ABSTRACT
We have used Leginon, a fully automatic system capable of acquiring cryo-electron micrographs, to collect data of single particles, specifically of the AAA ATPase p97. The images were acquired under low-dose conditions and required no operator intervention other than the initial setup and periodic refilling of the cold-stage dewar. Each image was acquired at two different defocus values. Two-dimensional projection maps of p97 were calculated from these data and compared to results previously obtained using the conventional manual data collection methods to film. The results demonstrate that Leginon performs as well as an experienced microscopist for the acquisition of single-particle data. The general advantages of automation are discussed.
Subject(s)
Adenosine Triphosphatases/chemistry , Cryoelectron Microscopy/methods , Image Processing, Computer-Assisted/methods , Nuclear Proteins/chemistry , Animals , Cattle , Cryoelectron Microscopy/instrumentation , Cryoelectron Microscopy/trends , Electronic Data Processing , Image Processing, Computer-Assisted/instrumentation , Image Processing, Computer-Assisted/standards , Particle Size , Protein ConformationABSTRACT
We have developed a system to automatically acquire cryo-electron micrographs. The system is designed to emulate all of the decisions and actions of a highly trained microscopist in collecting data from a vitreous ice specimen. These include identifying suitable areas of vitreous ice at low magnification, determining the presence and location of specimen on the grid, automatically adjusting imaging parameters (focus, astigmatism) under low-dose conditions, and acquiring images at high magnification to either film or a digital camera. This system is responsible for every aspect of image acquisition and can run unattended, other than requiring periodic refilling of the cryogens, for over 24 h. The system has been tested out on a variety of specimens that represent typical challenges in the field of cryo-electron microscopy. The results show that the overall performance of the system is equivalent to that of an experienced microscopist.
Subject(s)
Cryoelectron Microscopy/methods , Electronic Data Processing/instrumentation , Computer Graphics , Cryoelectron Microscopy/instrumentation , Cryoelectron Microscopy/standards , Electronic Data Processing/methods , Ice , Image Processing, Computer-Assisted/methods , Microtubules/ultrastructure , Mosaic Viruses/ultrastructure , User-Computer InterfaceABSTRACT
We have developed a system to automatically acquire large numbers of acceptable quality images from specimens of negatively stained catalase, a biological protein which forms crystals. In this paper we will describe the details of the system architecture and analyze the performance of the system as compared to a human operator. The ultimate goal of the system if to automate the process of acquiring cryo-electron micrographs.
Subject(s)
Automation/methods , Microscopy, Electron/methods , HumansABSTRACT
We have developed a method to improve the accuracy for absolute relocation of a target specimen using the goniometer on a Philips transmission electron microscope. We have achieved this by characterizing the performance of the Philips compustage, modeling its behavior, and using this model to calculate the goniometer movements required for accurate target relocation. This resulted in a 10-fold improvement in the positioning accuracy of the goniometer.
