National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)
R35 GM122588
United States
Citation
Journal: Structure / Year: 2019 Title: Collection of Continuous Rotation MicroED Data from Ion Beam-Milled Crystals of Any Size. Authors: Michael W Martynowycz / Wei Zhao / Johan Hattne / Grant J Jensen / Tamir Gonen / Abstract: Microcrystal electron diffraction (MicroED) allows for macromolecular structure solution from nanocrystals. To create crystals of suitable size for MicroED data collection, sample preparation ...Microcrystal electron diffraction (MicroED) allows for macromolecular structure solution from nanocrystals. To create crystals of suitable size for MicroED data collection, sample preparation typically involves sonication or pipetting a slurry of crystals from a crystallization drop. The resultant crystal fragments are fragile and the quality of the data that can be obtained from them is sensitive to subsequent sample preparation for cryoelectron microscopy as interactions in the water-air interface can damage crystals during blotting. Here, we demonstrate the use of a focused ion beam to generate lamellae of macromolecular protein crystals for continuous rotation MicroED that are of ideal thickness, easy to locate, and require no blotting optimization. In this manner, crystals of nearly any size may be scooped and milled to desired dimensions prior to data collection, thus streamlining the methodology for sample preparation for MicroED.
ProteinaseK / Endopeptidase K / Tritirachium alkaline proteinase
Mass: 28958.791 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Engyodontium album (fungus) / Gene: PROK / Production host: Engyodontium album (fungus) / References: UniProt: P06873, peptidase K
Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 90 K / Temperature (min): 77 K
Image recording
Average exposure time: 2 sec. / Electron dose: 0.04 e/Å2 / Film or detector model: FEI CETA (4k x 4k) / Num. of diffraction images: 100 / Num. of grids imaged: 1 Details: Continuous rotation from -30 to +30 with a rotation rate of 0.2 degrees per second and a readout every 3 seconds.
Image scans
Sampling size: 28 µm / Width: 4096 / Height: 4096
EM diffraction
Camera length: 1700 mm
EM diffraction shell
Resolution: 2.75→3.1483 Å / Fourier space coverage: 88.23 % / Multiplicity: 2.9 / Num. of structure factors: 1831 / Phase residual: 28.11 °
EM diffraction stats
Fourier space coverage: 88.4 % / High resolution: 2.75 Å / Num. of intensities measured: 31013 / Num. of structure factors: 10807 / Phase error: 22.41 ° / Phase residual: 22.41 ° / Phase error rejection criteria: 0 / Rmerge: 0.47 / Rsym: 0.39
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