RESUMEN
Translocation of nuclear-encoded preproteins across the outer membrane of mitochondria is mediated by the multicomponent transmembrane TOM complex. We have isolated the TOM core complex of Neurospora crassa by removing the receptors Tom70 and Tom20 from the isolated TOM holo complex by treatment with the detergent dodecyl maltoside. It consists of Tom40, Tom22, and the small Tom components, Tom6 and Tom7. This core complex was also purified directly from mitochondria after solubilization with dodecyl maltoside. The TOM core complex has the characteristics of the general insertion pore; it contains high-conductance channels and binds preprotein in a targeting sequence-dependent manner. It forms a double ring structure that, in contrast to the holo complex, lacks the third density seen in the latter particles. Three-dimensional reconstruction by electron tomography exhibits two open pores traversing the complex with a diameter of approximately 2.1 nm and a height of approximately 7 nm. Tom40 is the key structural element of the TOM core complex.
Asunto(s)
Proteínas Portadoras/química , Membranas Intracelulares/enzimología , Proteínas de la Membrana/química , Mitocondrias/enzimología , Proteínas Portadoras/aislamiento & purificación , Proteínas Portadoras/metabolismo , Proteínas Portadoras/ultraestructura , Proteínas Fúngicas/química , Proteínas Fúngicas/aislamiento & purificación , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/ultraestructura , Membranas Intracelulares/metabolismo , Membranas Intracelulares/fisiología , Membranas Intracelulares/ultraestructura , Canales Iónicos/química , Canales Iónicos/metabolismo , Canales Iónicos/fisiología , Canales Iónicos/ultraestructura , Proteínas de la Membrana/aislamiento & purificación , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/ultraestructura , Mitocondrias/metabolismo , Mitocondrias/fisiología , Mitocondrias/ultraestructura , Neurospora crassa/enzimología , Neurospora crassa/metabolismo , Neurospora crassa/fisiología , Neurospora crassa/ultraestructura , Unión Proteica , Precursores de Proteínas/metabolismo , Precursores de Proteínas/ultraestructuraRESUMEN
Single cystals of proteasomes from the archaebacterium Thermoplasma acidophilum were obtained using the hanging-drop vapor diffusion method. The crystals diffract to better than 3.0 A and belong to the orthorhombic space group P2(1)2(1)2(1) with unit cell dimensions a = 308.9 A, b = 208.8 A and c = 116.9 A. There is one molecular complex in the asymmetric unit. Two potentially useful heavy-atom derivatives have been obtained. The self-rotation function of the native Patterson map shows local sevenfold symmetry, consistent with the low-resolution structure obtained by electron microscopic techniques. The unit cell dimensions and crystal symmetry together with the shape and size of the proteasome suggest a packing arrangement of proteasome molecules in the unit cell, with their cylinder axis nearly parallel to the crystallographic a-axis.
Asunto(s)
Cisteína Endopeptidasas/química , Complejos Multienzimáticos/química , Conformación Proteica , Thermoplasma/enzimología , Cristalización , Cristalografía por Rayos X/métodos , Cisteína Endopeptidasas/aislamiento & purificación , Complejos Multienzimáticos/aislamiento & purificación , Complejo de la Endopetidasa ProteasomalRESUMEN
The photosystem 1 reaction center complex from the thermophilic cyanobacterium Synechococcus sp. was isolated by Triton X-100 solubilization and fractional precipitation with polyethylene glycol. As shown by gel electrophoresis, the isolated complex was composed of the 83 kDa subunits A and B, and at least six other subunits with molecular mass below 20 kDa. Electron transfer from the primary electron donor P700 to the FA/FB centers was demonstrated by flash-induced absorption change of the isolated complex, while electron paramagnetic resonance (EPR) spectroscopy showed that the complex contained a full set of Fe-S clusters. Isolated complexes were reconstituted into two-dimensional crystals in the presence of phospholipids and different cations. The crystals were found to be active by flash-induced separation and EPR spectroscopy. Electron microscopy and digital image processing of negatively stained and frozen-hydrated specimens revealed orthorhombic crystals with unit cell dimensions a = 138 A, b = 145 A and p12(1) symmetry. A three-dimensional map was calculated for negatively stained crystals to 19 A resolution, whereas the projection map of frozen-hydrated crystals exhibited 8 A resolution.
Asunto(s)
Cianobacterias/química , Proteínas del Complejo del Centro de Reacción Fotosintética/química , Proteínas del Complejo del Centro de Reacción Fotosintética/ultraestructura , Cristalización , Cristalografía por Rayos X , Espectroscopía de Resonancia por Spin del Electrón , Congelación , Procesamiento de Imagen Asistido por Computador , Membrana Dobles de Lípidos , Metabolismo de los Lípidos , Lípidos/química , Microscopía Electrónica/métodos , Modelos Químicos , Modelos Moleculares , Proteínas del Complejo del Centro de Reacción Fotosintética/metabolismo , SolubilidadRESUMEN
From 3-D reconstructions of automatically recorded tilt series of ice-embedded macromolecules, several hundred 3-D images of single particles can be extracted. Here we describe correlation-based techniques to align the particles with respect to translation and orientation in 3-D and the calculation of an averaged reconstruction after application of the correct weighting function to the particle projections. Multivariate statistical analysis and classification are applied to the set of three-dimensionally reconstructed particles to investigate interimage variations on the 3-D level. Copyright 1997 Academic Press. Copyright 1997 Academic Press
RESUMEN
Chaperonins are double-ring protein folding machines fueled by ATP binding and hydrolysis. Conformational rearrangements upon ATPase cycling of the group I chaperonins, typified by the Escherichia coli GroEL/GroES system, have been thoroughly investigated by cryo-electron microscopy and X-ray crystallography. For archaeal group II chaperonins, however, these methods have so far failed to provide a correlation between the structural and the functional states. Here, we show that the conformation of the native alphabeta-thermosome of Thermoplasma acidophilum in vitrified ice is strictly regulated by adenine nucleotides.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Proteínas Arqueales/química , Chaperoninas/química , Microscopía por Crioelectrón , Conformación ProteicaRESUMEN
Valosine-containing protein-like ATPase from Thermoplasma acidophilum is a member of the superfamily of ATPases associated with a diversity of cellular activities and is closely related to CDC48 from yeast and p97 from higher eukaryotes and more distantly to N-ethylmaleimide-sensitive fusion protein. We have used electron tomography to obtain low-resolution (2-2.5 nm) three-dimensional maps of both the whole 500 kDa complex and the N-terminally truncated valosine-containing protein-like ATPase from T. acidophilum complex lacking the putative substrate binding domain.
Asunto(s)
Adenosina Trifosfatasas/ultraestructura , Thermoplasma/enzimología , Proteínas Arqueales , Proteínas de Ciclo Celular , Microscopía Electrónica , Proteína que Contiene ValosinaRESUMEN
Using electron microscopy, the thickness of ice-embedded vesicles is estimated examining tilted and untilted views and assuming an ellipsoidal shape of the vesicles that appear to be circular in the untilted view. Another thickness measure is obtained from the ratio of the unfiltered and zero-loss-filtered image intensities of the vesicle. From these two measurements, the mean free path A for inelastic scattering of electrons in ice is calculated as 203 +/- 33 nm for 120 kV acceleration voltage. It is found that vesicles in thin ice films (< or = 1.5 lambda) significantly protrude out of the ice film. Due to surface tension the shape becomes an oblate ellipsoid. In holes covered with a thick ice film (> or = 3 lambda) and strong thickness gradients, vesicles are predominantly found in regions where the ice thickness is appropriate for their size. Also, a way of imaging the most probable loss under low-dose conditions involving thickness measurement is proposed. Even at large ice thicknesses zero-loss filtering always gives better image contrast. Most probable loss imaging can only help where there is no intensity in the zero-loss image, at very large thicknesses (lambda > 8).
Asunto(s)
Hielo , Procesamiento de Imagen Asistido por Computador/métodos , Membranas Artificiales , Microscopía Electrónica/métodos , Dimiristoilfosfatidilcolina , Procesamiento de Imagen Asistido por Computador/instrumentaciónAsunto(s)
Fibrinógeno , Rayos Láser , Cristalización , Humanos , Microscopía Electrónica , Modelos Moleculares , Dispersión de RadiaciónRESUMEN
Cryo-electron tomography was used to study the structural organization of whole frozen-hydrated mitochondria from Neurospora crassa. Unlike mitochondria from many other species and tissues, in this case the cristae form a three-dimensional network of interconnected lamellae. Basically, the three-dimensional structure of ice-embedded mitochondria from this species is consistent with previous descriptions of mitochondria prepared by chemical fixation and resin embedding. Nonetheless, ice-embedded mitochondria display some important differences: the outer surface of the mitochondria was found to be rather smooth, the intermembrane space was constant in width, and distinct contact sites between the membranes were clearly revealed. Furthermore ATP synthase particles on the outer surface of an "inside-out vesicle" were visible in 3-D reconstructions. Thus, cryo-electron tomography can provide detailed insights into these organelles with minimal perturbations of the physiological state. This indicates that it is a realistic goal to achieve "molecular resolution" with rather large biological specimens in the near future, ultimately allowing the identification and localization of macromolecules in their cellular context.
Asunto(s)
Microscopía por Crioelectrón , Mitocondrias/ultraestructura , Neurospora crassa/ultraestructura , Tomografía , Procesamiento de Imagen Asistido por Computador , Membranas Intracelulares/ultraestructuraRESUMEN
Thermotoga maritima, a thermophilic eubacterium, is motile at temperatures ranging from 50 to 105 degrees C. The cells are propelled by a single flagellum which most of the time spins clockwise. Changes in the swimming direction ("tumbles") are achieved by short reversals of the direction of filament rotation. The average speed of swimming cells depends on the temperature, reaching a maximum value of about 60 microns/s at 85 degrees C. The cells show a thermotactic response to temporal temperature changes. When the temperature is raised, the rate of tumbles is increased, while decreasing temperature decreases the tumbling rate.
Asunto(s)
Bacterias Anaerobias Gramnegativas/citología , Flagelos/ultraestructura , Bacterias Anaerobias Gramnegativas/fisiología , Calor , Procesamiento de Imagen Asistido por Computador , Microscopía por Video , MovimientoRESUMEN
Flagellated envelopes of Halobacterium salinarium cells were prepared by lysis with taurodeoxycholate. After solubilization of the envelopes with Triton X-100 at high ionic strength, flagella and round patches from which numerous flagella emerged were isolated by gel filtration chromatography. We conclude that the flagellar bundle of H. salinarium is inserted into a differentiated polar cap structure.
Asunto(s)
Flagelos/ultraestructura , Halobacterium salinarum/ultraestructura , Fraccionamiento Celular , Halobacterium salinarum/química , Microscopía Electrónica , Polietilenglicoles/química , Ácido Taurodesoxicólico/químicaRESUMEN
Electron tomography is the only technique available that allows us to visualize the three-dimensional structure of unfixed and unstained cells currently with a resolution of 6-8 nm, but with the prospect to reach 2-4 nm. This raises the possibility of detecting and identifying specific macromolecular complexes within their cellular context by virtue of their structural signature. Templates derived from the high-resolution structure of the molecule under scrutiny are used to search the reconstructed volume. Here we outline and test a computationally feasible two-step procedure: In a first step, mean-curvature motion is used for segmentation, yielding subvolumes that contain with a high probability macromolecules in the expected size range. Subsequently, the particles contained in the subvolumes are identified by cross-correlation, using a set of three-dimensional templates. With simulated and real tomographic data we demonstrate that such an approach is feasible and we explore the detection limits. Even structurally similar particles, such as the thermosome, GroEL, and the 20S proteasome can be identified with high fidelity. This opens up exciting prospects for mapping the territorial distribution of macromolecules and for analyzing molecular interactions in situ.
Asunto(s)
Chaperonina 60/química , Cisteína Endopeptidasas/química , Complejos Multienzimáticos/química , Algoritmos , Chaperonina 60/aislamiento & purificación , Cisteína Endopeptidasas/aislamiento & purificación , Sustancias Macromoleculares , Modelos Moleculares , Complejos Multienzimáticos/aislamiento & purificación , Complejo de la Endopetidasa Proteasomal , Conformación Proteica , Tomografía Computarizada por Rayos X/métodosRESUMEN
Whole cells of archaea were embedded in vitreous ice by plunge freezing and investigated by automated energy-filtered electron tomography at 120 kV. The embedded cells were between 300 and 750 nm thick, and their structures were reconstructed to a resolution of 20-40 nm from tilt series comprising 50-140 images. The dose was kept within tolerable limits. A resolution of 20 nm allowed visualization of the individual stalks of the S-layer of Pyrobaculum aerophilum cells, which had undergone partial lysis, in three dimensions. The attainable resolution for low-dose electron tomography under different experimental conditions was theoretically investigated in terms of the specimen thickness. To obtain 2-nm resolution at 120 kV (300 kV), the specimen must not be thicker than 100 nm (150 nm). For a resolution of 10 nm, the maximum thickness is 450 nm (700 nm). An accelerating voltage of 300 kV is advantageous, mainly for specimens thicker than 100 nm. Experimental investigations so far have resulted in a resolution that is worse by a factor of 2-5 as compared to theory.
Asunto(s)
Sulfolobus/ultraestructura , Thermoproteaceae/ultraestructura , Automatización , Congelación , Hielo , Procesamiento de Imagen Asistido por Computador , Microscopía Electrónica/métodos , Modelos Estructurales , Sensibilidad y EspecificidadRESUMEN
Semiautomatic single-axis tilt electron tomography has been used to visualize the three-dimensional organization of actin filaments in "phantom cells," i.e. lipid vesicles. The instrumentation consisted of a 120-kV electron microscope equipped with a postcolumn energy filter, which was used in the zero-loss imaging mode. Apart from changing the tilt angle, all steps required for automated tomography, such as recentering the image area, refocusing, and centering the energy-selecting slit, were performed by external computer control. This setup permitted imaging of ice-embedded samples up to a thickness of 800 nm with improved image contrast compared with that produced by tomography with a conventional electron microscope. In spite of the missing-wedge effect that is especially obvious in the study of membrane-filament interaction, single-axis tilt tomography was found to be an appropriate (in fact the only available) method for this kind of investigation. In contrast to random actin networks found in actin gels, actin filaments in and on vesicles with a bending radius of less than approximately 2 microns tend to be arranged in single layers of parallel filaments and often induce an elongated shape of the vesicles. Actin filaments located on the outside usually associate with the vesicle membrane.
Asunto(s)
Actinas/ultraestructura , Microscopía Electrónica/métodos , Tomografía/métodos , Actinas/química , Dimiristoilfosfatidilcolina , Hielo , Liposomas , Microscopía Electrónica/instrumentaciónRESUMEN
The clotting cascade system of the horseshoe crab (Limulus) is involved in both haemostasis and host defence. The cascade results in the conversion of coagulogen, a soluble protein, into an insoluble coagulin gel. The clotting enzyme excises the fragment peptide C from coagulogen, giving rise to aggregation of the monomers. The crystal structure of coagulogen reveals an elongated molecule that embraces the helical peptide C fragment. Cleavage and removal of the peptide C would expose an extended hydrophobic cove, which could interact with the hydrophobic edge of a second molecule, leading to a polymeric fibre. The C-terminal half of the coagulogen molecule exhibits a striking topological similarity to the neurotrophin nerve growth factor (NGF), providing the first evidence for a neurotrophin fold in invertebrates. Similarities between coagulogen and Spatzle, the Drosophila ligand of the receptor Toll, suggest that the neurotrophin fold might be considered more ancient and widespread than previously realized.
Asunto(s)
Proteínas Sanguíneas/química , Factores de Crecimiento Nervioso/química , Secuencia de Aminoácidos , Animales , Cristalografía por Rayos X , Cangrejos Herradura/metabolismo , Microscopía Electrónica , Datos de Secuencia Molecular , Conformación Proteica , Homología de Secuencia de AminoácidoRESUMEN
Using cryo-electron microscopy we have determined a projection map of the structure of the water selective pore aquaporin-1.
Asunto(s)
Acuaporinas , Canales Iónicos/química , Animales , Acuaporina 1 , Cristalografía/métodos , Congelación , Glucosa/química , Canales Iónicos/ultraestructura , Microscopía Electrónica/métodos , Conformación ProteicaRESUMEN
Automated electron tomography is shown to be a suitable means to visualize the shape of phospholipid vesicles embedded in vitrified ice. With a slow-scan charge-coupled device camera as a recording device, the cumulative electron dose needed to record a data set of 60 projections at a magnification of 20,000X can be kept as low as 15 e-/A2 (or 1500 electrons/nm2). The membrane of the three-dimensionally reconstructed vesicles is clearly visible in two-dimensional sections through the three-dimensionally reconstructed volume. Some edges indicating a polygonal shape of the vesicles, frozen from the gel phase, are also clearly recognized. Because of the presently limited tilt angle range (+/- 60 degrees), the upper and lower "caps" of the vesicles (representing about 35% of the surface of the ellipsoidal particles) remain invisible in the three-dimensional reconstruction.
Asunto(s)
Membrana Dobles de Lípidos/química , Microscopía Electrónica/métodos , 1,2-Dipalmitoilfosfatidilcolina/química , Fenómenos Biofísicos , Biofisica , Hielo , Procesamiento de Imagen Asistido por Computador , Técnicas In Vitro , Estructura MolecularRESUMEN
In 26S proteasomes, "19S cap complexes" associate with either one or both ends of the barrel-shaped 20S core complex. These regulatory complexes which comprise about 20 different subunits, including 6 ATPases of the AAA family, are thought to recognize ubiquitinated substrate proteins, to dissociate and unfold them before threading them into the 20S core where they are degraded. Here, we examine the structure of 26S proteasomes from Drosophila embryos and Xenopus oocytes by electron microscopy. Image analysis reveals a rather flexible linkage between the 19S caps and the 20S core, with a peculiar wagging-type movement of the caps relative to the core. At this stage of the analysis, it is not clear whether this movement is relevant in terms of function. Three-dimensional reconstructions, taking this into account, provide first insights into the remarkably complex structure of the 19S caps and allows us to put forward a composite model of the entire 26S complex.
Asunto(s)
Microscopía Electrónica/métodos , Péptido Hidrolasas/química , Péptido Hidrolasas/ultraestructura , Complejo de la Endopetidasa Proteasomal , Animales , Drosophila melanogaster/enzimología , Femenino , Procesamiento de Imagen Asistido por Computador , Sustancias Macromoleculares , Modelos Moleculares , Oocitos/enzimología , Conformación Proteica , XenopusRESUMEN
After a general introduction to three-dimensional electron microscopy and particularly to electron tomography (ET), the perspectives of applying ET to native (frozen-hydrated) cellular structures are discussed. In ET, a set of 2-D images of an object is recorded at different viewing directions and is then used for calculating a 3-D image. ET at a resolution of 2-5 nm would allow the 3-D organization of structural cellular components to be studied and would provide important information about spatial relationships and interactions. The question of whether it is a realistic long-term goal to visualize or--by sophisticated pattern recognition methods--identify macromolecules in cells frozen in toto or in frozen sections of cells is addressed. Because of the radiation sensitivity of biological specimens, a prerequisite of application of ET is the automation of the imaging process. Technical aspects of automated ET as realized in Martinsried and experiences are presented, and limitations of the technique are identified, both theoretically and experimentally. Possible improvements of instrumentation to overcome at least part of the limitations are discussed in some detail. Those means include increasing the accelerating voltage into the intermediate voltage range (300 to 500 kV), energy filtering, the use of a field emission gun, and a liquid-helium-cooled specimen stage. Two additional sections deal with ET of isolated macromolecules and of macromolecular structures in situ, and one section is devoted to possible methods for the detection of structures in volume data.