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

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

Frontiers in molecular biosciences (2020-08-28)
Emma V Beale, David G Waterman, Corey Hecksel, Jason van Rooyen, James B Gilchrist, James M Parkhurst, Felix de Haas, Bart Buijsse, Gwyndaf Evans, Peijun Zhang
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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Proteinase K from Tritirachium album, lyophilized powder, BioUltra, ≥30 units/mg protein, for molecular biology