ABSTRACT
Tilted electron microscope images are routinely collected for an ab initio structure reconstruction as a part of the Random Conical Tilt (RCT) or Orthogonal Tilt Reconstruction (OTR) methods, as well as for various applications using the "free-hand" procedure. These procedures all require identification of particle pairs in two corresponding images as well as accurate estimation of the tilt-axis used to rotate the electron microscope (EM) grid. Here we present a computational approach, PCT (particle correspondence from tilted pairs), based on tilt-invariant context and projection matching that addresses both problems. The method benefits from treating the two problems as a single optimization task. It automatically finds corresponding particle pairs and accurately computes tilt-axis direction even in the cases when EM grid is not perfectly planar.
Subject(s)
IMP Dehydrogenase/ultrastructure , Image Processing, Computer-Assisted/statistics & numerical data , Imaging, Three-Dimensional/statistics & numerical data , Ribosomes/ultrastructure , Cryoelectron Microscopy/instrumentation , Desulfovibrio vulgaris/chemistry , Escherichia coli/chemistry , Imaging, Three-Dimensional/instrumentation , Imaging, Three-Dimensional/methodsABSTRACT
An unbiased survey has been made of the stable, most abundant multi-protein complexes in Desulfovibrio vulgaris Hildenborough (DvH) that are larger than Mr approximately 400 k. The quaternary structures for 8 of the 16 complexes purified during this work were determined by single-particle reconstruction of negatively stained specimens, a success rate approximately 10 times greater than that of previous "proteomic" screens. In addition, the subunit compositions and stoichiometries of the remaining complexes were determined by biochemical methods. Our data show that the structures of only two of these large complexes, out of the 13 in this set that have recognizable functions, can be modeled with confidence based on the structures of known homologs. These results indicate that there is significantly greater variability in the way that homologous prokaryotic macromolecular complexes are assembled than has generally been appreciated. As a consequence, we suggest that relying solely on previously determined quaternary structures for homologous proteins may not be sufficient to properly understand their role in another cell of interest.
Subject(s)
Bacterial Proteins/chemistry , Desulfovibrio vulgaris/metabolism , Bacterial Proteins/isolation & purification , Crystallography, X-Ray , Databases, Protein , Desulfovibrio vulgaris/chemistry , Models, Molecular , Multiprotein Complexes/chemistry , Multiprotein Complexes/metabolism , Protein ConformationABSTRACT
The goal of this study is to evaluate the performance of software for automated particle-boxing, and in particular the performance of a new tool (TextonSVM) that recognizes the characteristic texture of particles of interest. As part of a high-throughput protocol, we use human editing that is based solely on class-average images to create final data sets that are enriched in what the investigator considers to be true-positive particles. The Fourier shell correlation (FSC) function is then used to characterize the homogeneity of different single-particle data sets that are derived from the same micrographs by two or more alternative methods. We find that the homogeneity is generally quite similar for class-edited data sets obtained by the texture-based method and by SIGNATURE, a cross-correlation-based method. The precision-recall characteristics of the texture-based method are, on the other hand, significantly better than those of the cross-correlation based method; that is to say, the texture-based approach produces a smaller fraction of false positives in the initial set of candidate particles. The computational efficiency of the two approaches is generally within a factor of two of one another. In situations when it is helpful to use a larger number of templates (exemplars), however, TextonSVM scales in a much more efficient way than do boxing programs that are based on localized cross-correlation.
Subject(s)
Algorithms , Software , Cryoelectron MicroscopyABSTRACT
Electron microscopy at a resolution of 0.4nm or better requires more careful adjustment of the illumination than is the case at a resolution of 0.8nm. The use of current-axis alignment is not always sufficient, for example, to avoid the introduction of large phase errors, at higher resolution, due to axial coma. In addition, one must also ensure that off-axis coma does not corrupt the data quality at the higher resolution. We particularly emphasize that the standard CTF correction does not account for the phase error associated with coma. We explain the cause of both axial coma and the typically most troublesome component of off-axis coma in terms of the well-known shift of the electron diffraction pattern relative to the optical axis that occurs when the illumination is not parallel to the axis. We review the experimental conditions under which coma causes unacceptably large phase errors, and we discuss steps that can be taken when setting up the conditions of illumination, so as to ensure that neither axial nor off-axis coma is a problem.
Subject(s)
Cryoelectron Microscopy/methodsABSTRACT
A lens system is proposed that not only provides spherical correction of the objective lens by charges that are induced on a thin foil, in the way proposed in a paper by Otto Scherzer [Optik 56(2), 133-147, 1980], but also provides Zernike phase contrast by means of an appropriate phase shift of the scattered electrons within the foil. This system has the potential to provide strong phase contrast from very low spatial frequencies to frequencies above 1/(100 pm).
ABSTRACT
Beam-induced specimen movement may be the major factor that limits the quality of high-resolution images of organic specimens. One of the possible measures to improve the situation that was proposed by Henderson and Glaeser [Ultramicroscopy 16 (1985) 139-150], which we refer to here as "stroboscopic image capture", is to divide the normal exposure into many successive frames, thus reducing the amount of electron exposure--and possibly the amount of beam-induced movement--per frame. The frames would then be aligned and summed. We have performed preliminary experiments on stroboscopic imaging using a 200-kV electron microscope that was equipped with a high dynamic range Charge-coupled device (CCD) camera for image recording and a liquid N2-cooled cryoholder. Single-layer paraffin crystals on carbon film were used as a test specimen. The ratio F(g)/F(0) of paraffin reflections, calculated from the images, serves as our criterion for the image quality. In the series that were evaluated, no significant improvement of the F(image)(g)/F(image)(0) ratio was found, even though the electron exposure per frame was reduced by a factor of 30. A frame-to-frame analysis of image distortions showed that considerable beam-induced movement had still occurred during each frame. In addition, the paraffin crystal lattice was observed to move relative to the supporting carbon film, a fact that cannot be explained as being an electron-optical effect caused by specimen charging. We conclude that a significant further reduction of the dose per frame (than was possible with this CCD detector) will be needed in order to test whether the frame-to-frame changes ultimately become small enough for stroboscopic image capture to show its potential.
Subject(s)
Image Processing, Computer-Assisted/methods , Microscopy, Electron/methods , Electrons , Paraffin , Photography/instrumentationABSTRACT
A miniature electrostatic element has been designed to selectively apply a 90 degrees phase shift to the unscattered beam in the back focal plane of the objective lens, in order to realize Zernike-type, in-focus phase contrast in an electron microscope. The design involves a cylindrically shaped, biased-voltage electrode, which is surrounded by a concentric grounded electrode. Electrostatic calculations have been used to determine that the fringing fields in the region of the scattered electron beams will cause a negligible phase shift as long as the ratio of electrode length to the transverse feature size is greater than 5:1. Unlike the planar, three-electrode einzel lens originally proposed by Boersch for the same purpose, this new design does not require insulating layers to separate the biased and grounded electrodes, and it can thus be produced by a very simple microfabrication process. Scanning electron microscope images confirm that mechanically robust devices with feature sizes of approximately 1 microm can be easily fabricated. Preliminary experimental images demonstrate that these devices do apply a 90 degrees phase shift between the scattered and unscattered electrons, as expected.
ABSTRACT
To go beyond the current structural consensus model of the nuclear pore complex (NPC), we performed cryo-electron tomography of fully native NPCs from Xenopus oocyte nuclear envelopes (NEs). The cytoplasmic face of the NPC revealed distinct anchoring sites for the cytoplasmic filaments, whereas the nuclear face was topped with a massive distal ring positioned above the central pore with indications of the anchoring sites for the nuclear basket filaments and putative intranuclear filaments. The rather "spongy" central framework of the NPC was perforated by an elaborate channel and void system, and at the membrane pore interface it exhibited distinct "handles" protruding into the lumen of the NE. The most variable structural moiety of the NPC was a rather tenuous central plug partially obstructing the central pore. Its mobile character was documented by time-lapse atomic force microscopy. Taken together, the new insights we gained into NPC structure support the notion that the NPC acts as a constrained diffusion pore for molecules and particles without retention signal and as an affinity gate for signal-bearing cargoes.
Subject(s)
Cell Nucleus/ultrastructure , Nuclear Pore/ultrastructure , Tomography/methods , Active Transport, Cell Nucleus , Animals , Cell Nucleus/chemistry , Cryoelectron Microscopy , Cytoplasm/ultrastructure , Female , Ice , Imaging, Three-Dimensional , Microscopy, Atomic Force , Models, Biological , Oocytes/ultrastructure , Xenopus laevisABSTRACT
We demonstrate two ways in which the Fourier transforms of images that consist solely of randomly distributed electrons (shot noise) can be used to compare the relative performance of different electronic cameras. The principle is to determine how closely the Fourier transform of a given image does, or does not, approach that of an image produced by an ideal camera, i.e. one for which single-electron events are modeled as Kronecker delta functions located at the same pixels where the electrons were incident on the camera. Experimentally, the average width of the single-electron response is characterized by fitting a single Lorentzian function to the azimuthally averaged amplitude of the Fourier transform. The reciprocal of the spatial frequency at which the Lorentzian function falls to a value of 0.5 provides an estimate of the number of pixels at which the corresponding line-spread function falls to a value of 1/e. In addition, the excess noise due to stochastic variations in the magnitude of the response of the camera (for single-electron events) is characterized by the amount to which the appropriately normalized power spectrum does, or does not, exceed the total number of electrons in the image. These simple measurements provide an easy way to evaluate the relative performance of different cameras. To illustrate this point we present data for three different types of scintillator-coupled camera plus a silicon-pixel (direct detection) camera.
Subject(s)
Electron Microscope Tomography/instrumentation , Electrons , Fourier Analysis , Image Processing, Computer-Assisted/instrumentation , NoiseABSTRACT
Microfabricated devices designed to provide phase contrast in the transmission electron microscope must be free of phase distortions caused by unexpected electrostatic effects. We find that such phase distortions occur even when a device is heated to 300 °C during use in order to avoid the formation of polymerized, carbonaceous contamination. Remaining factors that could cause unwanted phase distortions include patchy variations in the work function of a clean metal surface, radiation-induced formation of a localized oxide layer, and creation of a contact potential between an irradiated area and the surround due to radiation-induced structural changes. We show that coating a microfabricated device with evaporated carbon apparently eliminates the problem of patchy variation in the work function. Furthermore, we show that a carbon-coated titanium device is superior to a carbon-coated gold device, with respect to radiation-induced electrostatic effects. A carbon-coated, hybrid double-sideband/single-sideband aperture is used to record in-focus, cryo-EM images of monolayer crystals of streptavidin. Images showing no systematic phase error due to charging are achievable under conditions of low-dose data collection. The contrast in such in-focus images is sufficient that one can readily see individual streptavidin tetramer molecules. Nevertheless, these carbon-coated devices perform well for only a limited length of time, and the cause of failure is not yet understood.
Subject(s)
Microscopy, Electron, Transmission/instrumentation , Microscopy, Electron, Transmission/methods , Carbon/chemistry , Microtechnology , Protein Conformation , Static Electricity , Streptavidin/chemistryABSTRACT
Pyruvate-ferredoxin oxidoreductatse (PFOR) carries out the central step in oxidative decarboxylation of pyruvate to acetyl-CoA. We have purified this enzyme from Desulfovibrio vulgaris Hildenborough (DvH) as part of a systematic characterization of as many multiprotein complexes as possible for this organism, and the three-dimensional structure of this enzyme has been determined by a combination of electron microscopy (EM), single particle image analysis, homology modeling and computational molecular docking. Our results show that the 1MDa DvH PFOR complex is a homo-octomer, or more precisely, a tetramer of the dimeric form of the related enzyme found in Desulfovibrio africanus (Da), with which it shares a sequence identity of 69%. Our homology model of the DvH PFOR dimer is based on the Da PFOR X-ray structure. Docking of this model into our 17A resolution EM-reconstruction of negatively stained DvH PFOR octomers strongly suggests that the difference in oligomerization state for the two species is due to the insertion of a single valine residue (Val383) within a surface loop of the DvH enzyme. This study demonstrates that the strategy of intermediate resolution EM reconstruction coupled to homology modeling and docking can be powerful enough to infer the functionality of single amino acid residues.
Subject(s)
Desulfovibrio vulgaris/enzymology , Pyruvate Synthase/chemistry , Amino Acids , Computational Biology , Dimerization , Microscopy, Electron , Models, Molecular , Protein Conformation , Pyruvate Synthase/isolation & purification , ValineABSTRACT
A film-handling machine (robot) has been built which can, in conjunction with a commercially available film densitometer, exchange and digitize over 300 electron micrographs per day. Implementation of robotic film handling effectively eliminates the delay and tedium associated with digitizing images when data are initially recorded on photographic film. The modulation transfer function (MTF) of the commercially available densitometer is significantly worse than that of a high-end, scientific microdensitometer. Nevertheless, its signal-to-noise ratio (S/N) is quite excellent, allowing substantial restoration of the output to "near-to-perfect" performance. Due to the large area of the standard electron microscope film that can be digitized by the commercial densitometer (up to 10,000 x 13,680 pixels with an appropriately coded holder), automated film digitization offers a fast and inexpensive alternative to high-end CCD cameras as a means of acquiring large amounts of image data in electron microscopy.
Subject(s)
Densitometry/methods , Automation , Densitometry/instrumentation , Equipment Design , Image Processing, Computer-Assisted , Photography , Robotics , SoftwareABSTRACT
The resolution achieved in low-dose electron microscopy of biological macromolecules is significantly worse than what can be obtained on the same microscopes with more robust specimens. When two-dimensional crystals are used, it is also apparent that the high-resolution image contrast is much less than what it could be if the images were perfect. Because specimen charging is one factor that might limit the contrast and resolution achieved with biological specimens, we have investigated the use of holey support films that have been coated with a metallic film before depositing specimens onto a thin carbon film that is suspended over the holes. Monolayer crystals of paraffin (C44H90) are used as a test specimen for this work because of the relative ease in imaging Bragg spacings at 0.4 nm resolution, the relative ease of measuring the contrast in these images, and the similar degree of radiation sensitivity of these crystals when compared to biological macromolecules. A metallic coating on the surrounding support film does, indeed, produce a significant improvement in the high-resolution contrast for a small fraction of the images. The majority of images show little obvious improvement, however, and even the coated area of the support film continues to show a significant amount of beam-induced movement under low-dose conditions. The fact that the contrast in the best images can be as much as 25%-35% of what it would be in a perfect image is nevertheless encouraging, demonstrating that it should be possible, in principle, to achieve the same performance for every image. Routine data collection of this quality would make it possible to determine the structure of large, macromolecular complexes without the need to grow crystals of these difficult specimen materials.
Subject(s)
Macromolecular Substances , Microscopy, Electron/methods , Paraffin/analysis , Crystallization , Fourier Analysis , Image Enhancement , Image Processing, Computer-Assisted , Microscopy, Electron/instrumentationABSTRACT
Large nuclear ribonucleoprotein particles, which can be viewed as the naturally assembled precursor messenger RNA (pre-mRNA) processing machine, were analyzed in frozen-hydrated preparations by cryoelectron microscopy. A general and reproducible strategy for preparing ice-embedded large nuclear ribonucleoprotein (lnRNP) particles at sufficiently high concentration was developed. Taking advantage of their negatively charged components, the lnRNP particles are adsorbed and thus concentrated on a positively charged lipid monolayer while preserving their native structure. Using this approach we carried out cryoelectron tomography and three-dimensional image reconstruction of individual lnRNP particles. The study revealed a structure similar to that of negatively stained particles studied previously, yet with additional features. The small additional domain visualized in negative stain appeared to be larger in the ice preparations. In addition, using image restoration from focus series of ice-embedded lnRNP particles, new features such as holes within the subunits were visualized in two dimensions, and it was shown that the subunits are interconnected via a fiber, very likely formed by the pre-mRNA. This finding supports the model that each subunit represents a spliceosome that splices out the intron wound around it.
Subject(s)
Cell Nucleus Structures/ultrastructure , Cryoelectron Microscopy , Ribonucleoproteins/ultrastructure , Tomography, X-Ray Computed , HeLa Cells , Humans , Image Processing, Computer-Assisted , Imaging, Three-Dimensional/methods , Lipids/chemistry , RNA Precursors , RNA Processing, Post-Transcriptional , Ribonucleoproteins/chemistry , SpliceosomesABSTRACT
Gas vesicle formation in halophilic archaea is encoded by a DNA region (the vac region) containing 14 different genes: gvpACNO and gvpDEFGHIJKLM. In Halobacterium salinarum PHH1 (which expresses the p-vac region from plasmid pHH1), gas vesicles are spindle shaped, whereas predominantly cylindrical gas vesicles are synthesized by the chromosomal c-vac region of H. salinarum PHH4 and the single chromosomal mc-vac region of Haloferax mediterranei. Homologous complementation of gvp gene clusters derived from the chromosomal c-vac region led to cylindrical gas vesicles in transformants and proved that the activity of the c-gvpA promoter depended on a gene product from the c-gvpE-M DNA region. Heterologous complementation experiments with transcription units of different vac regions demonstrated that the formation of chimeric gas vesicles was possible. Comparison of micrographs of wild-type and chimeric gas vesicles indicated that the shape was not exclusively determined by GvpA, the major structural protein of the gas vesicle wall. More likely, a dynamic equilibrium of several gvp gene products was responsible for determination of the shape. Transmission electron microscopy of frozen hydrated, wild-type gas vesicles showed moiré patterns due to the superposition of the front and back parts of the ribbed gas vesicle envelope. Comparison of projections of model helices with the moiré pattern seen on the cylindrical part of the gas vesicles provided evidence that the ribs formed a helix of low pitch and not a stack of hoops.
Subject(s)
Halobacterium/genetics , Halobacterium/ultrastructure , Haloferax/genetics , Haloferax/ultrastructure , Vacuoles/ultrastructure , Archaeal Proteins/metabolism , DNA, Archaeal , Gene Expression Regulation, Archaeal , Genes, Archaeal , Genetic Complementation Test , Halobacterium/metabolism , Haloferax/metabolism , Image Processing, Computer-Assisted , Microscopy, Electron , Transformation, Genetic , Vacuoles/chemistryABSTRACT
Electron tomograms of intact frozen-hydrated cells are essentially three-dimensional images of the entire proteome of the cell, and they depict the whole network of macromolecular interactions. However, this information is not easily accessible because of the poor signal-to-noise ratio of the tomograms and the crowded nature of the cytoplasm. Here, we describe a template matching algorithm that is capable of detecting and identifying macromolecules in tomographic volumes in a fully automated manner. The algorithm is based on nonlinear cross correlation and incorporates elements of multivariate statistical analysis. Phantom cells, i.e., lipid vesicles filled with macromolecules, provide a realistic experimental scenario for an assessment of the fidelity of this approach. At the current resolution of approximately 4 nm, macromolecules in the size range of 0.5-1 MDa can be identified with good fidelity.