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.

Building bridges between cellular and molecular structural biology.

The integration of cellular and molecular structural data is key to understanding the function of macromolecular assemblies and complexes in their in vivo context. Here we report on the outcomes of a workshop that discussed how to integrate structural data from a range of public archives. The workshop identified two main priorities: the development of tools and file formats to support segmentation (that is, the decomposition of a three-dimensional volume into regions that can be associated with defined objects), and the development of tools to support the annotation of biological structures.

Practical Experience with Hole-Free Phase Plates for Cryo Electron Microscopy.

Phase plate (PP) imaging has proven to be valuable in transmission cryo electron microscopy of unstained, native-state biological specimens. Many PP types have been described, however until the recent implementation of the "hole-free" phase plate (HFPP), imaging has been challenging. We found the HFPP to be simple to construct and to set up in the transmission electron microscopy, but care in implementing automated data collection is needed.

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.

MRC2014: Extensions to the MRC format header for electron cryo-microscopy and tomography.

The MRC binary file format is widely used in the three-dimensional electron microscopy field for storing image and volume data. Files contain a header which describes the kind of data held, together with other important metadata. In response to advances in electron microscopy techniques, a number of variants to the file format have emerged which contain useful additional data, but which limit interoperability between different software packages.

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.

Data management challenges in three-dimensional EM.

This report describes the outcomes of the Data Management Challenges in 3D Electron Microscopy workshop. Key topics discussed include data models, validation and raw-data archiving. The meeting participants agreed that the EMDataBank should take the lead in addressing these issues, and concrete action points were agreed upon that will have a substantial impact on the accessibility of three-dimensional EM data in biology and medicine.

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.

Exploring the retinal connectome.

Purpose: A connectome is a comprehensive description of synaptic connectivity for a neural domain. Our goal was to produce a connectome data set for the inner plexiform layer of the mammalian retina. This paper describes our first retinal connectome, validates the method, and provides key initial findings.

Electron tomography of RabA4b- and PI-4Kβ1-labeled trans Golgi network compartments in Arabidopsis.

The trans Golgi network (TGN) of plant cells sorts and packages Golgi products into secretory (SV) and clathrin-coated (CCV) vesicles. We have analyzed of TGN cisternae in Arabidopsis root meristem cells by cell fractionation and electron microscopy/tomography to establish reliable criteria for identifying TGN cisternae in plant cells, and to define their functional attributes. Transformation of a trans Golgi cisterna into a Golgi-associated TGN cisterna begins with cisternal peeling, the formation of SV buds outside the plane of the cisterna and a 30-35% reduction in cisternal membrane area.

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.

Cryo-electron tomography of microtubule-kinesin motor complexes.

Microtubules complexed with molecular motors of the kinesin family or non-motor microtubule associated proteins (MAPs) such as tau or EB1 have been the subject of cryo-electron microcopy based 3-D studies for several years. Most of these studies that targeted complexes with intact microtubules have been carried out by helical 3-D reconstruction, while few were analyzed by single particle approaches or from 2-D crystalline arrays. Helical reconstruction of microtubule-MAP or motor complexes has been extremely successful but by definition, all helical 3-D reconstruction attempts require perfectly helical assemblies, which presents a serious limitation and confines the attempts to 15- or 16-protofilament microtubules, microtubule configurations that are very rare in nature.

A computational framework for ultrastructural mapping of neural circuitry.

Circuitry mapping of metazoan neural systems is difficult because canonical neural regions (regions containing one or more copies of all components) are large, regional borders are uncertain, neuronal diversity is high, and potential network topologies so numerous that only anatomical ground truth can resolve them. Complete mapping of a specific network requires synaptic resolution, canonical region coverage, and robust neuronal classification. Though transmission electron microscopy (TEM) remains the optimal tool for network mapping, the process of building large serial section TEM (ssTEM) image volumes is rendered difficult by the need to precisely mosaic distorted image tiles and register distorted mosaics.

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.

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.

Correction for non-perpendicularity of beam and tilt axis in tomographic reconstructions with the IMOD package.

A correction for non-perpendicularity between the beam axis and the tilt axis in electron tomographic tilt series has been implemented in the IMOD software package and its value and limitations have been explored. Correction for this effect can provide a significant improvement in the alignment error and the reconstruction quality in some cases. However, when the projection model being fit includes an anisotropic shrinkage (i.

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.

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.

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.

New views of cells in 3D: an introduction to electron tomography.

The most important goal of structural cell biology is to elucidate the mechanisms of the processes of life. The structure of a membrane system or fibrous array, the changes in such structures over time or the localizations of enzymes relative to organelle boundaries can often illuminate associated cellular functions. To be of maximal value, structural studies should provide isotropic, 3D information about well-preserved samples at the highest possible resolution.

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.

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.