SPIDER image processing for single-particle reconstruction of biological macromolecules from electron micrographs

Shaikh, Tanvir R.; Gao, Haixiao; Baxter, William T.; Asturias, Francisco J.; Boisset, Nicolas; Leith, A.; Frank, Joachim

doi:10.1038/nprot.2008.156

Cited by 453 publications

(398 citation statements)

References 35 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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“…Similarly, we decided to not pursue reconstruction of the "curved" receptor, because the biological significance of this configuration is currently unknown. The remaining classes (1-5) were sufficiently homogeneous and populated to allow 3D reconstruction by the random conical tilt method (20).…”

Section: Resultsmentioning

confidence: 99%

“…To extend this analysis, we manually selected 12,612 particle tilt pairs (at 0°and 55°) using WEB (20). Reference-free alignment and classification of the untilted views into 100 classes with IMAGIC-5 (21) showed that most receptor dimers adopted a preferred orientation, lying sideways on the carbon support film, and exposing the distinctive twofold symmetry previously seen in the crystal structure of SCF-induced dimeric KIT extracellular complex (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…For KIT monomer, 2,021 particles were selected from 400 micrographs using SIGNATURE (30). The chosen particles were normalized and low-pass filtered, followed by multivariate statistical analysis, classification, and alignment (20). After three rounds of multireference alignment, final classification resulted in 50 unique classes of monomeric KIT particles (Fig.…”

Section: Methodsmentioning

confidence: 99%

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Structure, domain organization, and different conformational states of stem cell factor-induced intact KIT dimers

Opatowsky

Lax

Tomé

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Using electron microscopy and fitting of crystal structures, we present the 3D reconstruction of ligand-induced dimers of intact receptor tyrosine kinase, KIT. We observe that KIT protomers form close contacts throughout the entire structure of ligand-bound receptor dimers, and that the dimeric receptors adopt multiple, defined conformational states. Interestingly, the homotypic interactions in the membrane proximal Ig-like domain of the extracellular region differ from those observed in the crystal structure of the unconstrained extracellular regions. We observe two prevalent conformations in which the tyrosine kinase domains interact asymmetrically. The asymmetric arrangement of the cytoplasmic regions may represent snapshots of molecular interactions occurring during trans autophosphorylation. Moreover, the asymmetric arrangements may facilitate specific intermolecular interactions necessary for trans phosphorylation of different KIT autophosphorylation sites that are required for stimulation of kinase activity and recruitment of signaling proteins by activated KIT.receptor tyrosine kinases | cell signaling | cancer | structure analysis | structural biology A broad range of fundamental cellular processes, including proliferation, differentiation, survival, and metabolism, are mediated by signaling pathways that are activated by the 58 members of the receptor tyrosine kinase (RTK) family of membrane receptors (1). The receptor tyrosine kinase KIT was initially discovered as the viral oncogenic protein of a feline sarcoma virus (2). Subsequent studies have demonstrated that KIT and its ligand stem cell factor (SCF) play important roles in the control of proliferation, differentiation, and survival of a variety of cell types, including germ cells, hematopoietic cells, melanocytes, intestinal pacemaker cells, and sensory neurons in the CNS (3-5).KIT and other members of type-III RTK family are composed of an extracellular ligand-binding region containing five Ig-like domains (designated D1-D5), followed by a single transmembrane (TM) spanning helix, a relatively large (35 aa) cytoplasmic juxtamembrane (JM) region, and a tyrosine kinase domain with a kinase insert region containing several autophosphorylation sites. Structural and biochemical studies have shown that the N-terminal region composed of D1, D2, and D3 of KIT extracellular region functions as a binding site for SCF, which, by virtue of its homodimeric structure, is responsible for bringing about KIT dimerization (6-11).SCF-induced KIT dimerization results in homotypic contacts between the membrane proximal Ig-like domains D4 and D5 of two neighboring KIT molecules. These homotypic contacts position the TM and the cytoplasmic regions of KIT and other RTKs at a distance and orientation that facilitate tyrosine autophosphorylation, stimulation of enzymatic activity, and cell signaling (11-13). Oncogenic gain-of-function mutations in KIT have been identified in various cancers, including gastrointestinal stromal tumors, acute myeloid leukemia, melanoma, an...

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“…were manually selected using the program boxer from the EMAN2 package (22); extracted into boxes of 500 by 500 pixels (full phage, coarsened by 2), 200 by 200 pixels (capsid), 100 by 100 pixels (connector), 80 by 80 pixels (tail), or 100 by 100 pixels (baseplate); and combined into the five different data sets (Table 1). The data sets were pretreated using the SPIDER package (23) and submitted to maximum likelihood (ML) classification and alignment (24) using the Xmipp package (25). The initial models were built to form a visually selected class average representing a side view imposing the corresponding symmetry (C6 for the full phage, tail, and baseplate; C12 for the connector; and icosahedral for the capsid).…”

Section: Methodsmentioning

confidence: 99%

The First Structure of a Mycobacteriophage, the Mycobacterium abscessus subsp. bolletii Phage Araucaria

Sassi

Bebeacua

Drancourt

et al. 2013

J Virol

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The unique characteristics of the waxy mycobacterial cell wall raise questions about specific structural features of their bacteriophages. No structure of any mycobacteriophage is available, although ϳ3,500 have been described to date. To fill this gap, we embarked in a genomic and structural study of a bacteriophage from Mycobacterium abscessus subsp. bolletii, a member of the Mycobacterium abscessus group. This opportunistic pathogen is responsible for respiratory tract infections in patients with lung disorders, particularly cystic fibrosis. M. abscessus subsp. bolletii was isolated from respiratory tract specimens, and bacteriophages were observed in the cultures. We report here the genome annotation and characterization of the M. abscessus subsp. bolletii prophage Araucaria, as well as the first single-particle electron microscopy reconstruction of the whole virion. Araucaria belongs to Siphoviridae and possesses a 64-kb genome containing 89 open reading frames (ORFs), among which 27 could be annotated with certainty. Although its capsid and connector share close similarity with those of several phages from Gram-negative (Gram ؊ ) or Gram ؉ bacteria, its most distinctive characteristic is the helical tail decorated by radial spikes, possibly host adhesion devices, according to which the phage name was chosen. Its host adsorption device, at the tail tip, assembles features observed in phages binding to protein receptors, such as phage SPP1. All together, these results suggest that Araucaria may infect its mycobacterial host using a mechanism involving adhesion to cell wall saccharides and protein, a feature that remains to be further explored.

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SPIDER image processing for single-particle reconstruction of biological macromolecules from electron micrographs

Cited by 453 publications

References 35 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

Structure, domain organization, and different conformational states of stem cell factor-induced intact KIT dimers

The First Structure of a Mycobacteriophage, the Mycobacterium abscessus subsp. bolletii Phage Araucaria

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