Cryo-correlative electron tomography for structural biology of complex mixtures

Join us on October 1 for a webinar presented by Dr. Nick Ariotti of the University of Queensland who will present his work creating a robust, structural analysis pipeline of macromolecules and lipid-protein complexes that does not require protein purification.

Using a cell-free expression system with cryogenic correlative light microscopy, Dr. Ariotti’s group can target fluorescently tagged protein complexes for subsequent cryo-electron tomography and structural analyses. Using this system, his team has characterized Leishmania tarentolae microtubules to 7.1 Å in the lysate. Through protein expression, cryo-fluorescence microscopy and cryo-ET, this system can be used to target integral membrane proteins inserting into native membrane as well as microtubule associated proteins. He will further demonstrate the utility of this system by showing the first full-length structural analysis of human Apoptosis-associated Speck like protein containing CARD (ASC) protein filament. 

The cell-free cryoCLEM platform, here termed CC-FLEXCET (Correlative Cell-Free Leishmania EXpression and Cryo-Electron Tomography) is a robust pipeline from DNA template to protein expression, plunge freezing, cryo-fluorescence microscopy and cryo-tilt series acquisition, through to structural analyses.  

This work was achieved with the Thermo Scientific Glacios Cryo-TEM, a 200 kV system designed to improve accessibility of cryo-EM. Following Dr. Ariotti’s talk, Dr. Lingbo Yu of Thermo Fisher Scientific will discuss 200 kV cryo-TEM technology and its capabilities.