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Project: Organization of alpha-Carboxysomes

Alpha-carboxysomes are a bacterial microcompartment responsible for carbon fixation in some chemoautotrophs and cyanobacteria. They are critical for their hosts' survival, but they can also be expressed and function in other bacteria and plant cells, and even function in vitro (outside of a cell). We have crystal structures of many of their proteins, but there are many unanswered questions about how the compartment is organized.

We performed cryo electron tomography with subtomogram averaging to look at the major enzyme cargo, Rubisco. We found that it polymerizes into long chains at high concentrations, and that those chains can arrange into a lattice. We did biophysical analyses on the data to look at binding partners, any conformational changes, interaction sites and more to understand how all of this works!

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Left: a carboxysome (4 nm orthoslice).  Center: subtomogram average of Rubisco.  Right: close-up on Rubisco side chains.  With this information, we can then go back and determine how the Rubisco is arranged and what it interacts with inside the carboxysome.

https://www.biorxiv.org/content/10.1101/2022.01.24.477598v1

 

The back-story:

  • This is a collaboration with Dave Savage's awesome group at UC-Berkeley, and is hopefully the first of several to follow! Cissi Blikstad and Tom Laughlin prepared the sample and shipped it to Lauren Ann Metskas, who did the tomography and subtomogram averaging. After the data were analyzed, Luke Oltrogge spent many hours with us working through the data interpretation, and Davi Ortega figured out the details for many of the calculations.
  • This work was funded by an NIH/NIGMS F32 postdoctoral fellowship to Dr. Metskas.
  • We did not know that we would find a Rubisco lattice in the carboxysomes - that was a complete surprise and led to a very different project than what we originally planned.
  • Submitted to Nature Communications in January 2022.
  • Time from first pipet tip to proof: counting from 3/2019