Biotechnology and Biological Sciences Research Council (BBSRC)
BB/S003339/1
United Kingdom
Wellcome Trust
202933/Z/16/Z
United Kingdom
Citation
Journal: Front Mol Biosci / Year: 2020 Title: A Workflow for Protein Structure Determination From Thin Crystal Lamella by Micro-Electron Diffraction. Authors: 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 ...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.
Mass: 18.015 Da / Num. of mol.: 6 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interest
N
-
Experimental details
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Experiment
Experiment
Method: ELECTRON CRYSTALLOGRAPHY
EM experiment
Aggregation state: 3D ARRAY / 3D reconstruction method: electron crystallography
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Sample preparation
Component
Name: proteinase K / Type: COMPLEX / Source: MULTIPLE SOURCES
Buffer solution
pH: 7.5
Buffer component
Conc.: 25 mM / Name: Tris
Specimen
Conc.: 50 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen support
Grid type: Quantifoil R1.2/1.3
Vitrification
Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 293.15 K Details: Excess liquid was removed by blotting for 4-6s with a Vitrobot
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Data collection
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
Microscopy
Model: FEI TALOS ARCTICA / Date: Apr 18, 2018
Electron gun
Electron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lens
Mode: DIFFRACTION / C2 aperture diameter: 20 µm
Image recording
Imaging-ID: 1 / Average exposure time: 0.85 sec. / Electron dose: 0.034 e/Å2 / Film or detector model: OTHER / Num. of grids imaged: 1 / Details: The images were recorded using a Ceta-D detector operated in rolling-shutter mode with 2x2 binning.
ID
Num. of diffraction images
1
44
2
78
3
45
4
139
EM diffraction
Camera length: 1350 mm
EM diffraction stats
Fourier space coverage: 100 % / High resolution: 2.4 Å / Num. of intensities measured: 121087 / Num. of structure factors: 10176 / Phase error: 0 ° / Phase residual: 0.1 ° / Phase error rejection criteria: none / Rmerge: 0.385 / Rsym: 0.385
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