A Workflow for Protein Structure Determination From Thin Crystal Lamella by Micro-Electron Diffraction

Abstract

MicroED has recently emerged as a powerful method for the analysis of biological structures at atomic resolution. This technique has been largely limited to protein nanocrystals which grow either as needles or plates measuring only a few hundred nanometers in thickness. Furthermore, traditional microED data processing uses established X-ray crystallography software that is not optimized for handling compound effects that are unique to electron diffraction data. Here, we present an integrated workflow for microED, from sample preparation by cryo-focused ion beam milling, through data collection with a standard Ceta-D detector, to data processing using the DIALS software suite, thus enabling routine atomic structure determination of protein crystals of any size and shape using microED. We demonstrate the effectiveness of the workflow by determining the structure of proteinase K to 2.0 Å resolution and show the advantage of using protein crystal lamellae over nanocrystals.

Keywords: cryoEM; cryoFIB; crystallography; lamella; microED; nanocrystals; proteinase K.

FIGURE 1

Electron diffraction of proteinase K crystals with and without cryoFIB milling. (A) An electron micrograph of proteinase K nanocrystals. (B) A light micrograph of proteinase K microcrystals. (C,D) Representative SEM images of proteinase K microcrystals before (C) and after (D) cryoFIB milling. (E) An ion beam image of the lamella after the final milling step illustrates the thickness of the lamella after the final milling step (dashed white lines). (F) A cryoEM image of the resultant proteinase K lamella at low magnification. The white arrows in panels (C–F) indicate the same object of interest. (G,H) Electron diffraction patterns recorded from proteinase K nanocrystals (G) and from crystal lamella (H). The dotted circle represents the 2.0 Å resolution shell. The scale bars, 10 μm in A, 20 μm in B, 5 μm in C,D, 1 μm in E, and 10 μm in F.

FIGURE 2

Overall model and example electrostatic potential maps for the proteinase K structures determined using electron diffraction data. (A) The models for the structures determined from nanocrystals (cyan) and the structures determined from lamella with the 20 μm (magenta) and 50 μm (green) condenser apertures are shown aligned by C-alpha residues in cartoon representation with the Ca2+ ion depicted as a sphere. (B–D) A section of the electrostatic potential maps around the disulfide bridge linking residues Cys34 and Cys123 is shown with the 2mFo – Fc maps contoured at 1.0 σ above the mean for the nancrystals (B), 20 μm C2 aperture lamella dataset (C) and the 50 μm C2 aperture lamella dataset (D).

FIGURE 3

F_o vs. F_c plots for the proteinase K structures. The F_o vs. F_c plots for the nanocrystals (A) and lamella structures with 20 μm (B) and 50 μm (C) apertures describe the correlation between F_o and F_c for each dataset. The |F_e| value indicates the y-intercept of the curve fitted to these plots and is inset into the bottom right corner of each graph.