Single-cell structural biology with intracellular electron crystallography

Abstract Intracellular crystallization is an emerging approach in structural biology that bypasses the need for protein purification. In 2024, the InCellCryst pipeline was introduced for structural studies of intracellular crystals by serial X-ray crystallography. Serial crystallography requires the exposure of tens of thousands of cells containing intracellular crystals, precluding high-resolution studies on proteins that crystallize only in a few cells. Here we introduce IncelluloED , a method that combines intracellular crystallization with in situ 3D electron diffraction in cells and achieves high-resolution structures from just one crystal inside one cell. Experiments on a microcrystal of the HEX-1 protein from Magnaporthe grisea , grown inside an insect cell, give a structure at 1.9 Å resolution from a volume of ~1.6 µm 3 as compared to 1.8 Å resolution achieved by serial X-ray crystallography from a combined volume exceeding eleven million µm 3 . IncelluloED uses widely available cryo-EM tools and brings high-resolution structural biology into home laboratories while also advancing a vision for a “single-cell structural laboratory”.