Putting It All Together

To identify what a macromolecular complex looks like in the cell, we can use Correlated Light and Electron Microscopy, or CLEM, as demonstrated by this example in Caulobacter crescentus [13]. A structure of interest, in this case a “stalk band” in a cellular appendage (more on that in Chapter 4), is genetically tagged with a fluorescent label. Cells are plunge-frozen on an EM grid and first imaged by light microscopy, to locate the tagged structure within the cell. The sample is then transferred to the TEM and landmarks such as large fluorescent beads are used to find the same location. Then we can zoom in and image that location by cryo-ET to reveal the structure in detail.

To identify the location of a component within a large macromolecular complex, difference mapping can be helpful. In this approach, the gene corresponding to that component is either knocked out or a tag, like GFP, is added that will make the protein larger. A sub-tomogram average of the complex is produced and compared to a sub-tomogram average of the complex from wild-type (unmodified) cells. Often, a difference in the structure is visible, corresponding to the missing or altered component. Here you see an example of how this was used to locate a component of the flagellar motor, a protein called FliI, in Campylobacter jejuni [14]. EMD-5300; EMD-10457