K2 summit direct electron detector camera

Manufactured by Ametek

Sourced in United States

The K2 Summit direct electron detector camera is a high-performance imaging device designed for use in electron microscopy applications. It features a direct electron detection technology that enables the capture of high-resolution images with improved signal-to-noise ratio and reduced radiation damage to samples. The camera's core function is to provide efficient and accurate data acquisition for various electron microscopy techniques.

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20 protocols using k2 summit direct electron detector camera

Cryo-EM Data Collection Workflow

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A 2.4 μl sample solution was applied onto a glow discharged holey carbon grid (Quantifoil R1.2/1.3 Mo grid, Quantifoil Micro Tools, Germany), and the grid was plunge-frozen into liquid ethane by Vitrobot mark IV (Thermo Fisher Science, USA) with a blotting time of 3 s at 18 °C and 90% humidity. All the data collection was performed on a prototype of CRYO ARM 200 (JEOL, Japan) equipped with a thermal field-emission electron gun operated at 200 kV, an Ω-type energy filter with a 20 eV slit width and a K2 Summit direct electron detector camera (Gatan, USA). An automated data acquisition program, JADAS (JEOL, Japan), was used to collect cryoEM image data, and pre-processing, motion correction, and CTF estimation were carried out in real-time by the Gwatch image processing pipeline software we developed. Movie frames were recorded using the K2 Summit camera at a calibrated magnification of ×45,579 corresponding to a pixel size of 1.097 Å with a defocus range from −0.6 to −1.8 μm. The data were collected with a total exposure of 10 s fractionated into 50 frames, with a total dose of ~50 electrons Å⁻² in counting mode. A total of 1702 movies were collected.

Kato T., Makino F., Miyata T., Horváth P, & Namba K. (2019). Structure of the native supercoiled flagellar hook as a universal joint. Nature Communications, 10, 5295.

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Cryo-EM Imaging of Ryanodine Receptors

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It should be noted that in this study, we used the cryo-EM micrographs from two previous studies^{22 (link),27 (link)}, in which the RyR-EGTA and RyR-30 μM Ca²⁺-ATP-caffeine samples were prepared on holey carbon grids (C-flat CF-1.2/1.3-2C-T, Protochips Inc, NC). In all, 3 μL of each sample was applied to holey-gold grids, blotted for 3.5–4 s and vitrified by rapidly plunging into liquid ethane with a Vitrobot (FEI). Data were acquired using an FEI Tecnai F30 Polara (FEI, Eindhoven) operating at 300 kV with the automated data collection software Leginon^{58 (link)} on a K2 Summit direct electron detector camera (Gatan, Pleasanton, CA) at a nominal magnification of ×31,000, corresponding to a calibrated pixel size of 1.255 Å. For experiments using carbon grids, images were recorded in dose-fractionated mode, each image being fractionated into 20 frames. The total exposure time was 4 s, yielding a total accumulated dose of 25 electrons/Å² on the specimen. The beam diameter was set at ~500 nm in order to capture two images per hole using the image shift. As normal in cryo-EM, only processes slow compared with the freezing time can be faithfully captured.

Dashti A., Mashayekhi G., Shekhar M., Ben Hail D., Salah S., Schwander P., des Georges A., Singharoy A., Frank J, & Ourmazd A. (2020). Retrieving functional pathways of biomolecules from single-particle snapshots. Nature Communications, 11, 4734.

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Freeze Substitution and Resin Embedding for Electron Microscopy

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Freeze substitution was performed as previously described^{36 (link)}. The mouse tissue was dissected, quickly transferred to 4% paraformaldehyde and 2% glutaraldehyde in phosphate buffer at pH 7.2 and allowed to fix for 2 h. The samples were slowly transitioned to 30% glycerol as a cryoprotectant and plunge-frozen in liquid ethane at −180 °C using a Leica Biosystems grid plunger. Samples were then freeze substituted in a Leica Biosystems AFS with 1.5% uranyl acetate in absolute methanol at −90 °C for 2 d and infiltrated with HM20 Lowicryl resin (Electron Microscopy Sciences) over the course of 2 d at −45 °C. The resin was ultraviolet polymerized for 3 d between −45 °C and 0 °C. Ultrathin sections were cut at 70–100 nm and collected on hexagonal 300-mesh Ni grids (Electron Microscopy Sciences). Samples were imaged at zero electron energy loss on a 200-kV JEOL 2200 FX equipped with an energy filter and a Gatan K2 Summit Direct Electron Detector camera. The acquisition software used was DigitalMicrograph (Gatan); processing was done with DigitalMicrograph and FIJI and was limited to cropping and adjustments to brightness and contrast. SGs from the salivary gland could not be analysed by TEM, as we could not obtain usable thin sections of this tissue due to problems with dehydration and plastic embedding of the acinar regions.

Ebrahim S., Chen D., Weiss M., Malec L., Ng Y., Rebustini I., Krystofiak E., Hu L., Liu J., Masedunskas A., Hardeman E., Gunning P., Kachar B, & Weigert R. (2019). Dynamic polyhedral actomyosin lattices remodel micron-scale curved membranes during exocytosis in live mice. Nature cell biology, 21(8), 933-939.

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Cryo-EM Sample Preparation Protocol

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Pre-irradiated 200 mesh Quantifoil R2/2 molybdenum grids were glow-discharged and 3 μl of proteoliposome solution added to the grid. Excess solution was blotted with a GP Leica (Leica) followed by plunge freezing into liquid ethane after blotting for 10 s at 80% humidity and 20 °C. Data collection was performed on a JEM-3000SFF (JEOL) electron microscope at 300 kV with a field emission gun and a magnification of ×40,500 . The specimen stage temperature was maintained at ~80–100 K. The images were recorded on a K2 summit direct electron detector camera (Gatan) operated in a counting mode with a pixel size of 1.235 Å at the specimen level. Each image included 24 fractioned frames and image stacks were binned 2 × 2 by Fourier cropping, resulting in a pixel size of 2.47 Å. The stacked frames were subjected to motion correction with MotionCor2^{61 (link)}.

Urbani A., Giorgio V., Carrer A., Franchin C., Arrigoni G., Jiko C., Abe K., Maeda S., Shinzawa-Itoh K., Bogers J.F., McMillan D.G., Gerle C., Szabò I, & Bernardi P. (2019). Purified F-ATP synthase forms a Ca2+-dependent high-conductance channel matching the mitochondrial permeability transition pore. Nature Communications, 10, 4341.

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Cryo-EM Imaging of GPI-TA Complex

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A total of 2.5 μL of the GPI-TA complex sample was applied to a glow-discharged holey carbon-film grid (Quantifoil, Au 1.2/1.3, 300 mesh) blotted with a Vitrobot system (FEI, Hillsboro, OR) using a 3.0 s blotting time with 100% humidity at 9 °C, then the sample was plunge-frozen in liquid ethane. Cryo-EM data collection was performed using a 300 kV Titan Krios microscope (FEI) equipped with a K2 summit direct electron detector camera (Gatan Inc., Pleasanton, CA, USA) set to super resolution mode, with a pixel size of 0.65 Å (a physical pixel size of 1.3 Å) and a defocus ranging from −1.5 µm to −2.3 µm. The specimen stage temperature was maintained at 80 K. The dose rate was 10 e⁻ s⁻¹, and each movie was 7.6 s long, dose-fractioned into 38 frames with an exposure of 1.18 e⁻ Å⁻² for each frame.

Liu S.S., Jin F., Liu Y.S., Murakami Y., Sugita Y., Kato T., Gao X.D., Kinoshita T., Hattori M, & Fujita M. (2021). Functional Analysis of the GPI Transamidase Complex by Screening for Amino Acid Mutations in Each Subunit. Molecules, 26(18), 5462.

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Cryo-EM Imaging of Biological Samples

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CryoEM images were recorded under a defocus range of 0.7–1.8 μm on a prototype of CRYO ARM 200 electron cryomicroscope (JEOL Ltd.), equipped with an Ω-type in-column energy filter and a Schottky-type field emission gun operated at 200 kV, using a K2 Summit direct electron detector camera (Gatan Inc.) with the counting mode at a nominal magnification of ×50 K, corresponding to a pixel size of 1.1 Å on the specimen. Each image was fractionated into 52 frames of every 0.3 s, at a dose rate of ~1.2 e⁻ pixel⁻¹ second⁻¹, to accumulate a total dose of ~65 e⁻ Å⁻². Dose fractionated cryoEM images were recorded using an automated data acquisition program, JADAS (JEOL Ltd.).

Yamada Y., Namba K, & Fujii T. (2020). Cardiac muscle thin filament structures reveal calcium regulatory mechanism. Nature Communications, 11, 153.

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Cryo-EM Analysis of AcrABZ-TolC Complexes

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For the AcrABZ-TolC/Puromycin sample, a 2.0 μl aliquot at 2 mg/ml was applied onto holey carbon film supported by a 200-mesh R2/1 Quantifoil grid (Quantifoil) that had been previously washed and glow discharged. The grid was blotted and rapidly frozen in liquid ethane using a Vitrobot IV (FEI) with constant temperature and humidity. The grid was stored in liquid nitrogen before imaging. Images of frozen-hydrated AcrABZ-TolC/puromycin particles were acquired on a FEI Tecnai G2 Polara electron microscope (FEI) operated at 300 kV using a K2 Summit direct electron detector camera (Gatan).
For the samples AcrABZ-TolC/MBX3132 and apo AcrAB-TolC, a 3 μl aliquot at a concentration of 2 mg ml⁻¹ was applied onto glow-discharged holey carbon grid (Quantifoil Au R1.2/1.3, 300 mesh). The grid was blotted and flash frozen in liquid ethane using a Vitrobot IV (FEI) with constant temperature and humidity. The grid was stored in liquid nitrogen before imaging. Zero-energy-loss images of frozen-hydrated AcrABZ-TolC/MBX3132 or apo AcrAB-TolC particles were recorded automatically on an FEI Titan Krios electron microscope at 300 kV, using a slit width of 20 eV on a GIF Quantum energy filter and a Gatan K2-Summit direct electron detector.
The data collection parameters for all three specimens are summarized in Supplementary file 1.

Wang Z., Fan G., Hryc C.F., Blaza J.N., Serysheva I.I., Schmid M.F., Chiu W., Luisi B.F, & Du D. (2017). An allosteric transport mechanism for the AcrAB-TolC multidrug efflux pump. eLife, 6, e24905.

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Cryo-EM Analysis of Protein Complex

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Images were collected on a Titan Krios FEG-TEM electron microscope (FEI) operated at 300 kV and recorded at a magnification of 105,000 using a K2 Summit direct electron detector camera (Gatan) in counting mode at a calibrated pixel size of 1.36 Å. In total, 1894 and 3175 dose-fractionated movies were recorded during two independent collections with the specimen subjected to total accumulated doses of 50.2 and 50.3 e⁻/Å² respectively. Individual movies were corrected for stage and beam-induced motion and CTF parameters calculated using SIMPLE3.0 (Figs. S3 and S4). Particles’ positions were automatically identified with template-based picking with the structure of SAGA resolution limited to 20 Å (EMD-10412) (25 (link)) and SIMPLE3.0. The initial set of 292,323 particles was refined to final set of 44,021 though iterative rounds of 2D classification (55 (link)). The class averages thus generated were used to determine an ab initio 3D model for further single particles refinement using RELION3.0 (Fig. S3, resolution limit of 30 Å) (56 (link), 57 ). A consensus 3D model was first obtained at 23.6 Å resolution (gold-standard FSC criterion of 0.143) and subjected to 3D classification to yield three classes populated by 16,623, 14,188, and 13,210 particles individually refined to resolutions 22.5, 29.1, and 29.1 Å respectively (Fig. 6, Figs. S3 and S4).

Adamus K., Reboul C., Voss J., Huang C., Schittenhelm R.B., Le S.N., Ellisdon A.M., Elmlund H., Boudes M, & Elmlund D. (2021). SAGA and SAGA-like SLIK transcriptional coactivators are structurally and biochemically equivalent. The Journal of Biological Chemistry, 296, 100671.

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Cryo-TEM Structural Analysis of Mla Complex

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Purified Mla complex at ~1 mg/ml was applied to glow-discharged holey grids, blotted for 6 s, and plunged in liquid ethane using a Vitrobot (FEI). Images were acquired on a FEI Tecnai G2 F20 200 kV Cryo-TEM equipped with a Gatan K-2 Summit Direct Electron Detector camera with a pixel size of 1.26 Å/pixel. 500 micrographs were collected using Leginon (Suloway et al., 2005 (link)) spanning a defocus range of −1 to −2 µm.
Movie frames were aligned with MotionCorr2 (Zheng et al., 2017 (link)) and the defocus parameters were estimated with CTFFIND4 (49). 333 high-quality micrographs were selected by manual inspection, from which ~ 55,000 particles were picked with DOG in Appion (Lander et al., 2009 (link)). Particle stacks were generated in Appion using a box size of 200 pixels. Several successive rounds of 2D and 3D classification were performed in Relion 2 (Scheres, 2012 (link); Kimanius et al., 2016 (link)) using an initial model generated by Common Lines in EMAN2 (53) leading to a final stack of ~14,000 particles for 3D structure refinement in Relion.

Kamischke C., Fan J., Bergeron J., Kulasekara H.D., Dalebroux Z.D., Burrell A., Kollman J.M, & Miller S.I. (2019). The Acinetobacter baumannii Mla system and glycerophospholipid transport to the outer membrane. eLife, 8, e40171.

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Cryo-ET Data Acquisition Protocol

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Similar to our previous experiments (Shi et al., 2019 (link)), the cryo-ET data acquisition uses a bidirectional tilt scheme. The frozen-hydrated samples were imaged on a 300 kV Titan Krios transmission electron microscope (FEI) using a K2 Summit direct electron detector camera (Gatan), with a magnification of 81,000x. The pixel size is calibrated to be 1.67 Å. The tilt-series images were acquired at −1 to −2 μm defocus range with an average cumulative dose of ~90 e⁻/Å² distributed over 34 images and covering an angular range of −51 to +51 degrees, with an angular increment of 3degree. 192 tilt series were collected in a 4-day imaging session.

Chen M., Shi X., Yu Z., Fan G., Serysheva I.I., Baker M.L., Luisi B.F., Ludtke S.J, & Wang Z. (2021). In situ structure of the AcrAB-TolC efflux pump at subnanometer resolution. Structure (London, England : 1993), 30(1), 107-113.e3.

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