Single-particle reconstruction from EM images of helical filaments

Egelman, Edward H

doi:10.1016/j.sbi.2007.07.006

Cited by 34 publications

(32 citation statements)

References 34 publications

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“…Image processing was carried out on a Linux cluster. EMAN software 17 was used for particle selection; CTFFIND 18 for contrast transfer function (CTF) determination, BSOFT 19 for the CTF correction; Imagic 20 for multivariate statistical analysis and classification; Spider 21,22 for multireference alignment, projection matching (PM) and 3D reconstruction; hsearch and himpose programs from the IHRSR package 23,24 for symmetry search and imposition; URO 25 and its graphical version VEDA http://mem.ibs.fr/VEDA, for crystal structure fitting; Pymol 26 and Chimera 27 for visualization. Supplementary Fig.…”

Section: Discussionmentioning

confidence: 99%

Self-organization of the vesicular stomatitis virus nucleocapsid into a bullet shape

et al. 2013

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The typical bullet shape of Rhabdoviruses is thought to rely on the matrix protein for stabilizing the nucleocapsid coil. Here we scrutinize the morphology of purified and recombinant nucleocapsids of vesicular stomatitis virus in vitro. We elucidate pH and ionic strength conditions for their folding into conical tips and further growth into whole bullets, and provide cryo-electron microscopy reconstructions of the bullet tip and the helical trunk. We address conformational variability of the reconstituted nucleocapsids and the issue of constraints imposed by the binding of matrix protein. Our findings bridge the gap between the isolated nucleoprotein-RNA string in its form of an undulating ribbon, and the tight bullet-shaped virion skeleton.

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Section: Discussionmentioning

confidence: 99%

Self-organization of the vesicular stomatitis virus nucleocapsid into a bullet shape

et al. 2013

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“…We performed single-particle based helical reconstruction of the PB1 domain samples using the previously described software package SPRING (Desfosses et al, 2014), which had been significantly modified from the original iterative helical real-space reconstruction procedure (Egelman, 2007). Processing details are summarized in Table S1.…”

Section: Image Processingmentioning

confidence: 99%

The Selective Autophagy Receptor p62 Forms a Flexible Filamentous Helical Scaffold

et al. 2015

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The scaffold protein p62/SQSTM1 is involved in protein turnover and signaling and is commonly found in dense protein bodies in eukaryotic cells. In autophagy, p62 acts as a selective autophagy receptor that recognizes and shuttles ubiquitinated proteins to the autophagosome for degradation. The structural organization of p62 in cellular bodies and the interplay of these assemblies with ubiquitin and the autophagic marker LC3 remain to be elucidated. Here, we present a cryo-EM structural analysis of p62. Together with structures of assemblies from the PB1 domain, we show that p62 is organized in flexible polymers with the PB1 domain constituting a helical scaffold. Filamentous p62 is capable of binding LC3 and addition of long ubiquitin chains induces disassembly and shortening of filaments. These studies explain how p62 assemblies provide a large molecular scaffold for the nascent autophagosome and reveal how they can bind ubiquitinated cargo.

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“…Here, EM images of filaments are divided into short segments that are treated as single particles. In the Egelman application of the method (IHRSR, "Iterative Helical Real Space Reconstruction") (44,45), ∼40 nm filament segments are matched by cross-correlation to models of F-actin structure with different rotational views. "Binned" segments now with known rotations are then combined by weighted back-projection to produce a 3D reconstruction of the actin filament.…”

Section: D Reconstruction Of Thin Filamentsmentioning

confidence: 99%

Thin Filament Structure and the Steric Blocking Model

Lehman

2016

Comprehensive Physiology

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By interacting with the troponin-tropomyosin complex on myofibrillar thin filaments, Ca2+ and myosin govern the regulatory switching processes influencing contractile activity of mammalian cardiac and skeletal muscles. A possible explanation of the roles played by Ca2+ and myosin emerged in the early 1970s when a compelling "steric model" began to gain traction as a likely mechanism accounting for muscle regulation. In its most simple form, the model holds that, under the control of Ca2+ binding to troponin and myosin binding to actin, tropomyosin strands running along thin filaments either block myosin-binding sites on actin when muscles are relaxed or move away from them when muscles are activated. Evidence for the steric model was initially based on interpretation of subtle changes observed in X-ray fiber diffraction patterns of intact skeletal muscle preparations. Over the past 25 years, electron microscopy coupled with three-dimensional reconstruction directly resolved thin filament organization under many experimental conditions and at increasingly higher resolution. At low-Ca2+, tropomyosin was shown to occupy a "blocked-state" position on the filament, and switched-on in a two-step process, involving first a movement of tropomyosin away from the majority of the myosin-binding site as Ca2+ binds to troponin and then a further movement to fully expose the site when small numbers of myosin heads bind to actin. In this contribution, basic information on Ca2+-regulation of muscle contraction is provided. A description is then given relating the voyage of discovery taken to arrive at the present understanding of the steric regulatory model.

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Single-particle reconstruction from EM images of helical filaments

Cited by 34 publications

References 34 publications

Self-organization of the vesicular stomatitis virus nucleocapsid into a bullet shape

Self-organization of the vesicular stomatitis virus nucleocapsid into a bullet shape

The Selective Autophagy Receptor p62 Forms a Flexible Filamentous Helical Scaffold

Thin Filament Structure and the Steric Blocking Model

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