Three-Dimensional Electron Microscopy of Macromolecules

Three-Dimensional Electron Microscopy of Macromolecules

Research Emphasis

Currrent Research

Technology and research development efforts are focused on extending the resolution, speed, and flexibility of electron cryomicroscopy for three-dimensional structure determination of biological macromolecular assemblies. The resource tackles structural problems that are too complex or too difficult for X-ray crystallography and nuclear magnetic resonance spectroscopy. In the center, researchers have demonstrated the feasibility of visualizing secondary structure elements such as alpha helices and beta sheets of protein components in several large assemblies. They are developing technology for routine structure determinations at subnanometer resolution, approaching a resolution sufficient for tracing a polypeptide backbone. Generally they focus on macromolecular assemblies ranging from 300 kDa to 30 MDa and can produce structures from very small quantities of purified specimens.

Experimentally, researchers are involved in evaluation of new instruments for single particle imaging, development of automation techniques for high-throughput data collection, and improvements to cryopreparation techniques. Computationally, they are developing algorithms and improving computational efficiency for the three-dimensional reconstruction of single particles toward atomic resolution. This software is embodied in EMAN and SAVR, which offer complete solutions for low symmetry and icosahedral single particles. In addition, they have produced SAIL, a set of specialized modules for producing professional-quality scientific animations. All three suites and several other tools are distributed free of charge.

Most efforts are focused on collaborative and service projects with a variety of groups around the world. Current biological projects include cytoskeletal filaments and bundles, ion channels, membrane transporters, chaperones, icosahedral viruses, and large oligomeric proteins. In addition, the resource sponsors workshops and symposia regularly to disseminate its imaging technology to a broader community.

Resource Capabilities

Methods

The resource is developing the experimental and computational infrastructure to facilitate the molecular structure determination of molecular machines by cryoelectron microscopy at subnanometer resolutions without crystallography. The determined structures should represent the solution conformations of the molecular machines at different functional states.

Instruments

The resource is uniquely equipped with four high-resolution intermediate-voltage electron cryomicroscopes (JEM2100, JEM2010F, JEM3000SFF, and JEM3200) operated at liquid nitrogen or at liquid helium temperatures. All of these instruments are equipped with 4K x 4K charge-coupled device (CCD) cameras. They have a variety of computational resources, including personal computer clusters with an aggregate peak computing power of ~1 teraflop, as well as a variety of personal computers, Macs, and Silicon Graphics workstations. The on-site database has ~20 terabytes of online storage for data archival. The National Center for Macromolecular Imaging has an OC-12 link to the Texas Gigapop, with an OC-3 connection to Abilene.

Software

See the NCMI Software Development page.

Special Features

The resource has developed unique image reconstruction software that is freely available on its Web site. It has also developed various computer driven data collection software to facilitate both expert and novice users for high-efficiency and high-quality data acquisition.

Available Resources

High-resolution electron cryomicroscopes including JEM3200, JEM3000SFF, JEM2010F, and JEM210. Each is equipped with a Gatan 4K x 4K CCD camera.

Training Opportunities and Workshops

The resource offers cryoelectron microscopy workshops once or twice a year and an image-reconstruction workshop once every two years. All presentations of the Workshops and Symposia have been archived on the resource's Web site.

Publications

Visis the Publications at NCMI page.

1. Jiang, W., Chang, J., Jakana, J., Weigele, P., King, J., and Chiu, W., Structure of epsilon15 bacteriophage reveals genome organization and DNA packaging/injection apparatus. Nature 439:612Ÿ616, 2006.

2. Ludtke, S. J., Serysheva, I. I., Hamilton, S. L., and Chiu, W., The pore structure of the closed RyR1 channel. Structure 13:1203–1211, 2005.

3. Ludtke, S. J., Chen, D. H., Song, J. L., Chuang, D. T., and Chiu, W., Seeing GroEL at 6 Å resolution by single particle electron cryomicroscopy. Structure 12:1129–1136, 2004.

4. Schmid, M. F., Sherman, M., Matsudaira, P., and Chiu, W., Structure of the acrosomal bundle. Nature 431:104–107, 2004.