Digitized precision measurements of the movements of sea urchin sperm flagella.

High speed cinemicrographs were made of sea urchin sperm at temperatures varying from 22 to 6 degrees C. Apparatus, combining a television camera and a video digitizer, was constructed to scan individual flagellar images and to digitize the flagellar waveforms. With appropriate smoothing and averaging procedures, the rough data were condensed by a microcomputer into the coordinates of 20 points along a flagellum, spaced 2 microns apart. The curvature of the flagellum at these points was also computed. The coordinates of the flagellar positions were obtained to an accuracy of approximately +/- 0.1 micron, flagellar curvature to an accuracy of approximately +/- 50 cm-1. At all temperatures the amplitude of the flagella was found to vary with time in a purely sinusoidal fashion to within +/- 2%. The local curvature of the flagella had basically a purely sinusoidal time course to within +/- 50 cm-1, but a varying amount of asymmetry was present in the distal and the proximal ends of the flagella. This asymmetry in the curvature was related to the radius of the circular path of the sperm. The flagellar waveforms can probably be summarized in simple algebraic functions.     

The structure of the archeabacterial flagellar filament of the extreme halophile Halobacterium salinarum R1M1 and its relation to eubacterial flagellar filaments and type IV pili

Although the phenomenology and mechanics of swimming are very similar in eubacteria and archaeabacteria (e.g. reversible rotation, helical polymorphism of the filament and formation of bundles), the dynamic flagellar filaments seem completely unrelated in terms of morphogenesis, structure and amino acid composition. Archeabacterial flagellar filaments share important features with type IV pili, which are components of retractable linear motors involved in twitching motility and cell adhesion. The archeabacterial filament is unique in: (1) having a relatively smooth surface and a small diameter of approximately 100A as compared to approximately 240A of eubacterial filaments and approximately 50A of type IV pili; (2) being glycosylated and sulfated in a pattern similar to the S-layer; (3) being synthesized as pre-flagellin with a signal-peptide cleavable by membrane peptidases upon transport; and (4) having an N terminus highly hydrophobic and homologous with that of the olygomerization domain of pilin.The synthesis of archeabacterial flagellin monomers as pre-flagellin and their post-translational, extracellular glycosylation suggest a different mode of monomer transport and polymerization at the cell-proximal end of the filament, similar to pili rather than to eubacterial flagellar filaments. The polymerization mode and small diameter may indicate the absence of a central channel in the filament.Using low-electron-dose images of cryo-negative-stained filaments, we determined the unique symmetry of the flagellar filament of the extreme halophile Halobacterium salinarum strain R1M1 and calculated a three-dimensional density map to a resolution of 19A. The map is based on layer-lines of order n=0, +10, -7, +3, -4, +6, and -1. The cross-section of the density map has a triskelion shape and is dominated by seven outer densities clustered into three groups, which are connected by lower-density arms to a dense central core surrounded by a lower-density shell. There is no evidence for a central channel. On the basis of the homology with the oligomerization domain of type IV pilin and the density distribution of the filament map, we propose a structure for the central core.     

Refining the structure of the Halobacterium salinarum flagellar filament using the iterative helical real space reconstruction method: insights into polymorphism

The eubacterial flagellar filament is an external, self-assembling, helical polymer approximately 220 A in diameter constructed from a highly conserved monomer, flagellin, which polymerizes externally at the distal end. The archaeal filament is only approximately 100 A in diameter, assembles at the proximal end and is constructed from different, glycosylated flagellins. Although the phenomenology of swimming is similar to that of eubacteria, the symmetry of the archebacterial filament is entirely different. Here, we extend our previous study on the flagellar coiled filament structure of strain R1M1 of Halobacterium salinarum. We use strain M175 of H.salinarum, which forms poly-flagellar bundles at high yield which, under conditions of relatively low ionic-strength (0.8 M versus 5 M) and low pH ( approximately 2.5 versus approximately 6.8), form straight filaments. We demonstrated previously that a single-particle approach to helical reconstruction has many advantages over conventional Fourier-Bessel methods when dealing with variable helical symmetry and heterogeneity. We show here that when this method is applied to the ordered helical structure of the archebacterial uncoiled flagellar filament, significant extensions in resolution can be obtained readily when compared to applying traditional helical techniques. The filament population can be separated into classes of different morphologies, which may represent polymorphic states. Using cryo-negatively stained images, a resolution of approximately 10-15 A has been achieved. Single alpha-helices can be fit into the reconstruction, supporting the proposed similarity of the structure to that of type IV bacterial pili.     

Structural studies of tropomyosin by cryoelectron microscopy and x-ray diffraction.

A comparison has been made between cryoelectron microscope images and the x-ray structure of one projection of the Bailey tropomyosin crystal. The computed transforms of the electron micrographs extend to a resolution of approximately 18 A compared with the reflections from x-ray crystallography which extend to 15 A. After correction of the images for lattice distortions and the contrast transfer function, the structure factors were constrained to the plane group (pmg) symmetry of this projection. Amplitude and phase data for five images were compared with the corresponding view from the three-dimensional x-ray diffraction data (Phillips, G.N., Jr., J.P. Fillers, and C. Cohen. 1986. J. Mol. Biol. 192: 111-131). The average R factor between the electron microscopy and x-ray amplitudes was 15%, with an amplitude-weighted mean phase difference of 4.8 degrees. The density maps derived from cryoelectron microscopy contain structural features similar to those from x-ray diffraction: these include the width and run of the filaments and their woven appearance at the crossover regions. Preliminary images obtained from frozen-hydrated tropomyosin/troponin cocrystals suggest that this approach may provide structural details not readily obtainable from x-ray diffraction studies.     

Building the atomic model for the bacterial flagellar filament by electron cryomicroscopy and image analysis

The bacterial flagellar filament is a helical propeller for bacterial locomotion. It is a well-ordered helical assembly of a single protein, flagellin, and its tubular structure is formed by 11 protofilaments, each in either of the two distinct conformations, L- and R-type, for supercoiling. We have been studying the three-dimensional structures of the flagellar filaments by electron cryomicroscopy and recently obtained a density map of the R-type filament up to 4 angstroms resolution from an image data set containing only about 41,000 molecular images. The density map showed the features of the alpha-helical backbone and some large side chains, which allowed us to build the complete atomic model as one of the first atomic models of macromolecules obtained solely by electron microscopy image analysis (Yonekura et al., 2003a). We briefly review the structure and the structure analysis, and point out essential techniques that have made this analysis possible.     

Three-dimensional structure of the frozen-hydrated flagellar filament. The left-handed filament of Salmonella typhimurium

Electron micrographs of frozen-hydrated preparations of flagellar filaments of Salmonella typhimurium were used to obtain a three-dimensional reconstruction of the structure. The filaments were obtained from the mutant SJW1660, which produces straight, left-handed filaments. The subunits in this filament are thought to be all in the L-state. The structure consists of a set of 11 longitudinal segmented rods of density that lie at a radius of 70 A. The outermost feature of the filament is a set of knobs of density that project outward from the rods. The interior of the filaments consists of arms that extend inward radially from the segmented rods. The 11 segmented rods and their interconnections are noteworthy because current theories regarding filament structure involve switching of subunits between the L and R states co-operatively along the directions of the rods.     

Solution x-ray scattering-based estimation of electron cryomicroscopy imaging parameters for reconstruction of virus particles

Structure factor amplitudes and phases can be computed directly from electron cryomicroscopy images. Inherent aberrations of the electromagnetic lenses and other instrumental factors affect the structure factors, however, resulting in decreased accuracy in the determined three-dimensional reconstruction. In contrast, solution x-ray scattering provides absolute and accurate measurement of spherically averaged structure factor amplitudes of particles in solution but does not provide information on the phases. In the present study, we explore the merits of using solution x-ray scattering data to estimate the imaging parameters necessary to make corrections to the structure factor amplitudes derived from electron cryomicroscopic images of icosahedral virus particles. Using 400-kV spot-scan images of the bacteriophage P22 procapsid, we have calculated an amplitude contrast of 8.0 +/- 5.2%. The amplitude decay parameter has been estimated to be 523 +/- 188 A2 with image noise compensation and 44 +/- 66 A2 without it. These results can also be used to estimate the minimum number of virus particles needed for reconstruction at different resolutions.     

Video-supported Analysis of Beggiatoa Filament Growth, Breakage, and Movement

A marine Beggiatoa sp. was cultured in semi-solid agar with opposing oxygen-sulfide gradients. Growth pattern, breakage of filaments for multiplication, and movement directions of Beggiatoa filaments in the transparent agar were investigated by time-lapse video recording. The initial doubling time of cells was 15.7 +/- 1.3 h (mean +/- SD) at room temperature. Filaments grew up to an average length of 1.7 +/- 0.2 mm, but filaments of up to approximately 6 mm were also present. First breakages of filaments occurred approximately 19 h after inoculation, and time-lapse movies illustrated that a parent filament could break into several daughter filaments within a few hours. In >20% of the cases, filament breakage occurred at the tip of a former loop. As filament breakage is accomplished by the presence of sacrificial cells, loop formation and the presence of sacrificial cells must coincide. We hypothesize that sacrificial cells enhance the chance of loop formation by interrupting the communication between two parts of one filament. With communication interrupted, these two parts of one filament can randomly move toward each other forming the tip of a loop at the sacrificial cell.     

Fibrinogen structure in projection at 18 A resolution. Electron density by co-ordinated cryo-electron microscopy and X-ray crystallography

Electron microscope images of frozen-hydrated crystals of a proteolytically modified fibrinogen show excellent preservation of the structure. An electron density map of the key centric projection of the crystal at 18 A resolution has been obtained by combining the phases derived from cryo-electron microscopy with X-ray amplitudes. Simulation methods developed in earlier studies have been used to interpret the map. In contrast to the earlier images, the map allows us to visualize the coiled-coil region of the molecule and possible substructure in the beta domains. The map also shows that there is a marked difference in density in the two regions corresponding to the molecular ends where the gamma domains interact. A possible interpretation of this finding is provided by assuming substructure in the gamma domains and the breaking of molecular symmetry where these domains interact. Some additional constraints useful for the determination of the three-dimensional structure were obtained from cryo-electron micrographs of a perpendicular view at 25 A resolution. Implications of this working model for the molecular length and contacts in the filaments in both the crystal and fibrin are described. The data used here will be valuable as a starting point for obtaining the three-dimensional structure.     

Quantitation of molecular densities by cryo-electron microscopy. Determination of the radial density distribution of tobacco mosaic virus.

We have determined the absolute mass and radial scattering density distribution of tobacco mosaic virus in the frozen-hydrated state by energy-filtered low-dose bright-field transmission electron microscopy. The absolute magnitude of electron scattering from tobacco mosaic virus in 150 nm of ice was within 3.0% of that predicted, with inelastic scattering accounting for approximately 80% of the scattering contrast. In order to test the accuracy of the radial reconstruction, a computer model of tobacco mosaic virus was built from the atomic co-ordinates assuming uniform solvent density. The validity of the model was confirmed by comparison of X-ray scattering and predictions of the model (R factor = 0.05). First-order corrections for the microscope contrast transfer function were necessary and sufficient for conversion of the cryo-electron microscopy images into accurate representations of the mass density. At 1.9 nm resolution the compensated reconstruction and model had density peaks of similar magnitude at 2.4, 4.2, 6.0 and 7.8 nm radius and a central hole of 2 nm radius. Equatorial Fourier transforms of the corrected electron images were in excellent agreement with predictions of the model (R factor = 0.12). Thus, the uniform solvent approximation was adequate at 1.9 nm resolution to describe quantitatively X-ray scattering in liquid water and electron imaging in vitreous ice. This is the first demonstration that cryo-electron microscopy images can be used to quantitate the absolute mass, mass per unit length and internal density distributions of proteins and nucleic acids.     

Position and orientation of phalloidin in F-actin determined by X-ray fiber diffraction analysis

Knowledge of the phalloidin binding position in F-actin and the relevant understanding of the mechanism of F-actin stabilization would help to define the structural characteristics of the F-actin filament. To determine the position of bound phalloidin experimentally, x-ray fiber diffraction data were obtained from well-oriented sols of F-actin and the phalloidin-F-actin complex. The differences in the layer-line intensity distributions, which were clearly observed even at low resolution (8 A), produced well-resolved peaks corresponding to interphalloidin vectors in the cylindrically averaged difference-Patterson map, from which the radial binding position was determined to be approximately 10 A from the filament axis. Then, the azimuthal and axial positions were determined by single isomorphous replacement phasing and a cross-Patterson map in radial projection to be approximately 84 degrees and 0.5 A relative to the actin mass center. The refined position was close to the position found by prior researchers. The position of rhodamine attached to phalloidin in the rhodamine-phalloidin-F-actin complex was also determined, in which the conjugated Leu(OH)(7) residue was found to face the outside of the filament. The position and orientation of the bound phalloidin so determined explain the increase in the interactions between long-pitch strands of F-actin and would also account for the inhibition of phosphate release, which might also contribute to the F-actin stabilization. The method of analysis developed in this study is applicable for the determination of binding positions of other drugs, such as jasplakinolide and dolastatin 11.     

Flexural rigidity of microtubules and actin filaments measured from thermal fluctuations in shape

Microtubules are long, proteinaceous filaments that perform structural functions in eukaryotic cells by defining cellular shape and serving as tracks for intracellular motor proteins. We report the first accurate measurements of the flexural rigidity of microtubules. By analyzing the thermally driven fluctuations in their shape, we estimated the mean flexural rigidity of taxol-stabilized microtubules to be 2.2 x 10(-23) Nm2 (with 6.4% uncertainty) for seven unlabeled microtubules and 2.1 x 10(-23) Nm2 (with 4.7% uncertainty) for eight rhodamine-labeled microtubules. These values are similar to earlier, less precise estimates of microtubule bending stiffness obtained by modeling flagellar motion. A similar analysis on seven rhodamine-phalloidin- labeled actin filaments gave a flexural rigidity of 7.3 x 10(-26) Nm2 (with 6% uncertainty), consistent with previously reported results. The flexural rigidity of these microtubules corresponds to a persistence length of 5,200 microns showing that a microtubule is rigid over cellular dimensions. By contrast, the persistence length of an actin filament is only approximately 17.7 microns, perhaps explaining why actin filaments within cells are usually cross-linked into bundles. The greater flexural rigidity of a microtubule compared to an actin filament mainly derives from the former's larger cross-section. If tubulin were homogeneous and isotropic, then the microtubule's Young's modulus would be approximately 1.2 GPa, similar to Plexiglas and rigid plastics. Microtubules are expected to be almost inextensible: the compliance of cells is due primarily to filament bending or sliding between filaments rather than the stretching of the filaments themselves.     

Three-dimensional structure of the truncated core of the Saccharomyces cerevisiae pyruvate dehydrogenase complex determined from negative stain and cryoelectron microscopy images.

Dihydrolipoamide acyltransferase (E2), a catalytic and structural component of the three functional classes of multienzyme complexes that catalyze the oxidative decarboxylation of alpha-keto acids, forms the central core to which the other components are attached. We have imaged by negative stain and cryoelectron microscopy the truncated dihydrolipoamide acetyltransferase core (60 subunits; M(r) = 2.7 x 10(6)) of the Saccharomyces cerevisiae pyruvate dehydrogenase complex. Using icosahedral particle reconstruction techniques, we determined its structure to 25 A resolution. Although the model derived from the negative stain reconstruction was approximately 20% smaller than the model derived from the frozen-hydrated data, when corrected for the effects of the electron microscope contrast transfer functions, the reconstructions showed excellent correspondence. The pentagonal dodecahedron-shaped macromolecule has a maximum diameter, as measured along the 3-fold axis, of approximately 226 A (frozen-hydrated value), and 12 large openings (approximately 63 A in diameter) on the 5-fold axes that lead into a large solvent-accessible cavity (approximately 76-140 A diameter). The 20 vertices consist of cone-shaped trimers, each with a flattened base on the outside of the structure and an apex directed toward the center. The trimers are interconnected by 20 A thick "bridges" on the 2-fold axes. These studies also show that the highest resolution features apparent in the frozen-hydrated reconstruction are revealed in a filtered reconstruction of the stained molecule.     

Zero-loss energy filtering as improved imaging mode in cryoelectronmicroscopy of frozen-hydrated specimens

One of the remaining problems in attaining higher structural resolution with cryoelectronmicroscopy of frozen-hydrated specimens is the low contrast of micrographs taken close to the electron optical focus. By measuring electron energy loss spectra (EELS) of ice layers we show that a large fraction of incident electrons undergoes an inelastic electron-plasmon scattering process. Thus these electrons do not carry structural information of the protein but increase the background of the electron image and therefore reduce the contrast of the negative. Here we report the improvement in contrast gained by filtering out inelastically scattered electrons using an energy-filtered transmission electron microscope (EFTEM). This gain in contrast permits a dramatic decrease in defocusing values, resulting in improved structural resolution. In addition, the increased signal to noise ratio allows the recording of micrographs at a reduced electron dose. This should result in less damage to vitrified and unstained proteins and other beam-sensitive specimens.     

Helical reconstruction of frozen-hydrated scallop myosin filaments.

Native myosin filaments from scallop striated muscle that have been rapidly frozen in relaxing solutions appear to be well preserved in vitreous ice. Electron micrographs of samples at -177 degrees C were recorded with an electron dose of 10 e/A2 at 1.5 microns defocus. After filament images were straightened by spline-fitting, several transforms showed well-defined layer-lines arising from the helical structure of the filament. A set of 17 near-meridional layer-lines has been collected and corrected for background and for phase and amplitude contrast functions. Preliminary helical reconstructions from this still incomplete data set reveal aspects of structure that were not apparent from earlier analysis of negatively stained filaments from scallop muscle. Individual pear-shaped myosin heads now appear to be well resolved from each other and from the filament backbone. The two heads of each myosin molecule appear to be splayed apart axially. The reconstructions also reveal that the filament backbone has a polygonal shape in cross-section, and that it appears to contain seven peripherally located subfilaments.     

Structure of CaATPase: electron microscopy of frozen-hydrated crystals at 6 A resolution in projection

Thin, three-dimensional crystals of CaATPase have been studied at high resolution by electron crystallography. These crystals were grown by adding purified CaATPase to appropriate concentrations of lipid, detergent and calcium. A thin film of crystals was then rapidly frozen and maintained in the frozen-hydrated state during electron microscopy. The resulting electron diffraction patterns extend to 4.1 A resolution and images contain phase data to 6 A resolution. By combining Fourier amplitudes from electron diffraction patterns with phases from images, a density map has been calculated in projection. Comparison of this map from unstained crystals with a previously determined map from negatively stained crystals reveals distinct contributions from intramembranous and extramembranous protein domains. On the basis of this distinction and of the packing of molecules in the crystal, we have proposed a specific arrangement for the ten alpha-helices that have been suggested as spanning the bilayer.     

A comparison of liquid nitrogen and liquid helium as cryogens for electron cryotomography

The principal resolution limitation in electron cryomicroscopy of frozen-hydrated biological samples is radiation damage. It has long been hoped that cooling such samples to just a few kelvins with liquid helium would slow this damage and allow statistically better-defined images to be recorded. A new "G2 Polara" microscope from FEI Company was used to image various biological samples cooled by either liquid nitrogen or liquid helium to approximately 82 or approximately 12 K, respectively, and the results were compared with particular interest in the doses (10-200 e-/A2) and resolutions (3-8 nm) typical for electron cryotomography. Simple dose series revealed a gradual loss of contrast at approximately 12K through the first several tens of e-/A2, after which small bubbles appeared. Single particle reconstructions from each image in a dose series showed no difference in the preservation of medium-resolution (3-5 nm) structural detail at the two temperatures. Tomographic reconstructions produced with total doses between 10 and 350 e-/A2 showed better results at approximately 82 K than approximately 12 K for every dose tested. Thus disappointingly, cooling with liquid helium is actually disadvantageous for cryotomography.     

Measuring optical and mechanical properties of a living cell with defocusing microscopy

Defocusing microscopy (DM) is a recently developed technique that allows quantitative analysis of membrane surface dynamics of living cells using a simple bright-field optical microscope. According to DM, the contrast of defocused images is proportional to cell surface curvature. Although, until now, this technique was used mainly to determine size and amount of membrane shape fluctuations, such as ruffles and small random membrane fluctuations, in macrophages, its applications on cell biology extend beyond that. We show how DM can be used to measure optical and mechanical properties of a living macrophage, such as cell refractive index n, membrane bending modulus K(c), and effective cell viscosity eta for membrane-actin meshwork relaxation. Experimental data collected from defocused images of bone marrow-derived macrophages were used to evaluate these parameters. The obtained values, averaged over several different macrophages, are n = (1.384 +/- 0.015), K(c) approximately 3.2 x 10(-19) J, and eta approximately 459 Pa.s. We also estimate the amplitude of the small fluctuations to be of the order of 3 nm, which is around the step size of a polymerizing actin filament.     

Micro-video study of discontinuous growth of bacterial flagellar filaments in vitro

In a microscope slide preparation, monomeric flagellins were found to polymerize into flagellar filaments spontaneously, without addition of seeds. Dynamic images of individual growing filaments in a dark-field light microscope were recorded throughout their growth by an ultrasensitive video camera. Each filament had its own unique growth curve. The growth curves consisted of two kinds of discrete phase; namely, the elongation and the rest phase. In the former, a filament elongates at a constant rate, fairly similar among all filaments. In the latter, elongation stops completely. Each filament exists in either of the two phases and alternates between them in a stochastic manner. A mean elongation rate of 89 + 15 nm per minute was obtained at the flagellin concentration of 2 mg/ml, for filaments in the elongation phase.     

Radial displacement of myosin cross-bridges in mouse myocardium due to ablation of myosin binding protein-C

Myosin binding protein-C (cMyBP-C) is a thick filament accessory protein, which in cardiac muscle functions to regulate the kinetics of cross-bridge interaction with actin; however, the underlying mechanism is not yet understood. To explore the structural basis for cMyBP-C function, we used synchrotron low-angle X-ray diffraction to measure interfilament lattice spacing and the equatorial intensity ratio, I(11)/I(10), in skinned myocardial preparations isolated from wild-type (WT) and cMyBP-C null (cMyBP-C(-/-)). In relaxed myocardium, ablation of cMyBP-C appeared to result in radial displacement of cross-bridges away from the thick filaments, as there was a significant increase ( approximately 30%) in the I(11)/I(10) ratio for cMyBP-C(-/-) (0.37+/-0.03) myocardium as compared to WT (0.28+/-0.01). While lattice spacing tended to be greater in cMyBP-C(-/-) myocardium (44.18+/-0.68 nm) when compared to WT (42.95+/-0.43 nm), the difference was not statistically significant. Furthermore, liquid-like disorder in the myofilament lattice was significantly greater ( approximately 40% greater) in cMyBP-C(-/-) myocardium as compared to WT. These results are consistent with our working hypothesis that cMyBP-C normally acts to tether myosin cross-bridges nearer to the thick filament backbone, thereby reducing the likelihood of cross-bridge binding to actin and limiting cooperative activation of the thin filament.