Maximum-likelihood Multi-reference Refinement for Electron Microscopy Images

Scheres, Sjors H.W.; Valle, Mikel; Nuñez, Rafael; Sorzano, Carlos Óscar S.; Marabini, Roberto; Herman, Gabor T.; Carazo, José-Marı́a

doi:10.1016/j.jmb.2005.02.031

Cited by 254 publications

(223 citation statements)

References 38 publications

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“…25 ML3D classification yielded two different maps that satisfactory explained all the reference-free averages described before ( Fig. 4c and Supplementary Fig.…”

Section: Electron Microscopy and Single Particle Reconstruction Of Sos1supporting

confidence: 66%

“…33 A total of 12,785 single images of SOS1 were manually extracted using EMAN. 24 2D reference-free classification and averaging were performed using maximum-likelihood methods 25 implemented in XMIPP package. 34 Some averages representing projections of the complex from different points of view were selected to build up initial models by common line methods 35 using StartAny algorithm.…”

Section: Electron Microscopy and 3d Reconstructionsmentioning

confidence: 99%

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Structural Insights on the Plant Salt-Overly-Sensitive 1 (SOS1) Na+/H+ Antiporter

Núñez-Ramírez

Sánchez-Barrena

Villalta

et al. 2012

Journal of Molecular Biology

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Highlights The plant Na + /H + antiporter SOS1 is critical for salt tolerance. SOS1 is a homodimer folded into a transmembrane and a cytosolic domain. The regulatory mechanism is based on the concerted rearrangement of these domains  The SOS1 structure provides a model valuable for biotechnological applications. Keywords: electron microscopy; membrane protein; plant salt tolerance; protein structure; Na + transport. SummaryThe Arabidopsis thaliana Na + /H + antiporter SOS1 is essential to maintain low intracellular levels of toxic Na + under salt stress. Available data show that the plant SOS2 protein kinase and its interacting activator, the SOS3 calcium-binding protein, function together in decoding calcium signals elicited by salt stress and regulating the phosphorylation state and the activity of SOS1. *Manuscript Click here to view linked References 2Molecular genetic studies have shown that the activation implies a domain reorganization of the antiporter cytosolic moiety indicating that there is a clear relationship between function and molecular structure of the antiporter. To provide information on this issue, we have carried out in vivo and in vitro studies on the oligomerization state of SOS1. In addition, we have performed electron microscopy and single particle reconstruction of negatively stained full length and active SOS1. Our studies show that the protein is a homodimer that contains a membrane domain similar to that found in other antiporters of the family, and an elongated, large and structured cytosolic domain.Both the transmembrane and cytosolic moieties contribute to the dimerization of the antiporter. The close contacts between the transmembrane and the cytosolic domains provide a link between regulation and transport activity of the antiporter.

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“…25 ML3D classification yielded two different maps that satisfactory explained all the reference-free averages described before ( Fig. 4c and Supplementary Fig.…”

Section: Electron Microscopy and Single Particle Reconstruction Of Sos1supporting

confidence: 66%

Section: Electron Microscopy and 3d Reconstructionsmentioning

confidence: 99%

Structural Insights on the Plant Salt-Overly-Sensitive 1 (SOS1) Na+/H+ Antiporter

Núñez-Ramírez

Sánchez-Barrena

Villalta

et al. 2012

Journal of Molecular Biology

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“…Low-dose images (<10e-A-2) of the complexes were taken on a FEI Tecnai G2 FEG200 electron microscope at 200 kV by using a Gatan side-entry cryo-holder with a nominal magnification of 62,000× and 1.8-to 3.0-μm underfocus. Images were classified by using reference-free methods (23), and the selected averages were used to build a reference volume using common lines (24), which was subsequently used for the 3D reconstruction procedure (25). Visualization and docking of the atomic structures were carried out by using Chimera (26).…”

Section: Methodsmentioning

confidence: 99%

Structures of the Gβ-CCT and PhLP1–Gβ-CCT complexes reveal a mechanism for G-protein β-subunit folding and Gβγ dimer assembly

Plimpton

Cuéllar

Lai

et al. 2015

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

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G-protein signaling depends on the ability of the individual subunits of the G-protein heterotrimer to assemble into a functional complex. Formation of the G-protein βγ (Gβγ) dimer is particularly challenging because it is an obligate dimer in which the individual subunits are unstable on their own. Recent studies have revealed an intricate chaperone system that brings Gβ and Gγ together. This system includes cytosolic chaperonin containing TCP-1 (CCT; also called TRiC) and its cochaperone phosducin-like protein 1 (PhLP1). Two key intermediates in the Gβγ assembly process, the Gβ-CCT and the PhLP1-Gβ-CCT complexes, were isolated and analyzed by a hybrid structural approach using cryo-electron microscopy, chemical cross-linking coupled with mass spectrometry, and unnatural amino acid cross-linking. The structures show that Gβ interacts with CCT in a near-native state through interactions of the Gγ-binding region of Gβ with the CCTγ subunit. PhLP1 binding stabilizes the Gβ fold, disrupting interactions with CCT and releasing a PhLP1-Gβ dimer for assembly with Gγ. This view provides unique insight into the interplay between CCT and a cochaperone to orchestrate the folding of a protein substrate.G-protein | chaperonin | phosducin-like protein | electron cryo-microscopy | cross-linking

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“…The images were initially analyzed using the ml_align2d program, a multi-reference 2D alignment routine with a maximum-likelihood target function 40 implemented in the Xmipp package 41 . Averages derived from the ml_align2d program were utilized to run iterative alternating rounds of supervised multi-reference alignment/classification and reference-free alignment as described 42 to improve the homogeneity of the image classes.…”

Section: Em Sample Preparation Data Collection and Image Analysismentioning

confidence: 99%

Architecture of the mediator head module

Imasaki¹,

Wang²,

Tsai³

et al. 2011

Acta Cryst Sect A

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Mediator is a key regulator of eukaryotic transcription 1 , connecting activators and repressors bound to regulatory DNA elements with RNA polymerase II (Pol II) 1-4 . In the yeast S. cerevisiae, Mediator comprises 25 subunits with a total mass over 1 MDa 5,6 , and is organized into three modules, termed Head, Middle/Arm and Tail 7-9 . Our understanding of Mediator assembly and its role in regulating transcription has been impeded to date by limited structural information. Here, we report the crystal structure of the essential Mediator Head module (seven subunits, 223 kDa) at 4.3 Å resolution. Our structure reveals three distinct domains with the integrity of the complex centered on a bundle of ten helices from five different Head subunits. An intricate pattern of interactions within this helical bundle ensures stable assembly of the Head subunits, and provides the binding sites for general transcription factors (GTFs) and Pol II. Our structural and functional data suggest the Head module to juxtapose TFIIH and the carboxyl-terminal domain (CTD) of the

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Maximum-likelihood Multi-reference Refinement for Electron Microscopy Images

Cited by 254 publications

References 38 publications

Structural Insights on the Plant Salt-Overly-Sensitive 1 (SOS1) Na+/H+ Antiporter

Structural Insights on the Plant Salt-Overly-Sensitive 1 (SOS1) Na+/H+ Antiporter

Structures of the Gβ-CCT and PhLP1–Gβ-CCT complexes reveal a mechanism for G-protein β-subunit folding and Gβγ dimer assembly

Architecture of the mediator head module

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