The aim of this proposal is to automate the process of recording and digitizing images obtained by electron cryomicroscopy (cryo-EM) of single particles. Automation is seen to be an essential step required for high throughput determination of structures of large, macromolecular assemblies, at high resolution. Our goal is to develop technology needed to record a data set consisting of -100,000 particles in a single 8-10 hour session. Images will initially be recorded on photographic film, and thus our first design objective is to record and develop -500 films per day. The function of the operator will be to prepare and evaluate the sample, initialize the data collection, exchange and develop film, and maintain liquid nitrogen levels in the microscope cold stage, etc. All remaining microscope operations and image capture operations will be performed automatically under control software developed as part of this technology development project. Digitization of films will also be automated. The plan in this case is to build a robot which will change films in a commercial, 6000-pixel (linear) CCD scanner. Other than loading negatives once a day, digitization will require no operator supervision. It is further anticipated that future development of large-array CCD cameras for use in electron microscopy will make it possible to record digital images directly, and conversion of our initial control software to add direct electronic readout as one option is intended later in this grant period. High throughput protocols will be developed and tested in the context of our own in-house research projects, after which the same technology will be installed for beta-testing at sites of collaborating research groups. Our goal is to make this technology easily available on any cryo-EM equipped with a TV camera and computer-controlled stage. High throughput determination of the structure of large macromolecular assemblies will greatly enhance our understanding of the ways in which complex biochemical operations are carried out at the subcellular level.
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| Typke, Dieter; Nordmeyer, Robert A; Jones, Arthur et al. (2005) High-throughput film-densitometry: an efficient approach to generate large data sets. J Struct Biol 149:17-29 |
| Rockel, Beate; Peters, Jurgen; Muller, Shirley A et al. (2005) Molecular architecture and assembly mechanism of Drosophila tripeptidyl peptidase II. Proc Natl Acad Sci U S A 102:10135-40 |
| Typke, Dieter; Downing, Kenneth H; Glaeser, Robert M (2004) Electron microscopy of biological macromolecules: bridging the gap between what physics allows and what we currently can get. Microsc Microanal 10:21-7 |
| Adiga, P S Umesh; Malladi, Ravi; Baxter, William et al. (2004) A binary segmentation approach for boxing ribosome particles in cryo EM micrographs. J Struct Biol 145:142-51 |
