Genomes to Life Contractor-Grantee Workshop III
February 6-9, 2005, Washington, D.C.
Technology Development and Use
Imaging, Molecular, and Cellular Analysis
96
Electron Tomography of Intact and Sectioned Microbial Cells
Kenneth H. Downing*1 (khdowning@lbl.gov), Luis Comolli1, Haixin Sui1, Hoi-Ying Holman1, Ellen Judd2, and Harley McAdams2
1Lawrence Berkeley National Laboratory, Berkeley, CA and 2Stanford University School of Medicine, Stanford, CA
Electron tomography is an effective tool for the study of subcellular structure at a range of resolutions. In many labs tomography is being used to understand the overall structure and interplay of sub-cellular organelles of eukaryotic cells. Such work is generally carried out on plastic-embedded, stained and sectioned samples. The resolution can be high enough to identify individual molecular complexes and even to understand conformational changes associated with their functions. When cells are suitably thin, they can also be examined without sectioning. The best preservation of intact cells is obtained by rapid freezing, which forms a vitreous embedding medium that maintains the cell in a lifelike state. Although this type of preparation generally preludes the use of contrast agents, the resolution is, in principal, sufficient to identify many of the major macromolecular complexes within the cell. Such information can give insights on localization and distribution of protein complexes and will be essential for the ultimate goals of understanding and building complete computational models of the microbes.
We have been studying several microbial cells, including Caulobacter crescentus, Magnetospirillum and Deinococcus radiodurans, in frozen-hydrated preparations. The work with Caulobacter provides a particularly interesting example of the type of information one can obtain by straightforward interpretation of the 3-D data in a tomogram. The cell membranes are very well resolved in thin slices extracted from the reconstructed volume, as shown in fig. 1. Using manual or automated procedures one can segment the volume to more clearly represent features such as the membranes, as shown in fig. 2. These features could not be unambiguously interpreted from simple projection images of the cells without having seen the structure in three dimensions. This work involved examining cells close to the time at which they completed division and revealed the differential closure of the inner and outer membranes.
Figure 1. A slice 1 nm thick through the 3-D tomographic reconstruction of a Caulobacter cell that has almost finished dividing. The membranes are well resolved, along with parts of the periodic S-layer and subcellular densities that correspond to large protein complexes.
Cell membranes are among the easiest features to identify in these reconstructions because they are continuous, extended structures in three dimensions. Our goal of identifying the major macromolecular complexes will require more sophisticated template matching tools as well as the best resolution we can achieve. As a test of the achievable resolution in such work, we have been investigating the structure of a large rhabdovirus, sonchus yellow net virus. Tomographic reconstructions show very clearly the 5-nm period structure of the coiled nucleoprotein core and even the trimeric structure of the ~70 kD glycoprotein that studs the surface of the virus. This work, along with results from a number of other labs, support the projection that we will be able to identify complexes within bacteria that have a molecular weight with a lower limit of 500 – 750 kD.
In other work, we have been using conventionally embedded microbial cells to monitor changes following a change in culture conditions. Desulfovibrio vulgaris cells, exposed to oxygen stress, were monitored by FTIR spectroscopy. At times where the IR spectra showed interpretable changes, cells were prepared for microscopy. Changes in the 3-D structure of the cells can be correlated with apparent metabolic changes indicated by spectroscopy.
Figure 2. Surface representation of the outer membrane of a different Caulobacter cell in a tomographic reconstruction, also showing some of the cell contents.
* Presenting author
