An efficient eukaryotic cell-free expression and correlative cryo-electron tomography platform

Cell-free expression using Leishmania tarentolae lysates allows rapid expression of eukaryotic proteins directly from DNA templates. We develop a pipeline that combines cell-free expression system with cryogenic fluorescence microscopy that we term CC-FLEXCET (Correlative Cell-Free Leishmania EXpression and Cryo-Electron Tomography), to target and visualize expressed protein complexes by cryo-electron tomography at high resolution. We demonstrate the utility of this method by structurally characterising the filaments of the full-length apoptosis-associated speck like protein containing CARD (ASC) protein. Cell-free expression of ASC results in a polymeric structure characteristic of its cellular speck assembly. Sub-tomogram averaging allows us to resolve both the pyrin domain (PYD) to medium resolution, and show, for the first time, the arrangement of the flexibly linked caspase recruitment domain (CARD). Finally, we observed an interaction between the ASC filament and the L. tarentolae ribosome. Using template matching and quantitative approaches, we characterise this interaction and determine that there is a random structural association between the filament and the ribosome, with 57% of ribosomes oriented with the LSU toward the ASC polymer. CC-FLEXCET facilitates structural analysis of macromolecules and protein-lipid assemblies without need of purification, providing a pipeline from DNA template to protein expression to cryo-tilt series acquisition, within a single day.