Accurate, automatic determination of astigmatism and phase with Ctfplotter in IMOD.

Ctfplotter in the IMOD software package is a flexible program for determination of CTF parameters in tilt series images. It uses a novel approach to find astigmatism by measuring defocus in one-dimensional power spectra rotationally averaged over a series of restricted angular ranges. Comparisons with Ctffind, Gctf, and Warp show that Ctfplotter's estimated astigmatism is generally more reliable than that found by these programs that fit CTF parameters to two-dimensional power spectra, especially at higher tilt angles. In addition to that intrinsic advantage, Ctfplotter can reduce the variability in astigmatism estimates further by summing results over multiple tilt angles (typically 5), while still finding defocus for each individual image. Its fitting strategy also produces better phase estimates. The program now includes features for tuning the sampling of the power spectrum so that it is well-represented for analysis, and for determining an appropriate fitting range that can vary with tilt angle. It can thus be used automatically in a variety of situations, not just for fitting tilt series, and has been integrated into the SerialEM acquisition software for real-time determination of focus and astigmatism.

Software tools for automated transmission electron microscopy.

The demand for high-throughput data collection in electron microscopy is increasing for applications in structural and cellular biology. Here we present a combination of software tools that enable automated acquisition guided by image analysis for a variety of transmission electron microscopy acquisition schemes. SerialEM controls microscopes and detectors and can trigger automated tasks at multiple positions with high flexibility. Py-EM interfaces with SerialEM to enact specimen-specific image-analysis pipelines that enable feedback microscopy. As example applications, we demonstrate dose reduction in cryo-electron microscopy experiments, fully automated acquisition of every cell in a plastic section and automated targeting on serial sections for 3D volume imaging across multiple grids.

Automated tilt series alignment and tomographic reconstruction in IMOD.

Automated tomographic reconstruction is now possible in the IMOD software package, including the merging of tomograms taken around two orthogonal axes. Several developments enable the production of high-quality tomograms. When using fiducial markers for alignment, the markers to be tracked through the series are chosen automatically; if there is an excess of markers available, a well-distributed subset is selected that is most likely to track well. Marker positions are refined by applying an edge-enhancing Sobel filter, which results in a 20% improvement in alignment error for plastic-embedded samples and 10% for frozen-hydrated samples. Robust fitting, in which outlying points are given less or no weight in computing the fitting error, is used to obtain an alignment solution, so that aberrant points from the automated tracking can have little effect on the alignment. When merging two dual-axis tomograms, the alignment between them is refined from correlations between local patches; a measure of structure was developed so that patches with insufficient structure to give accurate correlations can now be excluded automatically. We have also developed a script for running all steps in the reconstruction process with a flexible mechanism for setting parameters, and we have added a user interface for batch processing of tilt series to the Etomo program in IMOD. Batch processing is fully compatible with interactive processing and can increase efficiency even when the automation is not fully successful, because users can focus their effort on the steps that require manual intervention.

The challenge of determining handedness in electron tomography and the use of DNA origami gold nanoparticle helices as molecular standards.

The apparent handedness of an EM-tomography reconstruction depends on a number of conventions and can be confused in many ways. As the number of different hardware and software combinations being used for electron tomography continue to climb, and the reconstructions being produced reach higher and higher resolutions, the need to verify the hand of the results has increased. Here we enumerate various steps in a typical tomography experiment that affect handedness and show that DNA origami gold nanoparticle helices can be used as convenient and fail-safe handedness standards.

Clustering and variance maps for cryo-electron tomography using wedge-masked differences.

Cryo-electron tomography provides 3D imaging of frozen hydrated biological samples with nanometer resolution. Reconstructed volumes suffer from low signal-to-noise-ratio (SNR)(1) and artifacts caused by systematically missing tomographic data. Both problems can be overcome by combining multiple subvolumes with varying orientations, assuming they contain identical structures. Clustering (unsupervised classification) is required to ensure or verify population homogeneity, but this process is complicated by the problems of poor SNR and missing data, the factors that led to consideration of multiple subvolumes in the first place. Here, we describe a new approach to clustering and variance mapping in the face of these difficulties. The combined subvolume is taken as an estimate of the true subvolume, and the effect of missing data is computed for individual subvolumes. Clustering and variance mapping then proceed based on differences between expected and observed subvolumes. We show that this new method is faster and more accurate than two current, widely used techniques.

ER sliding dynamics and ER-mitochondrial contacts occur on acetylated microtubules.

The endoplasmic reticulum (ER) network is extremely dynamic in animal cells, yet little is known about the mechanism and function of its movements. The most common ER dynamic, termed ER sliding, involves ER tubule extension along stable microtubules (MTs). In this study, we show that ER sliding occurs on nocodazole-resistant MTs that are posttranslationally modified by acetylation. We demonstrate that high MT curvature is a good indicator of MT acetylation and show in live cells that ER sliding occurs predominantly on these curved, acetylated MTs. Furthermore, increasing MT acetylation by drug treatment increases the frequency of ER sliding. One purpose of the ER sliding on modified MT tracts could be to regulate its interorganelle contacts. We find that all mitochondria and many endosomes maintain contact with the ER despite the movements of each. However, mitochondria, but not endosomes, preferentially localize to acetylated MTs. Thus, different ER dynamics may occur on distinct MT populations to establish or maintain contacts with different organelles.

CTF determination and correction for low dose tomographic tilt series.

The resolution of cryo-electron tomography can be limited by the first zero of the microscope's contrast transfer function (CTF). To achieve higher resolution, it is critical to determine the CTF and correct its phase inversions. However, the extremely low signal-to-noise ratio (SNR) and the defocus gradient in the projections of tilted specimens make this process challenging. Two programs, CTFPLOTTER and CTFPHASEFLIP, have been developed to address these issues. CTFPLOTTER obtains a 1D power spectrum by periodogram averaging and rotational averaging and it estimates the noise background with a novel approach, which uses images taken with no specimen. The background-subtracted 1D power spectra from image regions at different defocus values are then shifted to align their first zeros and averaged together. This averaging improves the SNR sufficiently that it becomes possible to determine the defocus for subsets of the tilt series rather than just the entire series. CTFPHASEFLIP corrects images line-by-line by inverting phases appropriately in thin strips of the image at nearly constant defocus. CTF correction by these methods is shown to improve the resolution of aligned, averaged particles extracted from tomograms. However, some restoration of Fourier amplitudes at high frequencies is important for seeing the benefits from CTF correction.

Fibrils connect microtubule tips with kinetochores: a mechanism to couple tubulin dynamics to chromosome motion.

Kinetochores of mitotic chromosomes are coupled to spindle microtubules in ways that allow the energy from tubulin dynamics to drive chromosome motion. Most kinetochore-associated microtubule ends display curving "protofilaments," strands of tubulin dimers that bend away from the microtubule axis. Both a kinetochore "plate" and an encircling, ring-shaped protein complex have been proposed to link protofilament bending to poleward chromosome motion. Here we show by electron tomography that slender fibrils connect curved protofilaments directly to the inner kinetochore. Fibril-protofilament associations correlate with a local straightening of the flared protofilaments. Theoretical analysis reveals that protofilament-fibril connections would be efficient couplers for chromosome motion, and experimental work on two very different kinetochore components suggests that filamentous proteins can couple shortening microtubules to cargo movements. These analyses define a ring-independent mechanism for harnessing microtubule dynamics directly to chromosome movement.

Organization of interphase microtubules in fission yeast analyzed by electron tomography.

Polarized cells, such as neuronal, epithelial, and fungal cells, all display a specialized organization of their microtubules (MTs). The interphase MT cytoskeleton of the rod-shaped fission yeast, Schizosaccharomyces pombe, has been extensively described by fluorescence microscopy. Here, we describe a large-scale, electron tomography investigation of S. pombe, including a 3D reconstruction of a complete eukaryotic cell volume at sufficient resolution to show both how many MTs there are in a bundle and their detailed architecture. Most cytoplasmic MTs are open at one end and capped at the other, providing evidence about their polarity. Electron-dense bridges between the MTs themselves and between MTs and the nuclear envelope were frequently observed. Finally, we have investigated structure/function relationships between MTs and both mitochondria and vesicles. Our analysis shows that electron tomography of well-preserved cells is ideally suited for describing fine ultrastructural details that were not visible with previous techniques.

Automated electron microscope tomography using robust prediction of specimen movements.

A new method was developed to acquire images automatically at a series of specimen tilts, as required for tomographic reconstruction. The method uses changes in specimen position at previous tilt angles to predict the position at the current tilt angle. Actual measurement of the position or focus is skipped if the statistical error of the prediction is low enough. This method allows a tilt series to be acquired rapidly when conditions are good but falls back toward the traditional approach of taking focusing and tracking images when necessary. The method has been implemented in a program, SerialEM, that provides an efficient environment for data acquisition. This program includes control of an energy filter as well as a low-dose imaging mode, in which tracking and focusing occur away from the area of interest. The program can automatically acquire a montage of overlapping frames, allowing tomography of areas larger than the field of the CCD camera. It also includes tools for navigating between specimen positions and finding regions of interest.

The three-dimensional arrangement of intermediate filaments in Romney wool cortical cells.

The three-dimensional orientation and arrangement of intermediate filaments in Romney wool ortho-, meso-, and paracortical cells has been revealed using single axis high voltage electron tomography. Modelled tomograms confirm that intermediate filaments in orthocortical cells are arranged helically, with the helical angle progressively increasing from the centre to the periphery of macrofibrils. Intermediate filaments in meso- and paracortical cells display parallel arrangements differing mainly in packing density, with the mesocortex packed more tightly than the paracortex. The intermediate filament arrangements observed confirm expectations based on earlier two-dimensional transmission electron microscopy observations by the authors and other researchers. It is expected that these findings will contribute to a better understanding of the biological and structural basis of wool fibre curvature.

Three-dimensional ultrastructure of Saccharomyces cerevisiae meiotic spindles.

Meiotic chromosome segregation leads to the production of haploid germ cells. During meiosis I (MI), the paired homologous chromosomes are separated. Meiosis II (MII) segregation leads to the separation of paired sister chromatids. In the budding yeast Saccharomyces cerevisiae, both of these divisions take place in a single nucleus, giving rise to the four-spored ascus. We have modeled the microtubules in 20 MI and 15 MII spindles by using reconstruction from electron micrographs of serially sectioned meiotic cells. Meiotic spindles contain more microtubules than their mitotic counterparts, with the highest number in MI spindles. It is possible to differentiate between MI versus MII spindles based on microtubule numbers and organization. Similar to mitotic spindles, kinetochores in either MI or MII are attached by a single microtubule. The models indicate that the kinetochores of paired homologous chromosomes in MI or sister chromatids in MII are separated at metaphase, similar to mitotic cells. Examination of both MI and MII spindles reveals that anaphase A likely occurs in addition to anaphase B and that these movements are concurrent. This analysis offers a structural basis for considering meiotic segregation in yeast and for the analysis of mutants defective in this process.

A fast reconstruction algorithm for electron microscope tomography.

We have implemented a Fast Fourier Summation algorithm for tomographic reconstruction of three-dimensional biological data sets obtained via transmission electron microscopy. We designed the fast algorithm to reproduce results obtained by the direct summation algorithm (also known as filtered or R-weighted backprojection). For two-dimensional images, the new algorithm scales as O(N(theta)M log M)+O(MN log N) operations, where N(theta) is the number of projection angles and M x N is the size of the reconstructed image. Three-dimensional reconstructions are constructed from sequences of two-dimensional reconstructions. We demonstrate the algorithm on real data sets. For typical sizes of data sets, the new algorithm is 1.5-2.5 times faster than using direct summation in the space domain. The speed advantage is even greater as the size of the data sets grows. The new algorithm allows us to use higher order spline interpolation of the data without additional computational cost. The algorithm has been incorporated into a commonly used package for tomographic reconstruction.

Fast multiple alignment of ungapped DNA sequences using information theory and a relaxation method.

An information theory based multiple alignment ("Malign") method was used to align the DNA binding sequences of the OxyR and Fis proteins, whose sequence conservation is so spread out that it is difficult to identify the sites. In the algorithm described here, the information content of the sequences is used as a unique global criterion for the quality of the alignment. The algorithm uses look-up tables to avoid recalculating computationally expensive functions such as the logarithm. Because there are no arbitrary constants and because the results are reported in absolute units (bits), the best alignment can be chosen without ambiguity. Starting from randomly selected alignments, a hill-climbing algorithm can track through the immense space of s(n) combinations where s is the number of sequences and n is the number of positions possible for each sequence. Instead of producing a single alignment, the algorithm is fast enough that one can afford to use many start points and to classify the solutions. Good convergence is indicated by the presence of a single well-populated solution class having higher information content than other classes. The existence of several distinct classes for the Fis protein indicates that those binding sites have self-similar features.