National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
P41GM136508
United States
Citation
Journal: J Struct Biol / Year: 2022 Title: Electron-counting MicroED data with the K2 and K3 direct electron detectors. Authors: Max T B Clabbers / Michael W Martynowycz / Johan Hattne / Brent L Nannenga / Tamir Gonen / Abstract: Microcrystal electron diffraction (MicroED) uses electron cryo-microscopy (cryo-EM) to collect diffraction data from small crystals during continuous rotation of the sample. As a result of advances ...Microcrystal electron diffraction (MicroED) uses electron cryo-microscopy (cryo-EM) to collect diffraction data from small crystals during continuous rotation of the sample. As a result of advances in hardware as well as methods development, the data quality has continuously improved over the past decade, to the point where even macromolecular structures can be determined ab initio. Detectors suitable for electron diffraction should ideally have fast readout to record data in movie mode, and high sensitivity at low exposure rates to accurately report the intensities. Direct electron detectors are commonly used in cryo-EM imaging for their sensitivity and speed, but despite their availability are generally not used in diffraction. Primary concerns with diffraction experiments are the dynamic range and coincidence loss, which will corrupt the measurement if the flux exceeds the count rate of the detector. Here, we describe instrument setup and low-exposure MicroED data collection in electron-counting mode using K2 and K3 direct electron detectors and show that the integrated intensities can be effectively used to solve structures of two macromolecules between 1.2 Å and 2.8 Å resolution. Even though a beam stop was not used with the K3 studies we did not observe damage to the camera. As these cameras are already available in many cryo-EM facilities, this provides opportunities for users who do not have access to dedicated facilities for MicroED.
Name: CALCIUM ION / type: ligand / ID: 2 / Number of copies: 4 / Formula: CA
Molecular weight
Theoretical: 40.078 Da
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Experimental details
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Structure determination
Method
cryo EM
Processing
electron crystallography
Aggregation state
3D array
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Sample preparation
Concentration
50 mg/mL
Buffer
pH: 8
Grid
Model: Quantifoil R2/1 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 10.0 nm
Film or detector model: GATAN K2 BASE (4k x 4k) / Digitization - Dimensions - Width: 1650 pixel / Digitization - Dimensions - Height: 1470 pixel / Digitization - Sampling interval: 10.0 µm / Number grids imaged: 1 / Number real images: 1 / Number diffraction images: 1600 / Average exposure time: 0.025 sec. / Average electron dose: 0.01 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
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Image processing
Crystal parameters
Unit cell - A: 67.57 Å / Unit cell - B: 67.57 Å / Unit cell - C: 100.91 Å / Unit cell - γ: 90 ° / Unit cell - α: 90 ° / Unit cell - β: 90 ° / Space group: P 43 21 2
Crystallography statistics
Number intensities measured: 30326 / Number structure factors: 5453 / Fourier space coverage: 83 / R sym: 30 / R merge: 64 / Overall phase error: 28 / Overall phase residual: 0 / Phase error rejection criteria: None / High resolution: 2.7 Å / Shell - Shell ID: 1 / Shell - High resolution: 2.7 Å / Shell - Low resolution: 3.4 Å / Shell - Number structure factors: 2499 / Shell - Phase residual: 33 / Shell - Fourier space coverage: 83 / Shell - Multiplicity: 6
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Movie
Controller
Open data
Basic information
Map data
Sample
Function and homology information
proteolysis / extracellular region / 
Parengyodontium album (fungus)
Authors
United States, 2 items 
Citation
Structure visualization
Downloads & links
emd_27900.png
http://ftp.pdbj.org/pub/emdb/structures/EMD-27900
Sample components
Processing
Electron microscopy
