MicroED datasets from 3 proteinase K wedge-shaped lamellae

Plunge-frozen grids containing crystals of proteinase K with approximate dimensions of 12 x 10 x 10 µm were coated with a layer of organoplatinum using the in situ gas injection system of the Scios DualBeam cryoFIB instrument (Thermo Fisher Scientific). A series of initial milling steps produced lamellae of approximately 0.5 µm thickness. Wedged lamellae were then generated by tilting the stage through a defined angle away from the initial milling position before further milling from the underside only. The defined angle was calculated to produce a lamella where the leading edge measured approximately 200 nm and the back edge of the crystal within the lamella measured approximately 400 nm.

Electron diffraction data were collected using a Thermo Fisher Scientific Talos Arctica transmission electron microscope equipped with a Ceta-D camera. The microscope was operated at an accelerating voltage of 200 kV using gun lens 8, a spot size of 11 and a 50 µm C2 aperture. These conditions gave a beamsize of 1.3 µm and an approximate electron dose of 0.04 e/Å2/s at the sample position.

Diffraction images can be read in DIALS using the FormatMRC plug-in, after renaming the files to conform to a standard serial number template.