Fully automated, sequential tilt-series acquisition with Leginon

Suloway, Christian; Shi, Jing; Cheng, Anchi; Pulokas, James; Carragher, Bridget; Potter, Clinton S.; Zheng, Sijie; Agard, David A.; Jensen, Grant J.

doi:10.1016/j.jsb.2009.03.019

Cited by 190 publications

(147 citation statements)

References 25 publications

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“…Recent advances in cryo-ET suggest it can be used as a high-throughput method in a research laboratory (29)(30)(31), raising the possibility of its use in clinical laboratory. The increase in cell tomogram contrast using the Zernike phase plate technology in cryo-ET will likely ease the recognition and quantification of the observed subcellular features (32).…”

Section: Discussionmentioning

confidence: 99%

Electron cryotomography reveals ultrastructure alterations in platelets from patients with ovarian cancer

Wang

Stone

Kaelber

et al. 2015

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

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Thrombocytosis and platelet hyperreactivity are known to be associated with malignancy; however, there have been no ultrastructure studies of platelets from patients with ovarian cancer. Here, we used electron cryotomography (cryo-ET) to examine frozen-hydrated platelets from patients with invasive ovarian cancer (n = 12) and control subjects either with benign adnexal mass (n = 5) or free from disease (n = 6). Qualitative inspections of the tomograms indicate significant morphological differences between the cancer and control platelets, including disruption of the microtubule marginal band. Quantitative analysis of subcellular features in 120 platelet electron tomograms from these two groups showed statistically significant differences in mitochondria, as well as microtubules. These structural variations in the platelets from the patients with cancer may be correlated with the altered platelet functions associated with malignancy. Cryo-ET of platelets shows potential as a noninvasive biomarker technology for ovarian cancer and other platelet-related diseases.electron | cryotomography | platelet | microtubule | cancer

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Section: Discussionmentioning

confidence: 99%

Electron cryotomography reveals ultrastructure alterations in platelets from patients with ovarian cancer

Wang

Stone

Kaelber

et al. 2015

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

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“…Images were captured using a Gatan Ultrascan 4000 chargecoupled-device (CCD) camera at a nominal magnification of 50,000ϫ (and binned by a factor of 2, yielding a scaling of 4.4 Å per pixel), or using a Gatan K2 Summit direct detector in counting mode at a calibrated magnification of 11,500ϫ (yielding a scaling of 3.2 Å per pixel). Images were acquired at 2 to 4 m underfocus, and specimens were tilted in 2°s teps from 58°to Ϫ58°using the Leginon software package (34). A dose rate of 8 electrons/pixel/s and a 120 ms-exposure per frame were used in the counting mode with the K2 Summit detector.…”

Section: Methodsmentioning

confidence: 99%

Visualization and Sequencing of Membrane Remodeling Leading to Influenza Virus Fusion

Gui

Ebner

Mileant

et al. 2016

J Virol

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Protein-mediated membrane fusion is an essential step in many fundamental biological events, including enveloped virus infection. The nature of protein and membrane intermediates and the sequence of membrane remodeling during these essential processes remain poorly understood. Here we used cryo-electron tomography (cryo-ET) to image the interplay between influenza virus and vesicles with a range of lipid compositions. By following the population kinetics of membrane fusion intermediates imaged by cryo-ET, we found that membrane remodeling commenced with the hemagglutinin fusion protein spikes grappling onto the target membrane, followed by localized target membrane dimpling as local clusters of hemagglutinin started to undergo conformational refolding. The local dimples then transitioned to extended, tightly apposed contact zones where the two proximal membrane leaflets were in most cases indistinguishable from each other, suggesting significant dehydration and possible intermingling of the lipid head groups. Increasing the content of fusion-enhancing cholesterol or bis-monoacylglycerophosphate in the target membrane led to an increase in extended contact zone formation. Interestingly, hemifused intermediates were found to be extremely rare in the influenza virus fusion system studied here, most likely reflecting the instability of this state and its rapid conversion to postfusion complexes, which increased in population over time. By tracking the populations of fusion complexes over time, the architecture and sequence of membrane reorganization leading to efficient enveloped virus fusion were thus resolved. IMPORTANCEEnveloped viruses employ specialized surface proteins to mediate fusion of cellular and viral membranes that results in the formation of pores through which the viral genetic material is delivered to the cell. For influenza virus, the trimeric hemagglutinin (HA) glycoprotein spike mediates host cell attachment and membrane fusion. While structures of a subset of conformations and parts of the fusion machinery have been characterized, the nature and sequence of membrane deformations during fusion have largely eluded characterization. Building upon studies that focused on early stages of HA-mediated membrane remodeling, here cryo-electron tomography (cryo-ET) was used to image the three-dimensional organization of intact influenza virions at different stages of fusion with liposomes, leading all the way to completion of the fusion reaction. By monitoring the evolution of fusion intermediate populations over the course of acid-induced fusion, we identified the progression of membrane reorganization that leads to efficient fusion by an enveloped virus.

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“…For single-particle analysis, data were collected on a Tecnai F20 microscope (FEI Company, Hillsboro, OR) operated at 200 kV using the automated electron microscopy data acquisition system, Leginon (24), at the National Resource for Automated Molecular Microscopy (NRAMM). A single grid of the 135S preparation was used for imaging.…”

Section: Methodsmentioning

confidence: 99%

Cryo-Electron Microscopy Reconstruction Shows Poliovirus 135S Particles Poised for Membrane Interaction and RNA Release

Butan

Filman

Hogle

2014

J Virol

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During infection, binding of mature poliovirus to cell surface receptors induces an irreversible expansion of the capsid, to form an infectious cell-entry intermediate particle that sediments at 135S. In these expanded virions, the major capsid proteins (VP1 to VP3) adopt an altered icosahedral arrangement to open holes in the capsid at 2-fold and quasi-3-fold axes, and internal polypeptides VP4 and the N terminus of VP1, which can bind membranes, become externalized. Cryo-electron microscopy images for 117,330 particles were collected using Leginon and reconstructed using FREALIGN. Improved rigid-body positioning of major capsid proteins established reliably which polypeptide segments become disordered or rearranged. The virus-to-135S transition includes expansion of 4%, rearrangements of the GH loops of VP3 and VP1, and disordering of C-terminal extensions of VP1 and VP2. The N terminus of VP1 rearranges to become externalized near its quasi-3-fold exit, binds to rearranged GH loops of VP3 and VP1, and attaches to the top surface of VP2. These details improve our understanding of subsequent stages of infection, including endocytosis and RNA transfer into the cytoplasm. Poliovirus is the causative agent of poliomyelitis and the type member of the Enterovirus genus. As such, it shares structural and functional similarities with other members of the Picornaviridae family, including the coxsackievirus B viruses, which are associated with cardiomyopathies, central nervous system (CNS) infections, and diabetes; rhinoviruses, which are the most significant cause of the common cold; echoviruses, which can cause aseptic meningitis, gastroenteritis, and respiratory diseases; enterovirus 71 (EV71) and coxsackievirus A16 (CAV16), which have recently caused epidemics in Asia of hand-foot-and-mouth disease, with a high frequency of central nervous system involvement and with high morbidity and mortality; and the foot-and-mouth disease virus, which causes devastating outbreaks of foot-and-mouth disease in livestock (1).Mature poliovirus (which sediments at 160S) is a spherical, nonenveloped virus, with a diameter of approximately 30 nm. Its capsid consists of 60 copies each of 4 proteins (VP1 to VP4) arranged on a Tϭ1 (pseudo-Tϭ3) icosahedral lattice and encloses a 7,500-base positive-sense single-stranded RNA (ssRNA) viral genome (2, 3). The major proteins (VP1, VP2, and VP3) share a topology, which is an eight-stranded beta barrel. Each protein has a different set of loops connecting the strands in the barrel and unique N-and C-terminal extensions. The C-terminal extensions of the VP1 to -3 subunits are located on the exterior surface of the virus, whereas VP4 and the N-terminal extensions are intertwined on the interior of the shell, making contact with the RNA genome. VP4, which is on the inside surface of the capsid, folds into an elongated structure, due to its contacts on the inner surfaces of the capsid protein that make up the shell. The outer surface of the virus is dominated by star-shaped mesas surrounding each 5...

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Fully automated, sequential tilt-series acquisition with Leginon

Cited by 190 publications

References 25 publications

Electron cryotomography reveals ultrastructure alterations in platelets from patients with ovarian cancer

Electron cryotomography reveals ultrastructure alterations in platelets from patients with ovarian cancer

Visualization and Sequencing of Membrane Remodeling Leading to Influenza Virus Fusion

Cryo-Electron Microscopy Reconstruction Shows Poliovirus 135S Particles Poised for Membrane Interaction and RNA Release

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