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

Autor:	Beale EV; Diamond Light Source, Harwell Science and Innovation Campus, Didcot, United Kingdom., Waterman DG; STFC Rutherford Appleton Laboratory, Didcot, United Kingdom.; CCP4, Research Complex at Harwell, Rutherford Appleton Laboratory, Didcot, United Kingdom., Hecksel C; Electron Bio-Imaging Centre, Diamond Light Source, Harwell Science and Innovation Campus, Didcot, United Kingdom., van Rooyen J; Electron Bio-Imaging Centre, Diamond Light Source, Harwell Science and Innovation Campus, Didcot, United Kingdom., Gilchrist JB; Electron Bio-Imaging Centre, Diamond Light Source, Harwell Science and Innovation Campus, Didcot, United Kingdom., Parkhurst JM; Diamond Light Source, Harwell Science and Innovation Campus, Didcot, United Kingdom., de Haas F; Materials and Structural Analysis, Thermo Fisher Scientific, Eindhoven, Netherlands., Buijsse B; Materials and Structural Analysis, Thermo Fisher Scientific, Eindhoven, Netherlands., Evans G; Diamond Light Source, Harwell Science and Innovation Campus, Didcot, United Kingdom., Zhang P; Electron Bio-Imaging Centre, Diamond Light Source, Harwell Science and Innovation Campus, Didcot, United Kingdom.; Division of Structural Biology, Wellcome Centre for Human Genetics, University of Oxford, Oxford, United Kingdom.; Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, United States.
Jazyk:	angličtina
Zdroj:	Frontiers in molecular biosciences [Front Mol Biosci] 2020 Aug 04; Vol. 7, pp. 179. Date of Electronic Publication: 2020 Aug 04 (Print Publication: 2020).
DOI:	10.3389/fmolb.2020.00179
Abstrakt:	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. (Copyright © 2020 Beale, Waterman, Hecksel, van Rooyen, Gilchrist, Parkhurst, de Haas, Buijsse, Evans and Zhang.)
Databáze:	MEDLINE
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