@article {49087, title = {cisTEM, User-friendly software for single-particle image processing.}, journal = {eLife}, volume = {7}, year = {2018}, month = {2018 Mar 07}, abstract = {

We have developed new open-source software calledTEM (computational imaging system for transmission electron microscopy) for the processing of data for high-resolution electron cryo-microscopy and single-particle averaging.TEM features a graphical user interface that is used to submit jobs, monitor their progress, and display results. It implements a full processing pipeline including movie processing, image defocus determination, automatic particle picking, 2D classification, ab-initio 3D map generation from random parameters, 3D classification, and high-resolution refinement and reconstruction. Some of these steps implement newly-developed algorithms; others were adapted from previously published algorithms. The software is optimized to enable processing of typical datasets (2000 micrographs, 200k - 300k particles) on a high-end, CPU-based workstation in half a day or less, comparable to GPU-accelerated processing. Jobs can also be scheduled on large computer clusters using flexible run profiles that can be adapted for most computing environments.TEM is available for download from cistem.org.

}, issn = {2050-084X}, doi = {10.7554/eLife.35383}, author = {Grant, Timothy and Rohou, Alexis and Grigorieff, Nikolaus} } @article {46998, title = {CTFFIND4: Fast and accurate defocus estimation from electron micrographs.}, journal = {Journal of Structural Biology}, volume = {192}, year = {2015}, month = {2015 Aug 13}, pages = {216-21}, abstract = {

CTFFIND is a widely-used program for the estimation of objective lens defocus parameters from transmission electron micrographs. Defocus parameters are estimated by fitting a model of the microscope{\textquoteright}s contrast transfer function (CTF) to an image{\textquoteright}s amplitude spectrum. Here we describe modifications to the algorithm which make it significantly faster and more suitable for use with images collected using modern technologies such as dose fractionation and phase plates. We show that this new version preserves the accuracy of the original algorithm while allowing for higher throughput. We also describe a measure of the quality of the fit as a function of spatial frequency and suggest this can be used to define the highest resolution at which CTF oscillations were successfully modeled.

}, issn = {1095-8657}, doi = {10.1016/j.jsb.2015.08.008}, author = {Rohou, Alexis and Grigorieff, Nikolaus} } @article {48313, title = {Peptide dimer structure in an Aβ(1-42) fibril visualized with cryo-EM.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {112}, year = {2015}, month = {2015 Sep 22}, pages = {11858-63}, abstract = {

Alzheimer{\textquoteright}s disease (AD) is a fatal neurodegenerative disorder in humans and the main cause of dementia in aging societies. The disease is characterized by the aberrant formation of β-amyloid (Aβ) peptide oligomers and fibrils. These structures may damage the brain and give rise to cerebral amyloid angiopathy, neuronal dysfunction, and cellular toxicity. Although the connection between AD and Aβ fibrillation is extensively documented, much is still unknown about the formation of these Aβ aggregates and their structures at the molecular level. Here, we combined electron cryomicroscopy, 3D reconstruction, and integrative structural modeling methods to determine the molecular architecture of a fibril formed by Aβ(1-42), a particularly pathogenic variant of Aβ peptide. Our model reveals that the individual layers of the Aβ fibril are formed by peptide dimers with face-to-face packing. The two peptides forming the dimer possess identical tilde-shaped conformations and interact with each other by packing of their hydrophobic C-terminal β-strands. The peptide C termini are located close to the main fibril axis, where they produce a hydrophobic core and are surrounded by the structurally more flexible and charged segments of the peptide N termini. The observed molecular architecture is compatible with the general chemical properties of Aβ peptide and provides a structural basis for various biological observations that illuminate the molecular underpinnings of AD. Moreover, the structure provides direct evidence for a steric zipper within a fibril formed by full-length Aβ peptide.

}, keywords = {Amino Acid Sequence, Amyloid, Amyloid beta-Peptides, Cryoelectron Microscopy, Epitope Mapping, Image Processing, Computer-Assisted, Immunoglobulin Fab Fragments, Models, Molecular, Molecular Sequence Data, Peptide Fragments, Peptides, Protein Multimerization, Protein Structure, Secondary}, issn = {1091-6490}, doi = {10.1073/pnas.1503455112}, author = {Schmidt, Matthias and Rohou, Alexis and Lasker, Keren and Yadav, Jay K and Schiene-Fischer, Cordelia and F{\"a}ndrich, Marcus and Grigorieff, Nikolaus} } @article {47241, title = {Structure and conformational states of the bovine mitochondrial ATP synthase by cryo-EM.}, journal = {eLife}, volume = {4}, year = {2015}, month = {2015 Oct 06}, abstract = {

Adenosine triphosphate (ATP), the chemical energy currency of biology, is synthesized in eukaryotic cells primarily by the mitochondrial ATP synthase. ATP synthases operate by a rotary catalytic mechanism where proton translocation through the membrane-inserted FO region is coupled to ATP synthesis in the catalytic F1 region via rotation of a central rotor subcomplex. We report here single particle electron cryomicroscopy (cryo-EM) analysis of the bovine mitochondrial ATP synthase. Combining cryo-EM data with bioinformatic analysis allowed us to determine the fold of the a subunit, suggesting a proton translocation path through the FO region that involves both the a and b subunits. 3D classification of images revealed seven distinct states of the enzyme that show different modes of bending and twisting in the intact ATP synthase. Rotational fluctuations of the c8-ring within the FO region support a Brownian ratchet mechanism for proton-translocation-driven rotation in ATP synthases.

}, issn = {2050-084X}, doi = {10.7554/eLife.10180}, author = {Zhou, Anna and Rohou, Alexis and Schep, Daniel G and Bason, John V and Montgomery, Martin G and Walker, John E and Grigorieff, Nikolaus and Rubinstein, John L} } @article {6959, title = {Frealix: Model-based refinement of helical filament structures from electron micrographs.}, journal = {Journal of Structural Biology}, volume = {186}, year = {2014}, month = {2014 Mar 20}, pages = {234-44}, abstract = {

The structures of many helical protein filaments can be derived from electron micrographs of their suspensions in thin films of vitrified aqueous solutions. The most successful and generally-applicable approach treats short segments of these filaments as independent "single particles", yielding near-atomic resolution for rigid and well-ordered filaments. The single-particle approach can also accommodate filament deformations, yielding sub-nanometer resolution for more flexible filaments. However, in the case of thin and flexible filaments, such as some amyloid-β (Aβ) fibrils, the single-particle approach may fail because helical segments can be curved or otherwise distorted and their alignment can be inaccurate due to low contrast in the micrographs. We developed new software called Frealix that allows the use of arbitrarily short filament segments during alignment to approximate even high curvatures. All segments in a filament are aligned simultaneously with constraints that ensure that they connect to each other in space to form a continuous helical structure. In this paper, we describe the algorithm and benchmark it against datasets of Aβ(1-40) fibrils and tobacco mosaic virus (TMV), both analyzed in earlier work. In the case of TMV, our algorithm achieves similar results to single-particle analysis. In the case of Aβ(1-40) fibrils, we match the previously-obtained resolution but we are also able to obtain reliable alignments and \~{}8-{\r A} reconstructions from curved filaments. Our algorithm also offers a detailed characterization of filament deformations in three dimensions and enables a critical evaluation of the worm-like chain model for biological filaments.

}, issn = {1095-8657}, doi = {10.1016/j.jsb.2014.03.012}, author = {Rohou, Alexis and Grigorieff, Nikolaus} }