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.
Highlights
Electron diffraction has become a powerful method for structural biologists and complements well-established X-ray crystallography methods, such as rotation and serial data collection using synchrotrons and serial femtosecond crystallography with X-ray free-electron lasers (XFEL)
We demonstrated that the integrity of the crystal was maintained after cryo-focused ion beam (cryoFIB) milling, and more importantly, diffraction images from a crystal lamella displayed minimal dynamical scattering (Duyvesteyn et al, 2018)
Current microED data processing borrows software packages that were originally designed for X-ray diffraction experiments and are not optimized for handling systematic errors that are unique to electron diffraction data. To overcome these challenges and enable microED as a standard cryoEM method like single particle analysis (SPA) and cryo-electron tomography, we established an integrated workflow for routine microED of protein crystals for implementation in most standard cryoEM imaging facilities
Summary
Electron diffraction has become a powerful method for structural biologists and complements well-established X-ray crystallography methods, such as rotation and serial data collection using synchrotrons and serial femtosecond crystallography with X-ray free-electron lasers (XFEL). Current microED data processing borrows software packages that were originally designed for X-ray diffraction experiments and are not optimized for handling systematic errors that are unique to electron diffraction data To overcome these challenges and enable microED as a standard cryoEM method like single particle analysis (SPA) and cryo-electron tomography (cryoET), we established an integrated workflow for routine microED of protein crystals for implementation in most standard cryoEM imaging facilities. The workflow includes (1) cryoFIB milling to produce wellordered crystalline lamellae (200–300 nm) from larger protein crystals; (2) a standard Ceta-D detector from Thermo Fisher for microED data collection; (3) microED data analysis using DIALS (Winter et al, 2018) which has been optimized for electron diffraction (Clabbers et al, 2018) Using this workflow, the structure of proteinase K was determined to 2.0 Å resolution from a single protein crystal lamella. Future automated microED data collection strategies, similar to those for SPA and cryoET, will be built upon this integrated system
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