K2 direct electron detector

Manufactured by Ametek

Sourced in United States

The K2 direct electron detector is a high-performance imaging device designed for electron microscopy applications. It captures direct electron signals with high sensitivity and fast readout speeds, enabling high-resolution imaging and analysis of materials at the atomic scale.

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K2 Summit direct electron detector (187 citations) K2 Summit direct electron detector camera (20 citations) K2 detector (10 citations) Direct electron detector (7 citations) K2 Summit direct electron detector (Gatan), (4 citations) K2 direct electron detector camera (3 citations)

39 protocols using k2 direct electron detector

Cryo-EM Data Collection Protocols

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Single particle cryoEM data were collected on a Titan Krios microscope (‘Titan Krios 1’ at the Astbury Biostructure Laboratory) operating at 300 kV and Falcon III direct electron detector (Thermo Fisher Scientific) operating in integrating mode. Data were collected using EPU software with parameters as in Supplementary Table 1, based on a published protocol^{34 (link)}. Where the sample was tilted during collection, the autofocus and drift measurements were taken in line with the tilt axis.
Tilt series data were collected using a Titan Krios microscope (‘Titan Krios 2’) operating at 300 kV and Bioquantum energy filter (20 eV) K2 direct electron detector (Gatan) operating in counting mode, using Tomo software (Thermo Fisher Scientific). Data were collected with parameters as in Supplementary Table 2.

Byrne M.J., Iadanza M.G., Perez M.A., Maskell D.P., George R.M., Hesketh E.L., Beales P.A., Zack M.D., Berry C, & Thompson R.F. (2021). Cryo-EM structures of an insecticidal Bt toxin reveal its mechanism of action on the membrane. Nature Communications, 12, 2791.

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Cryo-EM Imaging of Macromolecular Complexes

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Frozen grids were imaged at −170 °C on a cryo-electron microscope (Polara, FEI Company) equipped with a K2 direct electron detector (Gatan) and operated at 300kV with a magnification of 15,500x, resulting in an effective pixel size of 2.6 Å. Images were collected automatically using SerialEM ^{52 (link)} in dose fraction mode. The cumulative electron dose for each single-axis tilt series was ~50e/Å^{2 (link)}, distributed over 35 images and covering an angular range of −51° to +51° with increments of 3°. Defocus ranged between −2 and −6 μm. For refinement of the trans-envelope structure, ~70 tilt series were collected on a Titan Krios, equipped with an energy filter (Gatan) with −3 μm defocus; the effective pixel size was 2.7 Å.

Wang C., Tu J., Liu J, & Molineux I.J. (2019). Structural Dynamics of Bacteriophage P22 Virions During the Initiation of Infection. Nature microbiology, 4(6), 1049-1056.

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Cryo-EM Data Collection with Direct Electron Detectors

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KCC4-MSP1D1 grids were transferred to a Talos Arctica cryo-electron microscope (FEI/Thermo Scientific, USA) operated at an acceleration voltage of 200 kV. Images were recorded in an automated fashion with SerialEM (Mastronarde, 2005 (link)) using image shift with a target defocus range of −0.7 ~ −2.2 µm over 5 s as 50 subframes with a K3 direct electron detector (Gatan, USA) in super-resolution mode with a super-resolution pixel size of 0.5685 Å. The electron dose was 9.333 e^- / Å (Marcoux et al., 2017 (link))/s (0.9333 e^-/ Å2/frame) at the detector level and total accumulated dose was 46.665 e-/Å2. KCC4-detergent grids were transferred to a Titan Krios cryo-electron microscope (FEI/Thermo Scientific, USA) operated at an acceleration voltage of 300 kV. Images were recorded in an automated fashion with SerialEM (Mastronarde, 2005 (link)) with a target defocus range of −0.7 to −2.2 µm over 9.6 s as 48 subframes with a K2 direct electron detector (Gatan, USA) in super-resolution mode with a super-resolution pixel size of 0.5746 Å. The electron dose was 6.092 e^- / Å (Marcoux et al., 2017 (link))/s (1.2184 e^- / Å (Marcoux et al., 2017 (link))/frame) at the detector level and total accumulated dose was 58.4832 e^-/Å (Marcoux et al., 2017 (link)). See also Table 1 for data collection statistics.

Reid M.S., Kern D.M, & Brohawn S.G. (2020). Cryo-EM structure of the potassium-chloride cotransporter KCC4 in lipid nanodiscs. eLife, 9, e52505.

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Cryo-EM structural analysis of AP2ΔμCSS:SMM tetherin-Nef complex

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AP2^ΔμCSS:SMM tetherin (4–26)-C2S-10aa-SIVsmm Nef (1–251)-C55A samples for data collection were prepared on 2/1-thick C-flat holey carbon copper 300-mesh grids that were prepared by plasma cleaning for 30 s at 25 mA using a Pelco easiGlow glow discharger (Ted Pella, Inc.). Complex at 0.6 mgmL-1 was applied in a 4 μL drop to one side of the grid, followed by a 30 second wait time, then plunge frozen into liquid ethane using a Vitrobot mark VI. The humidity was controlled at 100%, 22°C and the grids blotted with Whatman #1 paper using a blot force of 6 for 5 s. Two rounds of data collected (dataset 1 and 2) were performed on a Titan Krios (FEI; BACEM UCB) at a nominal magnification of 105,000 and a magnified pixel size of 1.149 Å pix-1. The dose rate was 6.08 e-/Å²/sec at the sample. Data collection was carried out using SerialEM. Dataset 1, consisting of 1647 movies, used stage shift navigation with 3 exposures per hole and focusing for each hole. Dataset 2 consisted of 10,248 micrographs using image shift navigation. 3 shots per hole were collected in a 3×3 grid of holes, focusing once per 9 holes. The defocus range was 1.00 to 2.5 m. Movies were acquired on a K2 direct electron detector (Gatan) in super-resolution counting mode with a dose fractionated frame rate of 250 ms and total collection time of 8250 ms.

Buffalo C.Z., Stürzel C.M., Heusinger E., Kmiec D., Kirchhoff F., Hurley J.H, & Ren X. (2019). Structural basis for tetherin antagonism as a barrier to zoonotic lentiviral transmission. Cell host & microbe, 26(3), 359-368.e8.

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Cryo-EM Imaging of Axonemes

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Vitrified samples were transferred into a Titan Krios transmission electron microscope (Thermo Fisher Scientific, Waltham, MA) operated at 300 keV. Axonemes that appeared well preserved (e.g., not compressed) by EM inspection were imaged using both a Volta-Phase-Plate at −0.5 µm defocus, making contrast transfer function correction unnecessary (Danev et al., 2014 (link)), and an energy filter in zero-loss mode (20-eV slit width; Gatan, Pleasanton, CA). Tilt series were recorded with a dose-symmetric scheme (Hagen et al., 2017 (link)) (−60° to +60° with 2° increments) using the microscope control software SerialEM (Mastronarde, 2005 (link)). The cumulative electron dose per tilt series was limited to ∼100 e/Å². All images were digitally recorded on a K2 direct electron detector (Gatan, Pleasanton, CA) with a dose rate of 8 electrons/pixel per second in counting mode and at a nominal magnification of 26,000×, resulting in a pixel size of 0.5 nm.

Dymek E.E., Lin J., Fu G., Porter M.E., Nicastro D, & Smith E.F. (2019). PACRG and FAP20 form the inner junction of axonemal doublet microtubules and regulate ciliary motility. Molecular Biology of the Cell, 30(15), 1805-1816.

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Cryo-EM Data Collection of PA7LF3

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Cryo-EM data sets of PA₇LF₃ were collected on a Titan Krios transmission electron microscope (FEI) equipped with a high-brightened field-emission gun (XFEG), operated at an acceleration voltage of 300 kV. Micrographs were recorded on a K2 direct electron detector (Gatan) at 130,000 x magnification in counting mode, corresponding to a pixel size of 1.07 Å. 40 frames taken at intervals of 375 ms (1.86 e^-/Å²) were collected during each exposure, resulting in a total exposure time of 15 s and total electron dose of 74.4 e^-/Å². Using the automated data collection software EPU (FEI), a total of 5238 micrographs with a defocus range between -1.2 and -2.6 μm was automatically collected.

Antoni C., Quentin D., Lang A.E., Aktories K., Gatsogiannis C, & Raunser S. (2020). Cryo-EM structure of the fully-loaded asymmetric anthrax lethal toxin in its heptameric pre-pore state. PLoS Pathogens, 16(8), e1008530.

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Cryo-EM Imaging of Mutant Huntingtin Aggregates

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Conditioned medium grids that were pre-screened on the Leica CLEM were loaded into a Talos Arctica microscope (Thermo Fisher Scientific) equipped with a post-column BioQuantum energy filter and K2 direct electron detector (Gatan, Inc.). A low-magnification (900x) montage of the entire grid was collected and manually aligned to the fluorescence image of the same grid to identify EGFP⁺ mHTT aggregates. 2D projection images and tilt series of target areas were collected at 31,000x magnification for EV-enclosed mHTTex1-EGFP densities with a pixel size of 4.30 Å/pixel. Uncoated mHTTex1-EGFP assemblies were imaged at 39,000x magnification with a corresponding pixel size of 3.49 Å/pixel. Imaging settings used were spot size 8, 100 μm condenser aperture, 100 μm objective aperture, 20 eV energy filter slit, and −5 μm defocus. Typically, a tilt series ranges from −60° to 60° at 3° step increments. The total dose is ~80–100 e/Å² per tilt series.
Tilt series for polyQ oligomer and liposome-polyQ samples were collected at 31,000x or 39,000x magnification, respectively, using the same imaging configurations.
Tilt series alignment and reconstruction were done in IMOD [17 (link)]. 3D map visualization and annotation were done using UCSF Chimera (University of California, San Francisco) [18 (link)].

Kuang X., Nunn K., Jiang J., Castellano P., Hardikar U., Horgan A., Kong J., Tan Z, & Dai W. (2021). Structural Insight into Transmissive Mutant Huntingtin Species by Correlative Light and Electron Microscopy and Cryo-electron Tomography. Biochemical and biophysical research communications, 560, 99-104.

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Cryo-EM Automated Data Acquisition and Processing

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Data were acquired using the Leginon automated data-acquisition program (Suloway et al., 2005 (link)). Image preprocessing (frame alignment with MotionCor2 (Zheng et al., 2017 (link)) and CTF estimation) were done using the Appion processing environment (Lander et al., 2009 (link)) for real-time feedback during data collection. Images were collected on a Talos Arctica transmission electron microscope (Thermo Fisher) operating at 200 keV with a gun lens of 6, a spot size of 6, 70 μm C2 aperture and 100 μm objective aperture. Movies were collected using a K2 direct electron detector (Gatan Inc.) operating in counting mode at 45,000x corresponding to a physical pixel size of 0.91 Å/pixel. The dose rate was 4.413 e/pix/sec for a 10 second exposure, which makes for a total dose of 44.13 e/Å² for the 1118 images collected at a defocus range of 0.8–2 μm.

Li Y., Cash J.N., Tesmer J.J, & Cianfrocco M.A. (2020). High-throughput cryo-EM enabled by user-free preprocessing routines. Structure (London, England : 1993), 28(7), 858-869.e3.

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Cryo-EM Sample Preparation for TFIID-IIA-SCP

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Cryo-EM samples were prepared on continuous carbon coated C-flat holey carbon grids (Protochips). Grids were plasma cleaned for 10 s in air using a Solarus Plasma Cleaner (Gatan) operating at 10 Watts. Immediately following crosslinking, 4 μl of purified TFIID-IIA-SCP complex or TAF-less PIC was added to the plasma-cleaned grid and loaded into a Vitrobot (FEI). The sample was incubated on the grid for 10 minutes at 4 °C and 100% relative humidity to enhance its absorption onto the carbon substrate, then was blotted and immediately plunge-frozen in liquid ethane. Frozen grids were transferred to a 626 Cryo-Transfer Holder (Gatan) and loaded into a Titan electron microscope (FEI) operating at 300 keV. Images were recorded with a K2 direct electron detector (Gatan) operating in counting mode at a calibrated magnification of 37,879 (1.32 Å pixel⁻¹) and a defocus range of −2 μm to −4 μm, using the Leginon data collection software for semi-automated acquisition targeting. 20-frame exposures were taken at 0.5 s per frame (10 s total exposure time), using a dose rate of 8 e⁻ pixel⁻¹ s⁻¹ (4.6 e⁻ Å⁻² s⁻¹ or 2.3 e⁻ Å⁻² per frame), corresponding to a total dose of 46 e⁻ Å⁻² per micrograph.

Louder R.K., He Y., López-Blanco J.R., Fang J., Chacón P, & Nogales E. (2016). Structure of promoter-bound TFIID and insight into human PIC assembly. Nature, 531(7596), 604-609.

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Cryo-EM Imaging of Macromolecular Complexes

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Wang C., Tu J., Liu J, & Molineux I.J. (2019). Structural Dynamics of Bacteriophage P22 Virions During the Initiation of Infection. Nature microbiology, 4(6), 1049-1056.

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