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

Abstract

Micro-electron diffraction (MicroED) 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 MicroED 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 MicroED to study many small macromolecular crystals with the size of 2-10 μm, which is too large for MicroED but quite small for conventional X-ray crystallography. We have applied this method to solve 2.5 Å crystal structure of lysozyme from its micro-crystal within the size of 10 × 10 × 10 μm³. Our work will greatly expand the availability space of crystals suitable for MicroED and fill up the gap between MicroED and X-ray crystallography.

Keywords: Cryo focused ion beam; Cryo-electron microscopy; Electron diffraction; In situ crystallization; Micro-crystal.

Fig. 1

In situ on-grid protein crystallization setup. A A magnified SEM image of the non-magnetic nickel grid coated with holy carbon film. Scale bar, 16.7 μm. B A further magnified SEM image in A showing the structural details of the holy carbon film. Scale bar, 1.7 μm. C The schematic diagram of the sitting drop vapor diffusion setup for in situ on-grid protein crystallization. The real photo of the micro-bridge is shown in right. Scale bar, 0.35 mm

Fig. 2

Photographs of the lysozyme crystals growing on different grids. A Copper grid. B Titanium grid. C Molybdenum grid. D Non-magnetic nickel grid. Scale bar, 500 μm

Fig. 3

FIB fabrication of frozen lysozyme crystals on grid. A and B SEM images (SE detector) of frozen lysozyme crystals on grid with low magnification (A) and high magnification (B). The areas with strong contrast indicate the positions of the crystals. The black arrows indicate the crystals close to the grid bars and the red arrows indicate the crystals trimmed by FIB. Scale bars, 50 μm. C TEM micrograph of the FIB fabricated crystal lamella. Scale bar, 500 nm. D Cryo-electron diffraction pattern of the FIB fabricated crystal lamella in C

Fig. 4

Statistic parameters during electron diffraction dataset processing by iMOSFLM. A A representative average spot profile of one diffraction image (up) and a representative standard profile for different regions of the detector (down). The red line indicates the profile is poor and averaged by including reflections from inner regions. B The crystal-to-detector distance changes with different diffraction images. C The crystal orientation changes with different diffraction images. D The electron beam position changes with different diffraction images. E The averaged SNR of diffraction spots changes with different diffraction images. The yellow curve represents all partial spots and the red one for all full spots

Fig. 5

Electron potential map determined by molecular replacement. A The overall map fitted with the whole lysozyme structural model. B A zoomed-in view of the electron potential map around a selected region showing the resolution and quality of the map