Image averaging of flexible fibrous macromolecules: The clathrin triskelion has an elastic proximal segment

Kocsis, Eva; Trus, Benes L.; Steer, Clifford J.; Bisher, M. E.; Steven, Alasdair C.

doi:10.1016/1047-8477(91)90025-r

Cited by 161 publications

(143 citation statements)

References 36 publications

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“…As the features in the fibroblast cells are not of welldefined sizes and shapes, it is hard to get a precise determination of the spatial resolution in the reconstructions, but an estimate can be made by considering the signal profile across the boundary of the cell nucleus that appears as a bright ring in NATURE COMMUNICATIONS | DOI: 10.1038/ncomms2640 ARTICLE the phase reconstructions. The Straighten plug-in for ImageJ 34 was used to select arcs about 40 pixels wide that straddled part of the nuclear perimeter, the arcs were straightened using the method described in Kocsis et al 36 and then line profiles perpendicular to the arc were used to estimate the sharpness of the edge. The separation of 10-90% contours suggested that a spatial resolution B250 nm was achieved in the reconstructions, but in practice this should be considered an upperlimit (worst-case) estimate, because the cell boundary may not be sharply defined, and the process of arc selection, interpolation and alignment may well result in some broadening of the edge.…”

Section: Methodsmentioning

confidence: 99%

Soft X-ray spectromicroscopy using ptychography with randomly phased illumination

et al. 2013

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Ptychography is a form of scanning diffractive imaging that can successfully retrieve the modulus and phase of both the sample transmission function and the illuminating probe. An experimental difficulty commonly encountered in diffractive imaging is the large dynamic range of the diffraction data. Here we report a novel ptychographic experiment using a randomly phased X-ray probe to considerably reduce the dynamic range of the recorded diffraction patterns. Images can be reconstructed reliably and robustly from this setup, even when scatter from the specimen is weak. A series of ptychographic reconstructions at X-ray energies around the L absorption edge of iron demonstrates the advantages of this method for soft X-ray spectromicroscopy, which can readily provide chemical sensitivity without the need for optical refocusing. In particular, the phase signal is in perfect registration with the modulus signal and provides complementary information that can be more sensitive to changes in the local chemical environment.

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Section: Methodsmentioning

confidence: 99%

Soft X-ray spectromicroscopy using ptychography with randomly phased illumination

et al. 2013

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“…Gray-scale images were captured for each color channel and then merged. Chromosome straightening was performed using the 'straighten-curved-objects' plug-in of Image J [45], and final image optimization was performed using Adobe Photoshop (Adobe Systems).…”

Section: Methodsmentioning

confidence: 99%

An integrated molecular cytogenetic map of Cucumis sativus L. chromosome 2

et al. 2011

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BackgroundIntegration of molecular, genetic and cytological maps is still a challenge for most plant species. Recent progress in molecular and cytogenetic studies created a basis for developing integrated maps in cucumber (Cucumis sativus L.).ResultsIn this study, eleven fosmid clones and three plasmids containing 45S rDNA, the centromeric satellite repeat Type III and the pericentriomeric repeat CsRP1 sequences respectively were hybridized to cucumber metaphase chromosomes to assign their cytological location on chromosome 2. Moreover, an integrated molecular cytogenetic map of cucumber chromosomes 2 was constructed by fluorescence in situ hybridization (FISH) mapping of 11 fosmid clones together with the cucumber centromere-specific Type III sequence on meiotic pachytene chromosomes. The cytogenetic map was fully integrated with genetic linkage map since each fosmid clone was anchored by a genetically mapped simple sequence repeat marker (SSR). The relationship between the genetic and physical distances along chromosome was analyzed.ConclusionsRecombination was not evenly distributed along the physical length of chromosome 2. Suppression of recombination was found in centromeric and pericentromeric regions. Our results also indicated that the molecular markers composing the linkage map for chromosome 2 provided excellent coverage of the chromosome.

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“…While our model differs from the protein structures described above, it still captures essential features and uses the most recent estimates of the dimensions of the clathrin heavy chain and terminal domain. Each leg was given a length of 52 nm, in correspondence with measurements made from transmission electron microscopy (TEM) images of clathrin (20)(21)(22). To model the solution structures for a comparison with DLS data, here, also, a bead diameter of 3.0 nm, derived from the partial crystal structure for the clathrin proximal leg 6 (17), was augmented by an additional 0.6 nm to approximate the effect of a layer of bound water needed when calculating R H .…”

Section: Modelingmentioning

confidence: 99%

Conformation of a Clathrin Triskelion in Solution

et al. 2006

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A principal component in the protein coats of certain post-golgi and endocytic vesicles is clathrin, which appears as a three-legged heteropolymer (known as a triskelion) that assembles into polyhedral cages principally made up of pentagonal and hexagonal faces. In vitro, this assembly depends upon the pH, with cages forming more readily at low pH and less readily at high pH. We have developed procedures, on the basis of static and dynamic light scattering, to determine the radius of gyration, R g , and hydrodynamic radius, R H , of isolated triskelia, under conditions where cage assembly occurs. Calculations based on rigid molecular bead models of a triskelion show that the measured values can be accounted for by bending the legs and a puckering at the vertex. We also show that the values of R g and R H measured for clathrin triskelia in solution are qualitatively consistent with the conformation of a triskelion in a "D 6 barrel" cage assembly measured by cryoelectron microscopy.A major component of the protein coats of certain endocytic vesicles is clathrin, a heteropolymer composed of a 192 kDa heavy chain and a variable, ca. 23-27 kDa, associated light chain (1). Three clathrin molecules join at a common hub to form a threelegged "triskelion", which is the basic building block of the coats. Each leg is approximately 3.0 nm thick and 52.0 nm in length and ends in a globular "terminal domain" of radius 5.0 nm. By convention, a leg is divided into a proximal segment adjoining the central hub, a linker region, and a distal segment that connects with the terminal domain. A clathrin heavy chain runs the entire length of the leg, with a light chain attached near the common hub (see Figure 1). In vitro, the triskelia assemble into polyhedral cages (or "baskets") composed of pentagonal and hexagonal faces (2), mimicking the structures seen on the outside of endocytic vesicles. One can dissociate baskets or uncoat vesicles by changing the properties † This work was supported by extramural grants from the National Institutes of Health and intramural funds from the National Institute HHS Public Access Author Manuscript Author ManuscriptAuthor ManuscriptAuthor Manuscript of the solution in which they are suspended (e.g., by changing pH and ionic strength) (3) or by adding an uncoating ATPase (4).Triskelia assemble at low pH to form baskets (3) or at higher pH, when clathrin assembly proteins (e.g., AP-2, AP-180, etc.) are added (5). It is not currently known why these conditions favor assembly. Possible mechanisms supporting assembly at low pH include conformational changes in the tertiary structure of the triskelia, which might relieve steric hindrances to basket assembly, and enhanced interactions between the intertwined triskelia that form the basket struts. The role of APs as assembly proteins may be to increase mechanical linkages between the triskelia (5-7).In this paper, we report light-scattering measurements on clathrin in solution, including conditions where assembly occurs. From static light scatteri...

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Image averaging of flexible fibrous macromolecules: The clathrin triskelion has an elastic proximal segment

Cited by 161 publications

References 36 publications

Soft X-ray spectromicroscopy using ptychography with randomly phased illumination

Soft X-ray spectromicroscopy using ptychography with randomly phased illumination

An integrated molecular cytogenetic map of Cucumis sativus L. chromosome 2

Conformation of a Clathrin Triskelion in Solution

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