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.

ACE: automated CTF estimation.

We present a completely automated algorithm for estimating the parameters of the contrast transfer function (CTF) of a transmission electron microscope. The primary contribution of this paper is the determination of the astigmatism prior to the estimation of the CTF parameters. The CTF parameter estimation is then reduced to a 1D problem using elliptical averaging. We have also implemented an automated method to calculate lower and upper cutoff frequencies to eliminate regions of the power spectrum which perturb the estimation of the CTF parameters. The algorithm comprises three optimization subproblems, two of which are proven to be convex. Results of the CTF estimation method are presented for images of carbon support films as well as for images of single particles embedded in ice and suspended over holes in the support film. A MATLAB implementation of the algorithm, called ACE, is freely available.

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.

Detecting particles in cryo-EM micrographs using learned features.

A new learning-based approach is presented for particle detection in cryo-electron micrographs using the Adaboost learning algorithm. The approach builds directly on the successful detectors developed for the domain of face detection. It is a discriminative algorithm which learns important features of the particle's appearance using a set of training examples of the particles and a set of images that do not contain particles. The algorithm is fast (10 s on a 1.3 GHz Pentium M processor), is generic, and is not limited to any particular shape or size of the particle to be detected. The method has been evaluated on a publicly available dataset of 82 cryoEM images of keyhole lympet hemocyanin (KLH). From 998 automatically extracted particle images, the 3-D structure of KLH has been reconstructed at a resolution of 23.2 A which is the same resolution as obtained using particles manually selected by a trained user.