Computer Averaging of Electron Micrographs of 40
            <i>S</i>
            Ribosomal Subunits

Frank, Joachim; Verschoor, Adriana; Boublík, M.

doi:10.1126/science.7313694

Cited by 373 publications

(115 citation statements)

References 14 publications

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“…Five hundred and five ring-like particles were processed in the same way, and three clusters were obtained; the main cluster had 321 ring-like particles. The resolutions of the average rectangular and ring-like particle maps were 3 and 3.4 nm, respectively, according to the Differential Phase Residual method (37).…”

Section: Methodsmentioning

confidence: 99%

Visualization of synaptotagmin I oligomers assembled onto lipid monolayers

Bai

et al. 2003

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

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Neuronal exocytosis is mediated by Ca 2؉ -triggered rearrangements between proteins and lipids that result in the opening and dilation of fusion pores. Synaptotagmin I (syt I) is a Ca 2؉ -sensing protein proposed to regulate fusion pore dynamics via Ca 2؉ -promoted binding of its cytoplasmic domain (C2A-C2B) to effector molecules, including anionic phospholipids and other copies of syt. Functional studies indicate that Ca 2؉ -triggered oligomerization of syt is a critical step in excitation-secretion coupling; however, this activity has recently been called into question. Here, we show that Ca 2؉ does not drive the oligomerization of C2A-C2B in solution. However, analysis of Ca 2؉ ⅐C2A-C2B bound to lipid monolayers, using electron microscopy, revealed the formation of ring-like heptameric oligomers that are Ϸ11 nm long and Ϸ11 nm in diameter. In some cases, C2A-C2B also assembled into long filaments. Oligomerization, but not membrane binding, was disrupted by neutralization of two lysine residues (K326,327) within the C2B domain of syt. These data indicate that Ca 2؉ first drives C2A-C2B⅐membrane interactions, resulting in conformational changes that trigger a subsequent C2B-mediated oligomerization step. Ca 2؉ -mediated rearrangements between syt subunits may regulate the opening or dilation kinetics of fusion pores or may play a role in endocytosis after fusion.C2 domain ͉ oligomerization ͉ calcium ͉ membrane

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Section: Methodsmentioning

confidence: 99%

Visualization of synaptotagmin I oligomers assembled onto lipid monolayers

Bai

et al. 2003

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

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“…Aligned particles were classified again, and cluster averages were calculated. The resolution of the calculated averages was estimated according to the Fourier ring correlation function (FRC) (38), the phase residual (PHR) (39), and the spectral signal to noise ratio (SSNR) (40).…”

Section: Methodsmentioning

confidence: 99%

“…The projection was calculated from 255 motifs of 4096, had a side length of 8.0 nm, and had a homogeneous density distribution over the whole particle. The resolution of the projection averages was determined using the following three criteria: (i) the FRC (38), (ii) the PHR (39), and (iii) the SSNR (40). The average of the larger and rather circular particle type (Fig.…”

Section: Fig 2 C18 Reverse-phase Chromatography Of Endoproteinase Lmentioning

confidence: 99%

Structural Characterization of Two Aquaporins Isolated from Native Spinach Leaf Plasma Membranes

Fotiadis

Jenö

Mini

et al. 2001

Journal of Biological Chemistry

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Two members of the aquaporin family, PM28A and a new one, PM28C, were isolated and shown to be the major constituents of spinach leaf plasma membranes. These two isoforms were identified and characterized by matrix-assisted laser desorption ionization-mass spectrometry. Edman degradation yielded the amino acid sequence of two domains belonging to the new isoform. PM28B, a previously described isoform, was not found in our preparations. Scanning transmission electron microscopy mass analysis revealed both PM28 isoforms to be tetrameric. Two types of particles, a larger and a smaller one, were found by transmission electron microscopy of negatively stained solubilized proteins and by atomic force microscopy of PM28 two-dimensional crystals. The ratio of larger to smaller particles observed by transmission electron microscopy and single particle analysis correlated with the ratio of PM28A to PM28C determined by matrix-assisted laser desorption ionization-mass spectrometry. The absence of PM28B and the ratio of PM28A to PM28C indicate that these plasma membrane intrinsic proteins are differentially expressed in spinach leaves. These findings suggest that differential expression of the various aquaporin isoforms may regulate the water flux across the plasma membrane, in addition to the known mechanism of regulation by phosphorylation.Water is the universal solvent and most important molecule for life. Immense water volumes pass across the membranes of all living cells, especially across the plasma membranes of plants (1). Simple diffusion of water through the lipid bilayer has an activation energy of Ͼ10 kcal/mol and cannot explain the rapid water flow through human red cell membranes found to exhibit an activation energy of Ͻ5 kcal/mol. This led to the hypothesis that water pores must exist (2). The discovery (3) and cloning (4) of the first known water channel, aquaporin-1 (AQP1), 1 from human erythrocytes and the demonstration of water transport in Xenopus oocytes expressing its complementary RNA (5) confirmed this hypothesis.Since then, a large number of channel-forming integral proteins homologous to AQP1 have been found in all forms of life (6). This membrane protein family was initially named the MIP family after its first sequenced member, the major intrinsic protein (MIP) of bovine lens fiber cells (7). Multiple sequence alignment and phylogenetic analysis of 164 members of the MIP family, now frequently referred to as aquaporin super family, revealed 16 subfamilies that form two distinct clusters, the aquaporin (AQP) cluster and the glycerol facilitator-like cluster (8). The AQPs are highly specific for water, whereas the glycerol facilitators allow the passage of small, nonionic molecules such as glycerol and urea (9). In addition, ovine MIP tetramers have been found to form a groove and tongue contact via their extracellular surfaces, lending support to a dual function of the protein, as a water channel and as a cell to cell adhesion molecule in the eye lens (10).Most members of the aquaporin super family...

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“…For comparison between observed projection averages and models, the phase residual criterion [11] was used. The phase residual is defined as:…”

Section: Methodsmentioning

confidence: 99%

Diameter of the mitochondrial outer membrane channel: Evidence from electron microscopy of frozen‐hydrated membrane crystals

Mannella¹,

Guo²,

Cognon³

1989

FEBS Letters

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The average projected density of mitochondrial outer membrane channels has been obtained from electron microscopic images of unstained, frozen-hydrated two-dimensional crystals. The density map is consistent with the projection of hollow, thin-walled cylinders aligned normal to the membrane plane with a diameter of 3.8 + 0.1 nm. This diameter closely matches that of the largest/~-barrel that can be formed from one copy of the 30-kDa mitochondrial outer membrane channel protein.

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Computer Averaging of Electron Micrographs of 40 S Ribosomal Subunits

Cited by 373 publications

References 14 publications

Visualization of synaptotagmin I oligomers assembled onto lipid monolayers

Visualization of synaptotagmin I oligomers assembled onto lipid monolayers

Structural Characterization of Two Aquaporins Isolated from Native Spinach Leaf Plasma Membranes

Diameter of the mitochondrial outer membrane channel: Evidence from electron microscopy of frozen‐hydrated membrane crystals

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