EMAN: Semiautomated Software for High-Resolution Single-Particle Reconstructions

Ludtke, Steven J.; Baldwin, Philip R.; Chiu, Wah

doi:10.1006/jsbi.1999.4174

Cited by 2,326 publications

(2,391 citation statements)

References 28 publications

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“…Images were recorded on a Gatan UltraScan 1000 2×2 CCD camera (Gatan Inc., Pleasanton, CA). Particles were selected using Boxer from EMAN 95 , normalized and then phase-flipped using ctfit from EMAN. In total, 34 class averages (selected classes shown in Extended Data Fig.…”

Section: Methodsmentioning

confidence: 99%

Integrative structure and functional anatomy of a nuclear pore complex

Kim

Fernández-Martı́nez

Nudelman

et al. 2018

Nature

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466

894

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Summary Despite the central role of Nuclear Pore Complexes (NPCs) as gatekeepers of RNA and protein transport between the cytoplasm and nucleoplasm, their large size and dynamic nature have impeded a full structural and functional elucidation. Here, we have determined a subnanometer precision structure for the entire 552-protein yeast NPC by satisfying diverse data including stoichiometry, a cryo-electron tomography map, and chemical cross-links. The structure reveals the NPC’s functional elements in unprecedented detail. The NPC is built of sturdy diagonal columns to which are attached connector cables, imbuing both strength and flexibility, while tying together all other elements of the NPC, including membrane-interacting regions and RNA processing platforms. Inwardly-directed anchors create a high density of transport factor-docking Phe-Gly repeats in the central channel, organized in distinct functional units. Taken together, this integrative structure allows us to rationalize the architecture, transport mechanism, and evolutionary origins of the NPC.

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Section: Methodsmentioning

confidence: 99%

Integrative structure and functional anatomy of a nuclear pore complex

Kim

Fernández-Martı́nez

Nudelman

et al. 2018

Nature

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466

894

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“…Single particles were collected from the digital data (see above) using the boxer module from the EMAN software package 17. Data processing, namely alignment steps, image averaging, multivariate statistical analysis, classification etc., was performed in the context of the IMAGIC 5 software package (Image Science GmbH, Berlin, Germany) 18…”

Section: Methodsmentioning

confidence: 99%

Hierarchically Ordered Self‐Assembly of Amphiphilic Bifullerenes

Wasserthal

Schade

Ludwig

et al. 2014

Chemistry A European J

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A series of novel functionalised dumbbell‐shaped bifullerenes in which two [5.0] pentakis‐adducts of C60 are covalently connected by cyclic bismalonates were synthesised. These dimeric compounds, carrying various combinations of hydrophilic and hydrophobic addends, self‐assemble in aqueous solution towards supramolecular architectures of different structural complexity as observed by cryogenic transmission electron microscopy (cryo‐TEM). The detailed analysis of the image data revealed an unprecedented hierarchical aggregation behaviour. Whereas completely hydrophilic substituted bifullerenes formed profoundly monodisperse populations of small oligomeric elementary micelles consisting of only three or four bifullerene molecules in a supposedly bent conformation, their amphiphilic equivalents underwent a hierarchical two‐step assembly process towards larger spherical and even rod‐like structures. The data suggest that the hierarchical assembly process is driven by hydrophobic interactions of preformed tetrameric elementary micelles.

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“…The first 3D ribosome structure using cryoEM at 40 Å resolution came in 1991 (Frank et al 1991). EMAN (Ludtke et al 1999;Tang et al 2007) and Frealign (Grigorieff, 1998(Grigorieff, , 2007 were developed later with explicit treatment of the CTF that would be required to reach high resolution. The first sub-nanometre single particle cryoEM map was obtained by Bottcher et al (1997) at 7.4 Å resolution and by Conway et al (1997) for the hepatitis B virus core, and these structures were calculated with icosahedral-specific programs.…”

Section: Computer-based Image Processingmentioning

confidence: 99%

“…EMAN (Ludtke et al 1999;Tang et al 2007) and Frealign (Grigorieff, 1998(Grigorieff, , 2007 were the first single particle computational approaches that explicitly included CTF correction at the heart of their operation, which is absolutely required to reach high resolution. The full range of point group symmetries was also embedded in both programs, a feature that had also been available in Imagic since the mid-1980s (van Heel et al 1996).…”

Section: Improved Computer Programsmentioning

confidence: 99%

Single particle electron cryomicroscopy: trends, issues and future perspective

Vinothkumar

Henderson

2016

Quart. Rev. Biophys.

166

115

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Abstract. There has been enormous progress during the last few years in the determination of three-dimensional biological structures by single particle electron cryomicroscopy (cryoEM), allowing maps to be obtained with higher resolution and from fewer images than required previously. This is due principally to the introduction of a new type of direct electron detector that has 2-to 3-fold higher detective quantum efficiency than available previously, and to the improvement of the computational algorithms for image processing. In spite of the great strides that have been made, quantitative analysis shows that there are still significant gains to be made provided that the problems associated with image degradation can be solved, possibly by minimising beam-induced specimen movement and charge build up during imaging. If this can be achieved, it should be possible to obtain near atomic resolution structures of smaller single particles, using fewer images and resolving more conformational states than at present, thus realising the full potential of the method. The recent popularity of cryoEM for molecular structure determination also highlights the need for lower cost microscopes, so we encourage development of an inexpensive, 100 keV electron cryomicroscope with a high-brightness field emission gun to make the method accessible to individual groups or institutions that cannot afford the investment and running costs of a state-of-the-art 300 keV installation. A key requisite for successful high-resolution structure determination by cryoEM includes interpretation of images and optimising the biochemistry and grid preparation to obtain nicely distributed macromolecules of interest. We thus include in this review a gallery of cryoEM micrographs that shows illustrative examples of single particle images of large and small macromolecular complexes.

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EMAN: Semiautomated Software for High-Resolution Single-Particle Reconstructions

Cited by 2,326 publications

References 28 publications

Integrative structure and functional anatomy of a nuclear pore complex

Integrative structure and functional anatomy of a nuclear pore complex

Hierarchically Ordered Self‐Assembly of Amphiphilic Bifullerenes

Single particle electron cryomicroscopy: trends, issues and future perspective

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