A Toolbox for ab initio 3-D reconstructions in single-particle electron microscopy (original) (raw)
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Bioinformatics, 2010
Motivation: Electron cryo-microscopy can be used to infer 3D structures of large macromolecules with high resolution, but the large amounts of data captured necessitate the development of appropriate statistical models to describe the data generation process, and to perform structure inference. We present a new method for performing ab initio inference of the 3D structures of macromolecules from single particle electron cryo-microscopy experiments using class average images. Results: We demonstrate this algorithm on one phantom, one synthetic dataset and three real (experimental) datasets (ATP synthase, V-type ATPase and GroEL). Structures consistent with the known structures were inferred for all datasets.
Journal of Synchrotron Radiation, 2004
Although the methodology of molecular microscopy has enormous potential, it is time consuming and labor intensive. The techniques required to produce a three dimensional (3D) electron density map of a macromolecular structure normally require manual operation of an electron microscope by a skilled operator and manual supervision of the sometimes complex software needed for analysis and calculation of 3D maps. We are developing systems to automate the process of data acquisition from an electron microscope and integrating these systems with specimen handling operations and post acquisition data processing. We report here on the current performance of our existing systems and the future challenges involved in substantially improving both the sustained throughput and the yield of automated data collection and analysis.
Bsoft: Image processing and molecular modeling for electron microscopy
Journal of Structural Biology, 2007
Bsoft is a software package written for image processing of electron micrographs, interpretation of reconstructions, molecular modeling, and general image processing. The code is modularized to allow for rapid testing and deployment of new processing algorithms, while also providing sufficient infrastructure to deal with many file formats and parametric data. The design is deliberately open to allow interchange of information with other image and molecular processing software through a standard parameter file (currently a text-based encoding of parameters in the STAR format) and its support of multiple image and molecular formats. It also allows shell scripting of processes and allows subtasks to be distributed across multiple computers for concurrent processing. Bsoft has undergone many modifications and advancements since its initial release [Heymann, J.B., 2001. Bsoft: image and molecular processing in electron microscopy. J. Struct. Biol. 133,[156][157][158][159][160][161][162][163][164][165][166][167][168][169]. Much of the emphasis is on single particle analysis and tomography, and sufficient functionality is available in the package to support most needed operations for these techniques. The key graphical user interface is the program bshow, which displays an image and is used for many interactive purposes such as fitting the contrast transfer function or picking particles. Bsoft also offers various tools to manipulate atomic structures and to refine the fit of a known molecular structure to a density in a reconstruction. Published by Elsevier Inc.
Proc Natl Acad Sci U S A., 2012
The method presented here refines molecular conformations directly against projections of single particles measured by electron microscopy. By optimizing the orientation of the projection at the same time as the conformation, the method is well-suited to two-dimensional class averages from cryoelectron microscopy. Such direct use of two-dimensional images circumvents the need for a three-dimensional density map, which may be difficult to reconstruct from projections due to structural heterogeneity or preferred ...
THREE DIMENSIONAL ELECTRON MICROSCOPY AND IN SILICO TOOLS FOR MACROMOLECULAR STRUCTURE DETERMINATION
Recently, structural biology witnessed a major tool -electron microscopy -in solving the structures of macromolecules in addition to the conventional techniques, X-ray crystallography and nuclear magnetic resonance (NMR). Three dimensional transmission electron microscopy (3DTEM) is one of the most sophisticated techniques for structure determination of molecular machines. Known to give the 3-dimensional structures in its native form with literally no upper limit on size of the macromolecule, this tool does not need the crystallization of the protein. Combining the 3DTEM data with in silico tools, one can have better refined structure of a desired complex. In this review we are discussing about the recent advancements in three dimensional electron microscopy and tools associated with it.
Determination of the ribosome structure to a resolution of 2.5 Å by single-particle cryo-EM
Protein science : a publication of the Protein Society, 2016
With the advance of new instruments and algorithms, and the accumulation of experience over decades, single-particle cryo-EM has become a pivotal part of structural biology. Recently, we determined the structure of a eukaryotic ribosome at 2.5 Å for the large subunit. The ribosome was derived from Trypanosoma cruzi, the protozoan pathogen of Chagas disease. The high-resolution density map allowed us to discern a large number of unprecedented details including rRNA modifications, water molecules, and ions such as Mg(2+) and Zn(2+) . In this paper, we focus on the procedures for data collection, image processing, and modeling, with particular emphasis on factors that contributed to the attainment of high resolution. The methods described here are readily applicable to other macromolecules for high-resolution reconstruction by single-particle cryo-EM. This article is protected by copyright. All rights reserved.
UROX 2.0: an interactive tool for fitting atomic models into electron-microscopy reconstructions
Acta Crystallographica Section D: Biological …, 2009
Electron microscopy of a macromolecular structure can lead to three-dimensional reconstructions with resolutions that are typically in the 30–10 Å range and sometimes even beyond 10 Å. Fitting atomic models of the individual components of the macromolecular structure (e.g. those obtained by X-ray crystallography or nuclear magnetic resonance) into an electron-microscopy map allows the interpretation of the latter at near-atomic resolution, providing insight into the interactions between the components. Graphical software is presented that was designed for the interactive fitting and refinement of atomic models into electron-microscopy reconstructions. Several characteristics enable it to be applied over a wide range of cases and resolutions. Firstly, calculations are performed in reciprocal space, which results in fast algorithms. This allows the entire reconstruction (or at least a sizeable portion of it) to be used by taking into account the symmetry of the reconstruction both in the calculations and in the graphical display. Secondly, atomic models can be placed graphically in the map while the correlation between the model-based electron density and the electron-microscopy reconstruction is computed and displayed in real time. The positions and orientations of the models are refined by a least-squares minimization. Thirdly, normal-mode calculations can be used to simulate conformational changes between the atomic model of an individual component and its corresponding density within a macromolecular complex determined by electron microscopy. These features are illustrated using three practical cases with different symmetries and resolutions. The software, together with examples and user instructions, is available free of charge at http://mem.ibs.fr/UROX/.
Image processing for electron microscopy single-particle analysis using XMIPP
Nature Protocols, 2008
We describe a collection of standardized image processing protocols for electron microscopy singleparticle analysis using the XMIPP software package. These protocols allow performing the entire processing workflow starting from digitized micrographs up to the final refinement and evaluation of 3D models. A particular emphasis has been placed on the treatment of structurally heterogeneous data through maximum-likelihood refinements and self-organizing maps as well as the generation of initial 3D models for such data sets through random conical tilt reconstruction methods. All protocols presented have been implemented as stand-alone, executable python scripts, for which a dedicated graphical user interface has been developed. Thereby, they may provide novice users with a convenient tool to quickly obtain useful results with minimum efforts in learning about the details of this comprehensive package. Examples of applications are presented for a negative stain random conical tilt data set on the hexameric helicase G40P and for a structurally heterogeneous data set on 70S Escherichia coli ribosomes embedded in vitrified ice.
Abstract Single particle reconstruction using Cryo-Electron Microscopy (cryo-EM) is an emerging technique in structural biology for estimating the 3-D structure (density) of protein macromolecules. Unlike tomography where a large number of images of a specimen can be acquired, the number of images of an individual particle is limited because of radiation damage. Instead, the specimen consists of identical copies of the same protein macro-molecule embedded in vitreous ice at random and unknown 3-D orientations.
Proceedings of The National Academy of Sciences, 2008
Electron cryomicroscopy (cryo-EM) yields images of macromolecular assemblies and their components, from which 3D structures can be determined, by using an image processing method commonly known as ''single-particle reconstruction.'' During the past two decades, this technique has become an important tool for 3D structure determination, but it generally has not been possible to determine atomic models. In principle, individual molecular images contain high-resolution information contaminated by a much higher level of noise. In practice, it has been unclear whether current averaging methods are adequate to extract this information from the background. We present here a reconstruction, obtained by using recently developed image processing methods, of the rotavirus inner capsid particle (''double-layer particle'' or DLP) at a resolution suitable for interpretation by an atomic model. The result establishes single-particle reconstruction as a highresolution technique. We show by direct comparison that the cryo-EM reconstruction of viral protein 6 (VP6) of the rotavirus DLP is similar in clarity to a 3.8-Å resolution map obtained from x-ray crystallography. At this resolution, most of the amino acid side chains produce recognizable density. The icosahedral symmetry of the particle was an important factor in achieving this resolution in the cryo-EM analysis, but as the size of recordable datasets increases, single-particle reconstruction also is likely to yield structures at comparable resolution from samples of much lower symmetry. This potential has broad implications for structural cell biology.