Biomedical subjects

Denis Fellmann

Publications and source records attributed to Denis Fellmann.

6 recordsLinked to original sources

PubMed2006

Automated cryoEM data acquisition and analysis of 284742 particles of GroEL.

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 describing an experiment in which we acquired images of more than 280,000 particles of GroEL in a single 25 h session at the microscope. We also demonstrate the potential for an automated pipeline for molecular microscopy by showing that these particles can be subjected to completely automated procedures to reconstruct a three-dimensional (3D) density map to a resolution better than 8 A. In generating the 3D maps, we used a variety of metadata associated with the data acquisition and processing steps to sort and select the particles. These metadata provide a number of insights into factors that affect the quality of the acquired images and the resulting reconstructions. In particular, we show that the resolution of the reconstructed 3D density maps improves with decreasing ice thickness. These data provide a basis for assessing the capabilities of high-throughput macromolecular microscopy.

Chaperonin 60↗

PubMed2006

Does contamination buildup limit throughput for automated cryoEM?

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 specimen limits the quality of the data that can be acquired during these long duration experiments and also whether the data acquisition session could be extended to allow acquisition of more than 1,000,000 particles. We report here a systematic characterization of contamination of specimens maintained for long periods of time at liquid nitrogen temperatures using standard side entry cryo stages. As part of this characterization we developed a more reliable method for accurately estimating specimen ice thickness. Using the method, we were able to calibrate image contrast against ice thickness under a variety of magnifications, objective aperture positions, and defoci, and demonstrated the strong dependence of the calibration curve on these parameters. The results show the anti-contamination aperture is, as expected, critical to the prevention of contamination and that loading film into the microscope dramatically increases the contamination rate, particularly in the first 3 h after the insertion of the film box. In the absence of film, we were able to reproducibly demonstrate that the contamination rate can be limited to a rate of approximately 1 angstrom/h providing reassurance that contamination will not be a major limiting factor for long term cryoEM experiments if a CCD camera is used for the imaging.

Algorithms↗

PubMed2005

Automated molecular microscopy: the new Leginon system.

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 new flexible architecture was designed to allow for the development of customized data collection protocols, several of which are described here. The system has been used to acquire data for approximately 150 experiments and we have demonstrated the capacity for high throughput data acquisition by acquiring images of more than 100,000 particles in a single session at the microscope.

Microscopy, Electron↗

PubMed2003

Rapid routine structure determination of macromolecular assemblies using electron microscopy: current progress and further challenges.

Although the methodology of molecular microscopy has enormous potential, it is time consuming and labor intensive. The techniques required to produce a three-dimensional (3D) electron density map of a macromolecular structure normally require manual operation of an electron microscope by a skilled operator and manual supervision of the sometimes complex software needed for analysis and calculation of 3D maps. Systems to automate the process of data acquisition from an electron microscope are being developing and these systems are being integrated with specimen handling operations and post acquisition data processing. Here, the current performance of our existing systems and the future challenges involved in substantially improving both the sustained throughput and the yield of automated data collection and analysis are reported.

Algorithms↗

PubMed2002

A relational database for cryoEM: experience at one year and 50 000 images.

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 database can be accessed using a variety of tools including specially developed Web-based interfaces that enable a user to annotate and categorize images using a Web-based form.

Algorithms↗

PubMedDate not supplied

Automatic particle selection: results of a comparative study.

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 objective in the cryoEM field. A number of research groups are making promising new advances in this area. Evaluation of algorithms using a standard set of cryoEM images is an essential aspect of this algorithm development. With this goal in mind, a particle selection "bakeoff" was included in the program of the Multidisciplinary Workshop on Automatic Particle Selection for cryoEM. Twelve groups participated by submitting the results of testing their own algorithms on a common dataset. The dataset consisted of 82 defocus pairs of high-magnification micrographs, containing keyhole limpet hemocyanin particles, acquired using cryoEM. The results of the bakeoff are presented in this paper along with a summary of the discussion from the workshop. It was agreed that establishing benchmark particles and using bakeoffs to evaluate algorithms are useful in promoting algorithm development for fully automated particle selection, and that the infrastructure set up to support the bakeoff should be maintained and extended to include larger and more varied datasets, and more criteria for future evaluations.

Algorithms↗