In situ protein crystallography: a single-crystal electron diffraction pipeline for structure determination inside living cells

Intracellular crystallization is an emerging approach in structural biology that bypasses the need for protein purification. An InCellCryst pipeline was recently established for structure determination by serial X-ray crystallography. Serial crystallography requires the exposure of tens of thousands of cells containing intracellular crystals, precluding high-resolution structural studies on proteins with low numbers of crystals. To overcome this limitation, we combined the InCellCryst approach with the advantages of 3D electron diffraction and established the new IncellED method. Using microcrystals of the HEX-1 protein from Magnaporthe grisea, grown inside High Five insect cells, we demonstrate that electron diffraction data collected from a single crystal yields a high-resolution structure at 1.9 Å resolution, comparable to 1.8 Å achieved by serial X-ray crystallography. The IncellED methodology opens structure determination for proteins that crystallize with low efficiency and use widely available electron cryo-microscope infrastructure, paving the way for intracellular macromolecular electron crystallography at high resolution.