Leginon: a system for fully automated acquisition of 1000 electron micrographs a day

Potter, C. S.; Chu, Hao-Hua; Frey, Brendan J.; Green, C; Kisseberth, N.; Madden, Timothy J.; Miller, Karla L.; Nahrstedt, Klara; Pulokas, James; Reilein, Amy; Tcheng, David; Weber, Daniel; Carragher, Bridget

doi:10.1016/s0304-3991(99)00043-1

Cited by 161 publications

(130 citation statements)

References 12 publications

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“…Samples were visualized in a Philips Technai F20 electron microscope (Mahwah, NJ) operating at 120 kV, and images were collected using Legion (Potter et al, 1999;Suloway et al, 2005) at 2.0-m underfocus with a 4K ϫ 4K Gatan CCD camera (Pleasanton, CA) at a nominal magnification of 50,000ϫ, corresponding to a resolution of 2.24 Å per pixel.…”

Section: Em Of Dynamin Interface Mutants Assembled In Vitromentioning

confidence: 99%

An Intramolecular Signaling Element that Modulates Dynamin Function In Vitro and In Vivo

Chappie

Acharya

Liu

et al. 2009

MBoC

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Dynamin exhibits a high basal rate of GTP hydrolysis that is enhanced by self-assembly on a lipid template. Dynamin's GTPase effector domain (GED) is required for this stimulation, though its mechanism of action is poorly understood. Recent structural work has suggested that GED may physically dock with the GTPase domain to exert its stimulatory effects. To examine how these interactions activate dynamin, we engineered a minimal GTPase-GED fusion protein (GG) that reconstitutes dynamin's basal GTPase activity and utilized it to define the structural framework that mediates GED's association with the GTPase domain. Chemical cross-linking of GG and mutagenesis of full-length dynamin establishes that the GTPase-GED interface is comprised of the N-and C-terminal helices of the GTPase domain and the C-terminus of GED. We further show that this interface is essential for structural stability in full-length dynamin. Finally, we identify mutations in this interface that disrupt assembly-stimulated GTP hydrolysis and dynamin-catalyzed membrane fission in vitro and impair the late stages of clathrin-mediated endocytosis in vivo. These data suggest that the components of the GTPase-GED interface act as an intramolecular signaling module, which we term the bundle signaling element, that can modulate dynamin function in vitro and in vivo.

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Section: Em Of Dynamin Interface Mutants Assembled In Vitromentioning

confidence: 99%

An Intramolecular Signaling Element that Modulates Dynamin Function In Vitro and In Vivo

Chappie

Acharya

Liu

et al. 2009

MBoC

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“…Notably, a robot has been developed by Potter and colleagues at the National Resource for Automated Molecular Microscopy at Scripps that is able to pick up grids one-by-one and to load them into the microscope for imaging (Potter et al, 2004;Stagg et al, 2006). When combined with the Leginon software for automated imaging of EM grids (Potter et al, 1999;Suloway et al, 2005), this robot is able to work through samples in an unattended manner, presenting the researcher with a series of digital images for later evaluation. Alternatively, the laboratory of Dr. Subramaniam (National Cancer Institute of National Institutes of Health, Bethesda MD) has collaborated with a commercial vendor (Gatan Inc.) to develop a cartridge-based specimen holder that simultaneously holds 100 samples within the electron microscope (Lefman et al, 2007).…”

Section: Discussionmentioning

confidence: 99%

A high-throughput strategy to screen 2D crystallization trials of membrane proteins

Vink

Derr

Love

et al. 2007

Journal of Structural Biology

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Electron microscopy of two-dimensional (2D) crystals has demonstrated potential for structure determination of membrane proteins. Technical limitations in large-scale crystallization screens have, however, prevented a major breakthrough in the routine application of this technology. Dialysis is generally used for detergent removal and reconstitution of the protein into a lipid bilayer, and devices for testing numerous conditions in parallel are not readily available. Furthermore, the small size of resulting 2D crystals requires electron microscopy to evaluate the results and automation of the necessary steps is essential to achieve a reasonable throughput. We have designed a crystallization block, using standard microplate dimensions, by which 96 unique samples can be dialyzed simultaneously against 96 different buffers and have demonstrated that the rate of detergent dialysis is comparable to those obtained with conventional dialysis devices. A liquid-handling robot was employed to set up 2D crystallization trials with the membrane proteins CopA from Archaeoglobus fulgidus and light-harvesting complex II (LH2) from Rhodobacter sphaeroides. For CopA, 1-week of dialysis yielded tubular crystals and, for LH2, large and well-ordered vesicular 2D crystals were obtained after 24-hours, illustrating the feasibility of this approach. Combined with a high-throughput procedure for preparation of EM-grids and automation of the subsequent negative staining step, the crystallization block offers a novel pipeline that promises to speed up considerably large-scale screening of 2D crystallization and to increase the likelihood of producing well-ordered crystals for analysis by electron crystallography.

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“…We have developed a completely automated system, called Leginon, for acquisition of images from specimens embedded in vitreous ice (Potter et al, 1999;Carragher et al, 2000). The Leginon system performs all the operations normally carried out by an experienced microscopist including the identification of ice of suitable thickness, centering specimen targets, and focusing.…”

Section: Introductionmentioning

confidence: 99%

Automated Image Acquisition for Single-Particle Reconstruction Using p97 as the Biological Sample

Rouiller

Pulokas

Butel

et al. 2001

Journal of Structural Biology

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Leginon: a system for fully automated acquisition of 1000 electron micrographs a day

Cited by 161 publications

References 12 publications

An Intramolecular Signaling Element that Modulates Dynamin Function In Vitro and In Vivo

An Intramolecular Signaling Element that Modulates Dynamin Function In Vitro and In Vivo

A high-throughput strategy to screen 2D crystallization trials of membrane proteins

Automated Image Acquisition for Single-Particle Reconstruction Using p97 as the Biological Sample

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