A workflow for protein structure determination from thin crystal lamella by micro-electron diffraction

DOI: 10.3389/fmolb.2020.00179

Authors: Emma V. Beale (Diamond Light Source) , David G. Waterman (STFC Rutherford Appleton Laboratory; CCP4, Research Complex at Harwell) , Corey Hecksel (Diamond Light Source) , Jason Van Rooyen (Diamond Light Source) , James B. Gilchrist (Diamond Light Source) , James M. Parkhurst (Diamond Light Source) , Felix De Haas (Thermo Fisher Scientific) , Bart Buijsse (Thermo Fisher Scientific) , Gwyndaf Evans (Diamond Light Source) , Peijun Zhang (Diamond Light Source; Wellcome Centre for Human Genetics, University of Oxford; University of Pittsburgh School of Medicine)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Frontiers In Molecular Biosciences , VOL 7

State: Published (Approved)
Published: August 2020
Diamond Proposal Number(s): 23169

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.

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

Subject Areas: Biology and Bio-materials, Technique Development

Diamond Offline Facilities: Electron Bio-Imaging Centre (eBIC)
Instruments: Krios I-Titan Krios I at Diamond

Added On: 03/09/2020 17:05

Documents:
fmolb-07-00179.pdf

Discipline Tags:

Technique Development - Life Sciences & Biotech Structural biology Life Sciences & Biotech

Technical Tags:

Microscopy Electron Microscopy (EM) Cryo Electron Microscopy (Cryo EM)