Optimization problems in electron microscopy of single particles (original) (raw)
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A multiresolution approach to orientation assignment in 3D electron microscopy of single particles
Journal of Structural Biology, 2004
Three-dimensional (3D) electron microscopy (3DEM) aims at the determination of the spatial distribution of the Coulomb potential of macromolecular complexes. The 3D reconstruction of a macromolecule using single-particle techniques involves thousands of 2D projections. One of the key parameters required to perform such a 3D reconstruction is the orientation of each projection image as well as its in-plane orientation. This information is unknown experimentally and must be determined using image-processing techniques. We propose the use of wavelets to match the experimental projections with those obtained from a reference 3D model. The wavelet decomposition of the projection images provides a framework for a multiscale matching algorithm in which speed and robustness to noise are gained. Furthermore, this multiresolution approach is combined with a novel orientation selection strategy. Results obtained from computer simulations as well as experimental data encourage the use of this approach.
A Toolbox for ab initio 3-D reconstructions in single-particle electron microscopy
Journal of Structural Biology, 2010
Structure determination of a novel macromolecular complex via single-particle electron microscopy depends upon overcoming the challenge of establishing a reliable 3-D reconstruction using only 2-D images. There are a variety of strategies that deal with this issue, but not all of them are readily accessible and straightforward to use. We have developed a ''toolbox" of ab initio reconstruction techniques that provide several options for calculating 3-D volumes in an easily managed and tightly controlled workflow that adheres to standard conventions and formats. This toolbox is designed to streamline the reconstruction process by removing the necessity for bookkeeping, while facilitating transparent data transfer between different software packages. It currently includes procedures for calculating ab initio reconstructions via random or orthogonal tilt geometry, tomograms, and common lines, all of which have been tested using the 50S ribosomal subunit. Our goal is that the accessibility of multiple independent reconstruction algorithms via this toolbox will improve the ease with which models can be generated, and provide a means of evaluating the confidence and reliability of the final reconstructed map.
Image processing and 3-D reconstruction in electron microscopy
IEEE Signal Processing Magazine, 2006
K nowledge of the structure of biological specimens is critical to understanding their functions at all scales [1] and is crucial in biosciences to complement biochemical studies. Electron microscopy (EM) enables the investigation of the structure of biological specimens over a wide range of sizes, from cellular structures to single macromolecules, providing information at low/medium/high resolution [2], [3] and, in some cases, at atomic resolution . The combination of EM with other high-resolution approaches, e.g., X-ray crystallography or nuclear magnetic resonance (NMR), allows integration of the structural information gathered at multiple levels of the biological complexity into a common framework, which is expected to provide a comprehensive description of the cellular function in molecular detail .
Nature Protocols, 2008
This protocol describes the reconstruction of biological molecules from the electron micrographs of single particles. Computation here is performed using the image-processing software SPIDER and can be managed using a graphical user interface, termed the SPIDER Reconstruction Engine. Two approaches are described to obtain an initial reconstruction: random-conical tilt and common lines. Once an existing model is available, reference-based alignment can be used, a procedure that can be iterated. Also described is supervised classification, a method to look for homogeneous subsets when multiple known conformations of the molecule may coexist.
Journal of Structural Biology, 2009
Solving the structure of macromolecular complexes using transmission electron microscopy can be an arduous task. Many of the steps in this process rely strongly on the aid of pre-existing structural knowledge, and are greatly complicated when this information is unavailable. Here we present two software tools meant to facilitate particle picking, an early stage in the single-particle processing of unknown macromolecules. The first tool, DoG Picker, is an efficient and reasonably general, particle picker based on the Difference of Gaussians (DoG) image transform. It can function alone, as a reference-free particle picker with the unique ability to sort particles based on size, or it can also be used as a way to bootstrap the creation of templates or training datasets for other particle pickers. The second tool is TiltPicker, an interactive graphical interface application designed to streamline the selection of particle pairs from tilted-pair datasets. In many respects, TiltPicker is a reimplementation of the SPIDER WEB tilted-particle picker, but built on modern computer frameworks making it easier to deploy and maintain. The TiltPicker program also includes several useful new features beyond those of its predecessor.
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.
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.
Journal of Scientific Computing, 2020
The single particle reconstruction (SPR) in cryogenic electron microscopy is considered in this paper. This is an emerging technique for determining the three-dimensional (3D) structure of biological specimens from a limited number of the micrographs. Because the micrographs are modulated by contrast transfer functions and corrupted by heavy noise, the number of micrographs might be limited, in general it is a serious ill-posed problem to reconstruct the original particle. In this paper, we propose a constrained total variation (TV) model for single particle reconstruction. The TV norm is represented by the dual formulation that changes the SPR problem into a minimax one. The primal-dual method is applied to find the saddle point of the minimax problem, and the convergence condition is given. Numerical results show that the proposed model is very effective in reconstructing the particle.
Single particle analysis of filamentous and highly elongated macromolecular assemblies
Journal of Structural Biology, 2004
The application of single particle techniques to the three-dimensional analysis of electron microscope images of elongated or filamentous macromolecular assemblies is evaluated, taking as an example the muscle thin filament. Although the thin filament contains local helical symmetry, because of the inherent variable twist along it, the helical coherence does not extend for large enough distances to allow the symmetry to be used for full reconstruction of the tropomyosin/troponin repeat along the filament. The muscle thin filament therefore represents a general case of a filamentous object in that it is not possible to exploit symmetry in a full analysis. Due to the nature of the imaging process in the electron microscope, only projections of the thin filament around its long axis are available without tilting the grid. Crucially, projection images around a single axis do not provide enough information to assign Euler angles ab initio using current methods. Tests with a model thin filament structure indicated that an out-of-plane tilt of 20°wasneededforabinitioangularassignmentofsufficientaccuracytocalculatea3Dstructuretoaresolutionof20°was needed for ab initio angular assignment of sufficient accuracy to calculate a 3D structure to a resolution of 20°wasneededforabinitioangularassignmentofsufficientaccuracytocalculatea3Dstructuretoaresolutionof25