In situ protein micro-crystal fabrication by cryo-FIB for electron diffraction

Micro-electron diffraction(Micro ED) is an emerging technique to use cryo-electron microscope to study the crystal structures of macromolecule from its micro-/nano-crystals, which are not suitable for conventional X-ray crystallography. However, this technique has been prevented for its wide application by the limited availability of producing good micro-/nano-crystals and the inappropriate transfer of crystals. Here, we developed a complete workflow to prepare suitable crystals efficiently for Micro ED experiment. This workflow includes in situ on-grid crystallization, single-side blotting, cryo-focus ion beam(cryo-FIB) fabrication, and cryo-electron diffraction of crystal cryo-lamella. This workflow enables us to apply Micro ED to study many small macromolecular crystals with the size of 2–10 μm, which is too large for Micro ED but quite small for conventional X-ray crystallography. We have applied this method to solve 2.5 ? crystal structure of lysozyme from its micro-crystal within the size of 10×10 ×10 lm~3. Our work will greatly expand the availability space of crystals suitable for Micro ED and fill up the gap between Micro ED and X-ray crystallography.