Automation of random conical tilt and orthogonal tilt data collection using feature-based correlation

Visualization by electron microscopy has provided many insights into the composition, quaternary structure, and mechanism of macromolecular assemblies. By preserving samples in stain or vitreous ice it is possible to image them as discrete particles, and from these images generate three-dimensional structures. This ‘single-particle’ approach suffers from two major shortcomings; it requires an initial model to reconstitute 2D data into a 3D volume, and it often fails when faced with conformational variability. Random conical tilt (RCT) and orthogonal tilt (OTR) are methods developed to overcome these problems, but the data collection required, particularly for vitreous ice specimens, is difficult and tedious. In this paper, we present an automated approach to RCT/OTR data collection that removes the burden of manual collection and offers higher quality and throughput than is otherwise possible. We show example datasets collected under stain and cryo conditions and provide statistics related to the efficiency and robustness of the process. Furthermore, we describe the new algorithms that make this method possible, which include new calibrations, improved targeting and feature-based tracking.     

The orthogonal tilt reconstruction method: an approach to generating single-class volumes with no missing cone for ab initio reconstruction of asymmetric particles

Generating reliable initial models is a critical step in the reconstruction of asymmetric single-particles by 3D electron microscopy. This is particularly difficult to do if heterogeneity is present in the sample. The Random Conical Tilt (RCT) method, arguably the most robust presently to accomplish this task, requires significant user intervention to solve the "missing cone" problem. We present here a novel approach, termed the orthogonal tilt reconstruction method, that eliminates the missing cone altogether, making it possible for single-class volumes to be used directly as initial references in refinement without further processing. The method involves collecting data at +45 degrees and -45 degrees tilts and only requires that particles adopt a relatively large number of orientations on the grid. One tilted data set is used for alignment and classification and the other set--which provides views orthogonal to those in the first--is used for reconstruction, resulting in the absence of a missing cone. We have tested this method with synthetic data and compared its performance to that of the RCT method. We also propose a way of increasing the level of homogeneity in individual 2D classes (and volumes) in a heterogeneous data set and identifying the most homogeneous volumes.     

Optimized Negative Staining: a High-throughput Protocol for Examining Small and Asymmetric Protein Structure by Electron Microscopy

Structural determination of proteins is rather challenging for proteins with molecular masses between 40 - 200 kDa. Considering that more than half of natural proteins have a molecular mass between 40 - 200 kDa^1,2, a robust and high-throughput method with a nanometer resolution capability is needed. Negative staining (NS) electron microscopy (EM) is an easy, rapid, and qualitative approach which has frequently been used in research laboratories to examine protein structure and protein-protein interactions. Unfortunately, conventional NS protocols often generate structural artifacts on proteins, especially with lipoproteins that usually form presenting rouleaux artifacts. By using images of lipoproteins from cryo-electron microscopy (cryo-EM) as a standard, the key parameters in NS specimen preparation conditions were recently screened and reported as the optimized NS protocol (OpNS), a modified conventional NS protocol ³ . Artifacts like rouleaux can be greatly limited by OpNS, additionally providing high contrast along with reasonably high‐resolution (near 1 nm) images of small and asymmetric proteins. These high-resolution and high contrast images are even favorable for an individual protein (a single object, no average) 3D reconstruction, such as a 160 kDa antibody, through the method of electron tomography^4,5. Moreover, OpNS can be a high‐throughput tool to examine hundreds of samples of small proteins. For example, the previously published mechanism of 53 kDa cholesteryl ester transfer protein (CETP) involved the screening and imaging of hundreds of samples ⁶. Considering cryo-EM rarely successfully images proteins less than 200 kDa has yet to publish any study involving screening over one hundred sample conditions, it is fair to call OpNS a high-throughput method for studying small proteins. Hopefully the OpNS protocol presented here can be a useful tool to push the boundaries of EM and accelerate EM studies into small protein structure, dynamics and mechanisms.     

TomoAlign: A novel approach to correcting sample motion and 3D CTF in CryoET

TomoAlign is a software package that integrates tools to mitigate two important resolution limiting factors in cryoET, namely the beam-induced sample motion and the contrast transfer function (CTF) of the microscope. The package is especially focused on cryoET of thick specimens where fiducial markers are required for accurate tilt-series alignment and sample motion estimation. TomoAlign models the beam-induced sample motion undergone during the tilt-series acquisition. The motion models are used to produce motion-corrected subtilt-series centered on the particles of interest. In addition, the defocus of each particle at each tilt image is determined and can be corrected, resulting in motion-corrected and CTF-corrected subtilt-series from which the subtomograms can be computed. Alternatively, the CTF information can be passed on so that CTF correction can be carried out entirely within external packages like Relion. TomoAlign serves as a versatile tool that can streamline the cryoET workflow from initial alignment of tilt-series to final subtomogram averaging during in situ structure determination.     

An octaheme c-type cytochrome from Shewanella oneidensis can reduce nitrite and hydroxylamine

A c-type cytochrome from Shewanella oneidensis MR-1, containing eight hemes, has been previously designated as an octaheme tetrathionate reductase (OTR). The structure of OTR revealed that the active site contains an unusual lysine-ligated heme, despite the presence of a CXXCH motif in the sequence that would predict histidine ligation. This lysine ligation has been previously observed only in the pentaheme nitrite reductases, suggesting that OTR may have a possible role in nitrite reduction. We have now shown that OTR is an efficient nitrite and hydroxylamine reductase and that ammonium ion is the product. These results indicate that OTR may have a role in the biological nitrogen cycle.     

Structure determination of membrane proteins in five easy pieces

Rotational Alignment (RA) solid-state NMR provides the basis for a general method for determining the structures of membrane proteins in phospholipid bilayers under physiological conditions. Membrane proteins are high priority targets for structure determination, and are challenging for existing experimental methods. Because membrane proteins reside in liquid crystalline phospholipid bilayer membranes it is important to study them in this type of environment. The RA solid-state NMR approach we have developed can be summarized in five steps, and incorporates methods of molecular biology, biochemistry, sample preparation, the implementation of NMR experiments, and structure calculations. It relies on solid-state NMR spectroscopy to obtain high-resolution spectra and residue-specific structural restraints for membrane proteins that undergo rotational diffusion around the membrane normal, but whose mobility is otherwise restricted by interactions with the membrane phospholipids. High resolution spectra of membrane proteins alone and in complex with other proteins and ligands set the stage for structure determination and functional studies of these proteins in their native, functional environment.     

An evaluation of least-squares fits to COSY spectra as a means of estimating proton—proton coupling constants. I. Simulated test problems

Summary A computational method is described that takes an initial estimate of the chemical shifts, line widths and scalar coupling constants for the protons in a molecule, and refines this estimate so as to improve the least-squares fit between an experimental COSY spectrum and the spectrum simulated from these parameters in the weak-coupling approximation. In order to evaluate the potential of such refinements for estimating these parameters from COSY experiments, the method has been applied to a large number of sample problems which were themselves simulated from standard conformations of the amino acids, along with 25 near-native conformations of the protein bovine pancreatic trypsin inhibitor. The results of this evaluation show that: (i) if the chemical shifts are known to within ca. 0.01 ppm and no noise or artifacts are present in the data, the method is capable of recovering the correct coupling constants, starting from essentially arbitrary values, to within 0.1 Hz in almost all cases. (ii) Although the precision of these estimates of the coupling constants is degraded by the limited resolution, noise and artifacts present in most experimental spectra, the large majority of coupling constants can still be recovered to within 1.0 Hz; the local minimum problem is not made significantly worse by such defects in the data. (iii) The method assigns an effective line width to all the resonances, and in the process can resolve overlapping cross peaks. (iv) The method is not capable of determining the chemical shifts a priori, due to the presence of numerous local minima in the least-squares residual as a function of these parameters.     

Use of confocal laser scanning microscopy on soil thin-sections for improved characterization of microbial growth in unconsolidated soils and aquifer materials

Confocal laser scanning microscopy (CLSM) was utilized to examine samples from an aquifer microcosm that was used to investigate microbially mediated losses in hydraulic conductivity. Samples were fixed, dehydrated and dried to prepare the biological material in a fashion similar to that used previously for viewing under the scanning electron microscope. Then, samples were prepared as thin-sections by employing soil micromorphological techniques. Serial images generated by the CLSM technique were visualized using computer three-dimensional rendering software. Results from the CLSM technique were compared with simple fluorescence microscopy of thin-sections and scanning electron microscopy (SEM) of samples from the microcosm. Computer visualization of serial sections with the CLSM technique provided images on a submicron scale in three dimensions. SEM has a much higher resolution, on a nanometer scale, but the results are not three dimensional. Artifacts associated with thin-section preparation are minimal for natural porous media composed mostly of sand, such as aquifer materials. Also, CLSM images are affected minimally by changes to biological material due to sample preparation, whereas artifacts associated with SEM images are very prominent, due to the higher magnification and resolution. CLSM of thin-sections and SEM are very powerful methods for viewing microbial growth in natural porous media, but CLSM is preferable because it allows three-dimensional visualization and measurements of cells and aggregates with few artifacts.     

Superb resolution and contrast of transmission electron microscopy images of unstained biological samples on graphene-coated grids

Background

In standard transmission electron microscopy (TEM), biological samples are supported on carbon films of nanometer thickness. Due to the similar electron scattering of protein samples and graphite supports, high quality images with structural details are obtained primarily by staining with heavy metals.

Methods

Single-layered graphene is used to support the protein self-assemblies of different molecular weights for qualitative and quantitative characterizations.

Results

We show unprecedented high resolution and contrast images of unstained samples on graphene on a low-end TEM. We show for the first time that the resolution and contrast of TEM images of unstained biological samples with high packing density in their native states supported on graphene can be comparable or superior to uranyl acetate-stained TEM images.

Conclusion

Our results demonstrate a novel technique for TEM structural characterization to circumvent the potential artifacts caused by staining agents without sacrificing image resolution or contrast, and eliminate the need for toxic metals. Moreover, this technique better preserves sample integrity for quantitative characterization by dark-field imaging with reduced beam damage.

General significance

This technique can be an effective alternative for bright-field qualitative characterization of biological samples with high packing density and those not amenable to the standard negative staining technique, in addition to providing high quality dark-field unstained images at reduced radiation damage to determine quantitative structural information of biological samples.     

A bioinformatic filter for improved base-call accuracy and polymorphism detection using the Affymetrix GeneChip whole-genome resequencing platform

DNA resequencing arrays enable rapid acquisition of high-quality sequence data. This technology represents a promising platform for rapid high-resolution genotyping of microorganisms. Traditional array-based resequencing methods have relied on the use of specific PCR-amplified fragments from the query samples as hybridization targets. While this specificity in the target DNA population reduces the potential for artifacts caused by cross-hybridization, the subsampling of the query genome limits the sequence coverage that can be obtained and therefore reduces the technique's resolution as a genotyping method. We have developed and validated an Affymetrix Inc. GeneChip(R) array-based, whole-genome resequencing platform for Francisella tularensis, the causative agent of tularemia. A set of bioinformatic filters that targeted systematic base-calling errors caused by cross-hybridization between the whole-genome sample and the array probes and by deletions in the sample DNA relative to the chip reference sequence were developed. Our approach eliminated 91% of the false-positive single-nucleotide polymorphism calls identified in the SCHU S4 query sample, at the cost of 10.7% of the true positives, yielding a total base-calling accuracy of 99.992%.     

Compensation of Missing Wedge Effects with Sequential Statistical Reconstruction in Electron Tomography

Electron tomography (ET) of biological samples is used to study the organization and the structure of the whole cell and subcellular complexes in great detail. However, projections cannot be acquired over full tilt angle range with biological samples in electron microscopy. ET image reconstruction can be considered an ill-posed problem because of this missing information. This results in artifacts, seen as the loss of three-dimensional (3D) resolution in the reconstructed images. The goal of this study was to achieve isotropic resolution with a statistical reconstruction method, sequential maximum a posteriori expectation maximization (sMAP-EM), using no prior morphological knowledge about the specimen. The missing wedge effects on sMAP-EM were examined with a synthetic cell phantom to assess the effects of noise. An experimental dataset of a multivesicular body was evaluated with a number of gold particles. An ellipsoid fitting based method was developed to realize the quantitative measures elongation and contrast in an automated, objective, and reliable way. The method statistically evaluates the sub-volumes containing gold particles randomly located in various parts of the whole volume, thus giving information about the robustness of the volume reconstruction. The quantitative results were also compared with reconstructions made with widely-used weighted backprojection and simultaneous iterative reconstruction technique methods. The results showed that the proposed sMAP-EM method significantly suppresses the effects of the missing information producing isotropic resolution. Furthermore, this method improves the contrast ratio, enhancing the applicability of further automatic and semi-automatic analysis. These improvements in ET reconstruction by sMAP-EM enable analysis of subcellular structures with higher three-dimensional resolution and contrast than conventional methods.     

Assessing the significance of conserved genomic aberrations using high resolution genomic microarrays

Genomic aberrations recurrent in a particular cancer type can be important prognostic markers for tumor progression. Typically in early tumorigenesis, cells incur a breakdown of the DNA replication machinery that results in an accumulation of genomic aberrations in the form of duplications, deletions, translocations, and other genomic alterations. Microarray methods allow for finer mapping of these aberrations than has previously been possible; however, data processing and analysis methods have not taken full advantage of this higher resolution. Attention has primarily been given to analysis on the single sample level, where multiple adjacent probes are necessarily used as replicates for the local region containing their target sequences. However, regions of concordant aberration can be short enough to be detected by only one, or very few, array elements. We describe a method called Multiple Sample Analysis for assessing the significance of concordant genomic aberrations across multiple experiments that does not require a-priori definition of aberration calls for each sample. If there are multiple samples, representing a class, then by exploiting the replication across samples our method can detect concordant aberrations at much higher resolution than can be derived from current single sample approaches. Additionally, this method provides a meaningful approach to addressing population-based questions such as determining important regions for a cancer subtype of interest or determining regions of copy number variation in a population. Multiple Sample Analysis also provides single sample aberration calls in the locations of significant concordance, producing high resolution calls per sample, in concordant regions. The approach is demonstrated on a dataset representing a challenging but important resource: breast tumors that have been formalin-fixed, paraffin-embedded, archived, and subsequently UV-laser capture microdissected and hybridized to two-channel BAC arrays using an amplification protocol. We demonstrate the accurate detection on simulated data, and on real datasets involving known regions of aberration within subtypes of breast cancer at a resolution consistent with that of the array. Similarly, we apply our method to previously published datasets, including a 250K SNP array, and verify known results as well as detect novel regions of concordant aberration. The algorithm has been fully implemented and tested and is freely available as a Java application at http://www.cbil.upenn.edu/MSA.     

Spatial resolution of the variable-period x-ray standing-wave method as applied to model membranes.

A series of model membranes as Langmuir-Blodgett (LB) films composed of long-chain zinc alkanoates (saturated fatty acid salts) was used to evaluate the spatial resolution of the variable-period x-ray standing-wave (XSW) technique. The chain length dependence of the zinc mean position (z) above the supporting substrate demonstrates that it is possible to detect differences in (z) of 1-2 A. Thus 1-2 A is the spatial resolution of the method in the current application. The data show that the chain tilt angle is chain length dependent, varying from 40 degrees to 0 degrees for alkanoates 18 and 24 carbon atoms long, respectively. The spread about the mean position of the zinc in the film, sigma(in), was found to be independent of chain length at 10.0 A for all members of the series. Sigma(in) was shown to be insensitive to the presence of a "spacer" omega-tricosenoic acid (omegaTA) bilayer placed between the zinc alkanoate LB film and the coated gold mirror. However, an overlayer of omegaTA sharpened the zinc ion distribution and lowered the chain tilt angle. This study provides important information regarding sample composition and constitution that facilitates membrane structure determination by XSWs.     

High resolution detection of uncoated metaphase chromosomes by means of field emission scanning electron microscopy

HeLa metaphase chromosomes were exammed by means of in lens field emission scanning electron microscopy, which permits high resolution detection of uncoated biological samples. By using uncoated chromosomes as a model for comparison we report evidence of how traditional scanning electron microscopy techniques such as metal coating and conductive methods can generate errors in chromosome structure evaluation, since both give rise to morphological artifacts. By comparing the morphology of uncoated chromosomes obtained by two different isolation procedures, such as that utilized in standard cytogenetics and the polyamine method, we have drawn the following conclusions: (a) the standard cytogenetic method gives rise to a chromosome structure consisting of a flattened network of 10 nm fibers, in which higher order chromatin organization is absent. (b) Chromosomes obtained by the polyamine method show both three-dimensional profile and higher level folding of chromatin fibers, supporting the loop chromosome organization previously suggested by scanning electron microscopy observation of hexylene glycol isolated chromosomes.     

Exploratory data analysis of DNA microarrays by multivariate curve resolution

In this work, the application of a multivariate curve resolution procedure based on alternating least squares optimization (MCR-ALS) for the analysis of data from DNA microarrays is proposed. For this purpose, simulated and publicly available experimental data sets have been analyzed. Application of MCR-ALS, a method that operates without the use of any training set, has enabled the resolution of the relevant information about different cancer lines classification using a set of few components; each of these defined by a sample and a pure gene expression profile. From resolved sample profiles, a classification of samples according to their origin is proposed. From the resolved pure gene expression profiles, a set of over- or underexpressed genes that could be related to the development of cancer diseases has been selected. Advantages of the MCR-ALS procedure in relation to other previously proposed procedures such as principal component analysis are discussed.     

Predictive metabolite profiling applying hierarchical multivariate curve resolution to GC-MS data--a potential tool for multi-parametric diagnosis

A method for predictive metabolite profiling based on resolution of GC-MS data followed by multivariate data analysis is presented and applied to three different biofluid data sets (rat urine, aspen leaf extracts, and human blood plasma). Hierarchical multivariate curve resolution (H-MCR) was used to simultaneously resolve the GC-MS data into pure profiles, describing the relative metabolite concentrations between samples, for multivariate analysis. Here, we present an extension of the H-MCR method allowing treatment of independent samples according to processing parameters estimated from a set of training samples. Predictions or inclusion of the new samples, based on their metabolite profiles, into an existing model could then be carried out, which is a requirement for a working application within, e.g., clinical diagnosis. Apart from allowing treatment and prediction of independent samples the proposed method also reduces the time for the curve resolution process since only a subset of representative samples have to be processed while the remaining samples can be treated according to the obtained processing parameters. The time required for resolving the 30 training samples in the rat urine example was approximately 13 h, while the treatment of the 30 test samples according to the training parameters required only approximately 30 s per sample (approximately 15 min in total). In addition, the presented results show that the suggested approach works for describing metabolic changes in different biofluids, indicating that this is a general approach for high-throughput predictive metabolite profiling, which could have important applications in areas such as plant functional genomics, drug toxicity, treatment efficacy and early disease diagnosis.     

Tilt-series and electron microscope alignment for the correction of the non-perpendicularity of beam and tilt-axis

In electron tomography the sample is tilted in the electron microscope and projections are recorded at different viewing angles. In the correct geometric setting, the tilt-axis of the object under scrutiny is perpendicular to the beam direction. However, we will demonstrate that this does not necessarily apply to all electron microscopes equipped with the default column alignment. The resulting effect is that a conical tilt is performed, which has to be considered in the reconstruction to avoid artifacts and to improve the resolution. A novel solution, with significantly improved convergence properties, will be introduced for calculating the three-dimensional marker model, which is necessary for the alignment of the tilt-series. Thereby, the angle between the beam direction and the tilt-axis is calculated, together with other geometrical distortions, like magnification and rotation changes, and incorporated in the reconstruction. Hereby, artifacts can be eliminated at the image processing basis, and the resolution can be significantly improved at the medium to high range frequencies. Synthetical and real data are used to demonstrate the obstructions caused by this effect and the quality improvement of the reconstructions. Finally, we also present a way to align the hardware of the microscope to correct for the non-perpendicularity between the beam direction and the tilt-axis, which is specifically tailored for tomographic applications.     

A bioinformatic filter for improved base-call accuracy and polymorphism detection using the Affymetrix GeneChip® whole-genome resequencing platform

DNA resequencing arrays enable rapid acquisition of high-quality sequence data. This technology represents a promising platform for rapid high-resolution genotyping of microorganisms. Traditional array-based resequencing methods have relied on the use of specific PCR-amplified fragments from the query samples as hybridization targets. While this specificity in the target DNA population reduces the potential for artifacts caused by cross-hybridization, the subsampling of the query genome limits the sequence coverage that can be obtained and therefore reduces the technique's resolution as a genotyping method. We have developed and validated an Affymetrix Inc. GeneChip® array-based, whole-genome resequencing platform for Francisella tularensis, the causative agent of tularemia. A set of bioinformatic filters that targeted systematic base-calling errors caused by cross-hybridization between the whole-genome sample and the array probes and by deletions in the sample DNA relative to the chip reference sequence were developed. Our approach eliminated 91% of the false-positive single-nucleotide polymorphism calls identified in the SCHU S4 query sample, at the cost of 10.7% of the true positives, yielding a total base-calling accuracy of 99.992%.     

Differences in uterine immunoexpression of PR, ERα and OTR when comparing prostaglandin- to progestagen-based protocols for ovine estrus synchronization

The aim of this work was to compare PR, ERα and OTR uterine expression between days 9 and 21 of pregnancy in ewes whose estrus had been synchronized with two different protocols. Sixty-four adult Manchega ewes were synchronized with either conventional progestagens (P) or prostaglandin analogues (PG), and mated. Uterine samples were obtained from pregnant animals (group P, n=24; group PG, n=25) on days 9 post coitus (pc), 13pc, 15pc, 17pc and 21pc. Immunohistochemical detection of progesterone receptor (PR), estrogen receptor-α (ERα) and oxytocin receptor (OTR) was assessed in different uterine cell compartments including luminal and glandular epithelium, stroma and myometrium. Interaction day × treatment was obtained when assessing PR expression in the caruncular stroma (P=0.027) and myometrium (P=0.000), as well as for ERα in the superficial stroma (P=0.05). Significant "day post coitus" effect was found regarding to PR (P<0.01, with the exception of the superficial stroma, deep stroma and myometrium), ERα (P<0.01), and OTR (P<0.05, except in the deep compartments). No significant "treatment" effect was found for PR, ERα or OTR protein immunoexpression. This study supports the implication of PR, ERα and OTR within days 9-21 of the ovine pregnancy. Moreover, different expression pattern of PR and ERα proteins has been found between treatments in various compartments studied. Collectively, these results indicate that PR, ERα and OTR expression during early pregnancy is similar between ewes treated with either progestagens or prostaglandin analogues-based protocols for estrus synchronization.     

Reconstruction of the probe angular distribution from a series of electron spin resonance spectra of tilted oriented samples.

Model-independent methods for the reconstruction of the nitroxide spin probe angular distribution of labeled oriented biological assemblies from electron spin resonance (ESR) spectra were investigated. We found that accurate probe angular distribution information could be obtained from the simultaneous consideration of a series of ESR spectra originating from a sample at differing tilt angles relative to the Zeeman magnetic field. Using simulated tilt series data sets, we developed a consistent criteria for judging the reliability of the simulated fit to the data as a function of the free spectral parameters and thereby have increased the significance of the model-independent reconstruction of the probe angular distribution derived from the fit. We have also enhanced the angular resolution measurable with the model-independent methodology by increasing the rank of the order parameters that we can reliably deduce from a spectrum. This enhancement allows us to accurately deduce higher resolution features of the spin probe distribution. Finally we investigated the usefulness of fitting the tilt series data in multiple data sets such that tilt series data from many identical sample preparations are fitted simultaneously. This method proved to be useful in rapidly reducing a large amount of data by eliminating any redundant computations in the application of the enhanced model-independent analysis to identical sets of tilt series data. We applied the methodology developed here to ESR spectra from probe labeled muscle fibers to study the orientation of myosin cross-bridges in fibers. This application is described in the accompanying paper.