UCSF tomography: An integrated software suite for real-time electron microscopic tomographic data collection, alignment, and reconstruction

Zheng, Sijie; Keszthelyi, Bettina; Branlund, Eric; Lyle, John M.; Braunfeld, Michael B.; Sedat, John W.; Agard, David A.

doi:10.1016/j.jsb.2006.06.005

Cited by 222 publications

(163 citation statements)

References 18 publications

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“…Images were recorded with a Gatan K2 Summit direct electron detector in integration mode, with single frames taken at each tilt with UCSF Tomo 73 , at a nominal spacing of 5.6 Å per pixel. A total of 253 tilt series were collected in steps between −60°, 0° and 60° in increments of 2.5–4° for different tilt series.…”

Section: Methodsmentioning

confidence: 99%

Integrative structure and functional anatomy of a nuclear pore complex

Kim

Fernández-Martı́nez

Nudelman

et al. 2018

Nature

490

894

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Summary Despite the central role of Nuclear Pore Complexes (NPCs) as gatekeepers of RNA and protein transport between the cytoplasm and nucleoplasm, their large size and dynamic nature have impeded a full structural and functional elucidation. Here, we have determined a subnanometer precision structure for the entire 552-protein yeast NPC by satisfying diverse data including stoichiometry, a cryo-electron tomography map, and chemical cross-links. The structure reveals the NPC’s functional elements in unprecedented detail. The NPC is built of sturdy diagonal columns to which are attached connector cables, imbuing both strength and flexibility, while tying together all other elements of the NPC, including membrane-interacting regions and RNA processing platforms. Inwardly-directed anchors create a high density of transport factor-docking Phe-Gly repeats in the central channel, organized in distinct functional units. Taken together, this integrative structure allows us to rationalize the architecture, transport mechanism, and evolutionary origins of the NPC.

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Section: Methodsmentioning

confidence: 99%

Integrative structure and functional anatomy of a nuclear pore complex

Kim

Fernández-Martı́nez

Nudelman

et al. 2018

Nature

490

894

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“…The resulting frozen-hydrated specimens were imaged at −170°C using a Polara G2 electron microscope (FEI Company) equipped with a field emission gun and a Direct Detection Camera (Gatan K2 Summit). Because the camera was recently integrated into our EM system, two tomographic packages [UCSF Tomography (42) and SerialEM (43)] and different procedures were used to collect low-dose tilt series.…”

Section: Methodsmentioning

confidence: 99%

Visualization of the type III secretion sorting platform of Shigella flexneri

Hu¹,

Morado²,

Margolin³

et al. 2015

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

204

300

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Bacterial type III secretion machines are widely used to inject virulence proteins into eukaryotic host cells. These secretion machines are evolutionarily related to bacterial flagella and consist of a large cytoplasmic complex, a transmembrane basal body, and an extracellular needle. The cytoplasmic complex forms a sorting platform essential for effector selection and needle assembly, but it remains largely uncharacterized. Here we use high-throughput cryoelectron tomography (cryo-ET) to visualize intact machines in a virulent Shigella flexneri strain genetically modified to produce minicells capable of interaction with host cells. A high-resolution in situ structure of the intact machine determined by subtomogram averaging reveals the cytoplasmic sorting platform, which consists of a central hub and six spokes, with a pod-like structure at the terminus of each spoke. Molecular modeling of wild-type and mutant machines allowed us to propose a model of the sorting platform in which the hub consists mainly of a hexamer of the Spa47 ATPase, whereas the MxiN protein comprises the spokes and the Spa33 protein forms the pods. Multiple contacts among those components are essential to align the Spa47 ATPase with the central channel of the MxiA protein export gate to form a unique nanomachine. The molecular architecture of the Shigella type III secretion machine and its sorting platform provide the structural foundation for further dissecting the mechanisms underlying type III secretion and pathogenesis and also highlight the major structural distinctions from bacterial flagella.nanomachine | injectisome | pathogen-host interaction | cryo-electron tomography | protein secretion

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“…Single-axis tilt series of desmosomal regions in the epidermis covering an angular range from −65°to +65°with 1°or 2°angular increment were recorded in a Tecnai G2 Polara microscope (FEI) that had been cooled to liquid nitrogen temperature and equipped with a postcolumn GIF 2002 energy filter (Gatan). The tilt series were recorded under low-dose conditions by using the UCSF tomography software (36). Images were recorded on a 2,000 × 2,000 pixel CCD camera at a defocus level between −9 and −4 μm (pixel sizes are 1 nm and 0.6 nm at the specimen level; Table S1).…”

Section: Methodsmentioning

confidence: 99%

The three-dimensional molecular structure of the desmosomal plaque

Al‐Amoudi

Castaño-Díez

Devos

et al. 2011

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

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The cytoplasmic surface of intercellular junctions is a complex network of molecular interactions that link the extracellular region of the desmosomal cadherins with the cytoskeletal intermediate filaments. Although 3D structures of the major plaque components are known, the overall architecture remains unknown. We used cryoelectron tomography of vitreous sections from human epidermis to record 3D images of desmosomes in vivo and in situ at molecular resolution. Our results show that the architecture of the cytoplasmic surface of the desmosome is a 2D interconnected quasiperiodic lattice, with a similar spatial organization to the extracellular side. Subtomogram averaging of the plaque region reveals two distinct layers of the desmosomal plaque: a lowdensity layer closer to the membrane and a high-density layer further away from the membrane. When combined with a heuristic, allowing simultaneous constrained fitting of the high-resolution structures of the major plaque proteins (desmoplakin, plakophilin, and plakoglobin), it reveals their mutual molecular interactions and explains their stoichiometry. The arrangement suggests that alternate plakoglobin-desmoplakin complexes create a template on which desmosomal cadherins cluster before they stabilize extracellularly by binding at their N-terminal tips. Plakophilins are added as a molecular reinforcement to fill the gap between the formed plaque complexes and the plasma membrane.vitreous sectioning | image processing | genetic algorithms | multi-protein fitting C ell junctions are widely distributed in animal tissues and are most abundant in tissues that are subjected to considerable mechanical stress, such as heart, epidermis, and muscle. These junctions are composed of two main classes of proteins: transmembrane adhesion proteins with an extracellular domain that interacts with either the ECM (e.g., focal adhesion and hemidesmosome) or the extracellular domains of specific transmembrane adhesion proteins from the juxtaposed cell (e.g., adherens junctions and desmosomes); and a cytoplasmic tail that binds to one or more intracellular anchor proteins. These form a distinct plaque on the cytoplasmic face of the plasma membrane and connect the junctional complexes to actin filaments or intermediate filaments (IFs). When they have been assembled, these junctions play essential roles in tissue morphogenesis and maintenance; plaque disruption is a hallmark of many blistering and cancer diseases (1, 2). Biochemical and mutagenesis studies have revealed many interactions between the plaque components and their connections to the extracellular space (3-8). However, the organization and interactions of the constituent proteins in the context of the assembled junctions remain poorly understood. For example, whether or how actin filaments bind in situ to the adherens junctions and how the anchoring proteins in hemidesmosome plaque connect integrin to the IFs is still unclear.In the current study, we present the 3D cytoplasmic surface of the desmosome-the desmosomal plaque-fr...

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UCSF tomography: An integrated software suite for real-time electron microscopic tomographic data collection, alignment, and reconstruction

Cited by 222 publications

References 18 publications

Integrative structure and functional anatomy of a nuclear pore complex

Integrative structure and functional anatomy of a nuclear pore complex

Visualization of the type III secretion sorting platform of Shigella flexneri

The three-dimensional molecular structure of the desmosomal plaque

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