1.10 Putting It All Together Atlas of Bacterial and Archaeal Cell Structure Home
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CLEM

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 imaged first 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.

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Mapping

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

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Putting It All Together

Science benefits from collaboration, and structural biology is no exception. This extends to our tools. To understand cells across their full length scale, we need to combine what we learn from different techniques. Consider this Bdellovibrio bacteriovorus cell. To visualize its overall structure, we can use cryo-ET. To identify a particular structure in the cell, we can alter its abundance (by genetically deleting it or overexpressing it) or use a fluorescent tag (⇩). Once we have identified a structure, we can get a higher-resolution view of it by sub-tomogram averaging. Then we can again use genetics to locate the positions of individual pieces in the structure (⇩). Combining this information with clues from other biochemistry methods, we can place high-resolution structures of components solved by X-ray crystallography and single particle reconstruction into their correct context. In this way, we can begin to build up a full picture, from individual atoms to entire cells. We still have a long way to go, but someday we hope to be able to map the location and interactions of every protein in a bacterium or archaeon, creating a true, molecular atlas of the cell.

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