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We report here on the current state of our efforts in automated molecular microscopy. Our primary automated data acquisition software system, Leginon, has been completely redesigned over the past two years. The new distributed system has been developed using the Python programming language and is compatible with both Linux and Windows operating systems. The(More)
The use of cryoEM and three-dimensional image reconstruction is becoming increasingly common. Our vision for this technique is to provide a straightforward manner in which users can proceed from raw data to a reliable 3D reconstruction through a pipeline that both facilitates management of the processing steps and makes the results at each step more(More)
Manual selection of single particles in images acquired using cryo-electron microscopy (cryoEM) will become a significant bottleneck when datasets of a hundred thousand or even a million particles are required for structure determination at near atomic resolution. Algorithm development of fully automated particle selection is thus an important research(More)
One of the goals in developing our automated electron microscopy data acquisition system, Leginon, was to improve both the ease of use and the throughput of the process of acquiring low dose images of macromolecular specimens embedded in vitreous ice. In this article, we demonstrate the potential of the Leginon system for high-throughput data acquisition by(More)
Structure determination of a novel macromolecular complex via single-particle electron microscopy depends upon overcoming the challenge of establishing a reliable 3-D reconstruction using only 2-D images. There are a variety of strategies that deal with this issue, but not all of them are readily accessible and straightforward to use. We have developed a(More)
Screening trials to determine the presence of two-dimensional (2D) protein crystals suitable for three-dimensional structure determination using electron crystallography is a very labor-intensive process. Methods compatible with fully automated screening have been developed for the process of crystal production by dialysis and for producing negatively(More)
Visualization by electron microscopy has provided many insights into the composition, quaternary structure, and mechanism of macromolecular assemblies. By preserving samples in stain or vitreous ice it is possible to image them as discrete particles, and from these images generate three-dimensional structures. This 'single-particle' approach suffers from(More)
Two issues that often impact the cryo-electron microscopy (cryoEM) specimen preparation process are agglomeration of particles near hole edges in holey carbon films and variations in vitreous ice thickness. In many cases, the source of these issues was identified to be the residues and topography often seen in commercially available films. To study and(More)
For the past year we have been using a relational database as part of an automated data collection system for cryoEM. The database is vital for keeping track of the very large number of images collected and analyzed by the automated system and essential for quantitatively evaluating the utility of methods and algorithms used in the data collection. The(More)
The development of automated systems for data acquisition in cryo electron microscopy has enabled the possibility of acquiring very large number of images from a single specimen grid. We have demonstrated that over images of 250,000 single particles can be acquired in a 24 h period. This has raised questions as to whether contamination buildup on the(More)