor, or B-factor. Standard restoration procedures usually sharpen the experimental maps either by applying a Gaussian function with an inverse ad hoc B-factor, or according to the amplitude decay of a reference structure. EM-BFACTOR is a program that has been designed to widely facilitate the use of the novel method for objective B-factor determination and contrast restoration introduced by Rosenthal and Henderson [Rosenthal, P.B., Henderson, R., 2003. Optimal determination of particle orientation, absolute hand, and contrast loss in single-particle electron cryomicroscopy. J. Mol. Biol. 333, 721-745]. The program has been developed to interact with the most common packages for single particle electron cryomicroscopy. This sharpening method has been further investigated via EM-BFACTOR, concluding that it helps to unravel the high resolution molecular features concealed in experimental density maps, thereby making them better suited for interpretation. Therefore, the method may facilitate the analysis of experimental data in high resolution single particle electron cryomicroscopy.     

Structural analysis of membrane protein complexes by single particle electron microscopy

Single particle electron microscopy (EM) is an increasingly important tool for the structural analysis of macromolecular complexes. The main advantage of the technique over other methods is that it is not necessary to precede the analysis with the growth of crystals of the sample. This advantage is particularly important for membrane proteins and large protein complexes where generating crystals is often the main barrier to structure determination. Therefore, single particle EM can be employed with great utility in the study of large membrane protein complexes. Although the construction of atomic resolution models by single particle EM is possible in theory, currently the highest resolution maps are still limited to approximately 7-10A resolution and 15-30 A resolution is more typical. However, by combining single particle EM maps with high-resolution models of subunits or subcomplexes from X-ray crystallography and NMR spectroscopy it is possible to build up an atomic model of a macromolecular assembly. Image analysis procedures are almost identical for micrographs of soluble protein complexes and detergent solubilized membrane protein complexes. However, electron microscopists attempting to prepare specimens of a membrane protein complex for imaging may find that these complexes require different handling than soluble protein complexes. This paper seeks to explain how high-quality specimen grids of membrane protein complexes may be prepared to allow for the determination of their structure by EM and image analysis.     

Electron microscopic study of free and ribosome bound rRNAs from E. coli

The morphology of ribosomal subunits, nucleoprotein cores, and free rRNAs from Escherichia coli has been studied by two high resolution electron microscopic techniques. Conventional transmission electron micrographs showed unfolding of 30S and 50S ribosomal subunits with increasing depletion of proteins. With dedicated (0.3 nm resolution) scanning transmission electron microscopy we were able to visualize unstained freeze dried ribosomal subunits and free rRNAs without artifacts of staining and structural distortion by air drying. By this technique, we have also determined the mass of individual ribosomal particles and rRNA molecules. While the ribosomal subunits displayed their characteristic structural features and mass, free rRNAs appeared unfolded and polydisperse. These results provide direct evidence for distinct conformational differences between free rRNAs and native ribosomal subunits of E. coli.     

Focus gradient correction applied to tilt series image data used in electron tomography

The resolution in 3D reconstructions from tilt series is limited to the information below the first zero of the contrast transfer function unless the signal is corrected computationally. The restoration is usually based on the assumption of a linear space-invariant system and a linear relationship between object mass density and observed image contrast. The space-invariant model is no longer valid when applied to tilted micrographs because the defocus varies in a direction perpendicular to the tilt axis and with it the shape of the associated point spread function. In this paper, a method is presented for determining the defocus gradient in thin specimens such as sections and 2D crystals, and for restoration of the images subsequently used for 3D reconstruction. The alignment procedure for 3D reconstruction includes area matching and tilt geometry refinement. A map with limited resolution computed from uncorrected micrographs is compared to a volume computed from corrected micrographs with extended resolution.     

Fast rotational matching of single-particle images

The presence of noise and absence of contrast in electron micrographs lead to a reduced resolution of the final 3D reconstruction, due to the inherent limitations of single-particle image alignment. The fast rotational matching (FRM) algorithm was introduced recently for an accurate alignment of 2D images under such challenging conditions. Here, we implemented this algorithm for the first time in a standard 3D reconstruction package used in electron microscopy. This allowed us to carry out exhaustive tests of the robustness and reliability in iterative orientation determination, classification, and 3D reconstruction on simulated and experimental image data. A classification test on GroEL chaperonin images demonstrates that FRM assigns up to 13% more images to their correct reference orientation, compared to the classical self-correlation function method. Moreover, at sub-nanometer resolution, GroEL and rice dwarf virus reconstructions exhibit a remarkable resolution gain of 10-20% that is attributed to the novel image alignment kernel.     

A binary segmentation approach for boxing ribosome particles in cryo EM micrographs

Three-dimensional reconstruction of ribosome particles from electron micrographs requires selection of many single-particle images. Roughly 100,000 particles are required to achieve approximately 10 A resolution. Manual selection of particles, by visual observation of the micrographs on a computer screen, is recognized as a bottleneck in automated single-particle reconstruction. This paper describes an efficient approach for automated boxing of ribosome particles in micrographs. Use of a fast, anisotropic non-linear reaction-diffusion method to pre-process micrographs and rank-leveling to enhance the contrast between particles and the background, followed by binary and morphological segmentation constitute the core of this technique. Modifying the shape of the particles to facilitate segmentation of individual particles within clusters and boxing the isolated particles is successfully attempted. Tests on a limited number of micrographs have shown that over 80% success is achieved in automatic particle picking.     

Advances in Structural Biology and the Application to Biological Filament Systems

Structural biology has experienced several transformative technological advances in recent years. These include: development of extremely bright X‐ray sources (microfocus synchrotron beamlines and free electron lasers) and the use of electrons to extend protein crystallography to ever decreasing crystal sizes; and an increase in the resolution attainable by cryo‐electron microscopy. Here we discuss the use of these techniques in general terms and highlight their application for biological filament systems, an area that is severely underrepresented in atomic resolution structures. We assemble a model of a capped tropomyosin‐actin minifilament to demonstrate the utility of combining structures determined by different techniques. Finally, we survey the methods that attempt to transform high resolution structural biology into more physiological environments, such as the cell. Together these techniques promise a compelling decade for structural biology and, more importantly, they will provide exciting discoveries in understanding the designs and purposes of biological machines.     

Glutaraldehyde: Role in electron microscopy

In spite of the inherent limitations of chemical fixation, glutaraldehyde is unsurpassed in its ability to preserve cell ultrastructure. This achievement is due to the introduction of irreversible intra-and intermolecular cross-links into cellular proteins by the dialdehyde. Glutaraldehyde is very effective in stabilizing surface as well as intracellular structures for conventional scanning and transmission electron microscopy and high voltage electron microscopy. Even in immunocytochemical and autoradiographical studies, glutaradehyde plays a dominant role. Furthermore, prior to freeze-substitution, freeze-drying and freeze-fracturing, specimens often are stabilized with this dialdehyde. Glutaraldehyde efficiency can be increased by adding appropriate cross-linking and rapidly penetrating reagents as well as contrast enhancing reagents to this dialdehyde. Improved preservation and staining, for example, of ionic sites, soluble inorganic phosphate, lipids, biogenic amines, actin filaments, spermatozoa and phage-infected bacteria can be accomplished by adding polyethyleneimine, lead acetate, malachite green, potassium dichromate, tannic acid, trinitro compounds and uranyl acetate, respectively, to glutaraldehyde. Other refinements include the use of low concentrations of glutaraldehyde, short durations of cross-linking, minimum radiation exposure, and low temperature electron microscopy. The usefulness of glutaraldehyde in high resolution electron microscopy is limited because chemical fixation inevitably causes chemical and structural alterations in the specimen. However, fixation with glutaraldehyde or its mixture with formaldehyde has served immeasurably the progress in the understanding of cell ultrastructure and function. Preservation of specimens with glutaraldehyde for electron microscopy is expected to continue. Therefore, attempts must continue to be made to interpret the dynamics of the living cell from the static electron micrographs.     

Effects of radiation damage with 400-kV electrons on frozen, hydrated actin bundles.

Electron images can be used to provide amplitudes and phases for the structural determination of biological specimens. Radiation damage limits the amount of structural information retrievable by computer processing. A 400-kV electron microscope was used to investigate radiation damage effects on frozen, hydrated actin bundles kept at -168 degrees C. The quality of phases within and among images in a damage series was evaluated quantitatively out to 16 A resolution. It was found that the phases of structure factors with good signal-to-noise ratio (IQ less than or equal to 4) can be reliably retrieved from images taken at a cumulative dose of at least 25 electrons/A2.     

Glycolipid storage material in Fabry's disease: a study by electron microscopy, freeze-fracture, and digital image analysis

The glycolipid storage material in Fabry's disease was studied by electron microscopy of thin-sectioned (TS) and freeze-fractured (FF) specimens. In the kidney all deposits were found to be located in lysosomes, arranged as lamellar stacks. Deposits in the heart consisted of intracytoplasmic concentric whirls or folded lamellar structures. High resolution TS micrographs disclosed various defects in the lamellar structure. For stabilization, such defects require additional amphiphilic, surface-active molecules. These molecules could interact with other cellular constituents. The lamellar periodicity of the deposits in FF specimens was determined by reconstruction of the three-dimensional fracture face by digital image analysis. Homogeneous multilamellar deposits exhibited a periodicity of 14-15 nm, contrasting with the conventional estimates of 4-5 nm on TS micrographs. This difference is explained by better preservation of the physiologic hydrated state in FF specimens, with 1 vol of lipids binding 2 vol of water. Inhomogeneous structures with an even higher state of hydration included water lenses between the sheets. The strong hydration obviously contributes to the enlargement of the intracellular glycolipid deposits.     

The ups and downs of beam damage,contrast and noise in biological electron microscopy

A personal account of the early problems associated with contrast in images obtained by electron microscopy from biological specimens is presented, together with the effects of electron beam damage. The author's experiences with different types of electron microscope as well as problems of contrast enhancement is described. A short account is given of the physical effects occuring during specimen preparation and their relation to structural preservation when attempting to achieve atomic resolution. Recent developments in biological electron microscopy are also discussed with a view to future trends.     

Structural basis and kinetics of DsbD-dependent cytochrome c maturation

DsbD from Escherichia coli transports two electrons from cytoplasmic thioredoxin to the periplasmic substrate proteins DsbC, DsbG and CcmG. DsbD consists of an N-terminal periplasmic domain (nDsbD), a C-terminal periplasmic domain, and a central transmembrane domain. Each domain possesses two cysteines required for electron transport. Herein, we demonstrate fast (3.9 x 10(5) M(-1)s(-1)) and direct disulfide exchange between nDsbD and CcmG, a highly specific disulfide reductase essential for cytochrome c maturation. We determined the crystal structure of the disulfide-linked complex between nDsbD and the soluble part of CcmG at 1.94 A resolution. In contrast to the other two known complexes of nDsbD with target proteins, the N-terminal segment of nDsbD contributes to specific recognition of CcmG. This and other features, like the possibility of using an additional interaction surface, constitute the structural basis for the adaptability of nDsbD to different protein substrates.     

Difference imaging of adenovirus: bridging the resolution gap between X-ray crystallography and electron microscopy.

While X-ray crystallography provides atomic resolution structures of proteins and small viruses, electron microscopy provides complementary structural information on the organization of larger assemblies at lower resolution. A novel combination of these two techniques has bridged this resolution gap and revealed the various structural components forming the capsid of human type 2 adenovirus. An image reconstruction of the intact virus, derived from cryo-electron micrographs, was deconvolved with an approximate contrast transfer function to mitigate microscope distortions. A model capsid was calculated from 240 copies of the crystallographic structure of the major capsid protein and filtered to the correct resolution. Subtraction of the calculated capsid from the corrected reconstruction gave a three-dimensional difference map revealing the minor proteins that stabilize the virion. Elongated density penetrating the hexon capsid at the facet edges was ascribed to polypeptide IIIa, a component required for virion assembly. Density on the inner surface of the capsid, connecting the ring of peripentonal hexons, was assigned as polypeptide VI, a component that binds DNA. Identification of the regions of hexon that contact the penton base suggests a structural mechanism for previously proposed events during cell entry.     

An automatic particle pickup method using a neural network applicable to low-contrast electron micrographs

Three-dimensional reconstruction from electron micrographs requires the selection of many single-particle projection images; more than 10 000 are generally required to obtain 5- to 10-A structural resolution. Consequently, various automatic detection algorithms have been developed and successfully applied to large symmetric protein complexes. This paper presents a new automated particle recognition and pickup procedure based on the three-layer neural network that has a large application range than other automated procedures. Its use for both faint and noisy electron micrographs is demonstrated. The method requires only 200 selected particles as learning data and is able to detect images of proteins as small as 200 kDa.