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The Protein Complex Analysis Project (PCAP) has two major goals: 1. to develop an integrated set of high throughput pipelines to identify and characterize multi-protein complexes in a microbe more swiftly and comprehensively than currently possible and 2. to use these pipelines to elucidate and model the protein interaction networks regulating stress responses in Desulfovibrio vulgaris with the aim of understanding how this and similar microbes can be used in bioremediation of metal and radionuclides found in U.S. Department of Energy (DOE) contaminated sites. PCAP builds on the established research and infrastructure of another Genomics: GTL initiative conducted by the Environmental Stress Pathways Project (ESPP). ESPP has developed D. vulgaris as a model for stress responses and has used gene expression profiling to define specific sets of proteins whose expression changes after application of a stressor. Proteins, however, do not act in isolation. They participate in intricate networks of protein/protein interactions that regulate cellular metabolism. To understand and model how these identified genes affect the organism, therefore, it is essential to establish not only the other proteins that they directly contact, but the full repertoire of protein/protein interactions within the cell. In addition, there may well be genes whose activity is changed in response to stress not by regulating their expression level but by altering the protein partners that they bind, by modifying their structures, or by changing their subcellular locations. There may also be differences in the way proteins within individual cells respond to stress that are not apparent in assays that examine the average change in a population of cells. Therefore, we are extending ESPP's analysis to characterize the polypeptide composition of as many multi-protein complexes in the cell as possible and determine their stoichiometries, their quaternary structures, and their locations in planktonic cells and in individual cells within biofilms. PCAP will characterize complexes in wild type cells grown under normal conditions and also examine how these complexes are affected in cells perturbed by stress or by mutation of key stress regulatory genes. These data will all be combined with those of the ongoing work of the ESPP to understand, from a physical-chemical, control-theoretical, and evolutionary point of view, the role of multi-protein complexes in stress pathways involved in the biogeochemistry of soil microbes under a wide variety of conditions. Essential to this endeavor is the development of automated high throughput methods that are robust and allow for the comprehensive analysis of many protein complexes. Biochemical purification of endogenous complexes and identification by mass spectrometry is being coupled with in vitro and in vivo EM molecular imaging methods. Because no single method can isolate all complexes, we are developing two protein purification pipelines, one the current standard Tandem Affinity Purification approach, the other a novel tagless strategy. Specific variants ofeach of these are being developed for water soluble and membrane proteins. Our Bioinstrumentation group is developing highly parallel micro-scale protein purification and protein sample preparation platforms, and mass spectrometry data analysis is being automated to allow the throughput required. The stoichiometries of the purified complexes are being determined and the quaternary structures of complexes larger than 250 kDa are being solved by single particle EM. We are developing EM tomography approaches to examine whole cells and sectioned, stained material to detect complexes in cells and determine their localization and structures. New image analysis methods will be applied to speed determination of quaternary structures from EM data. Once key components in the interaction network are defined, to test and validate our pathway models, mutant strains not expressing these

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., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (© 2013 Elsevier B.V. All rights reserved.)

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., (Copyright © 2013 Elsevier B.V. All rights reserved.)

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., (Copyright © 2011 Elsevier Inc. All rights reserved.)

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., (Copyright © 2011 Elsevier Inc. All rights reserved.)

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).

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.

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.

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.

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.

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.