K2 summit direct detector

Manufactured by Ametek

Sourced in United States

The K2 Summit direct detector is a high-performance electron microscopy detector from Ametek. It is designed to capture high-quality images and data from electron microscopes. The K2 Summit uses direct electron detection technology to provide low-noise and high-resolution imaging.

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31 protocols using k2 summit direct detector

Cryo-EM Data Collection Protocol

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Cryo-EM data were collected in movie mode on an FEI Krios microscope (FEI Company, Hillsboro, OR) operating at 300 kV and equipped with a K2 Summit direct detector (Gatan Inc., Pleasanton, CA) operating in super-resolution mode with pixel size of 0.82 Å per super-resolution pixel. Each movie consisted of 50 frames collected over 18.8 s with an exposure per frame of 1.4 e-/Å2 as shown by Digital Micrograph (Gatan Inc., Pleasanton, CA), giving a total exposure of 70 e-/Å2. The defocus ranged between ~0.7 to ~2.5 µm underfocus.

Abeyrathne P.D., Koh C.S., Grant T., Grigorieff N, & Korostelev A.A. (2016). Ensemble cryo-EM uncovers inchworm-like translocation of a viral IRES through the ribosome. eLife, 5, e14874.

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Cryo-EM Data Collection on PIC-cMed

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Cryo-EM data collection was performed on R1.2/1.3 gold grids (Quantifoil, Großlöbichau, Germany). Grids were glow-discharged for 45 seconds before application of 5 µL concentrated PIC-cMed sample, blotted for 5 seconds and vitrified by plunging into liquid ethane with a Vitrobot Mark IV (FEI Company, Hillsboro, US) operated at 4°C and 100% humidity. Cryo-EM data were acquired on a FEI Titan Krios G2 transmission electron microscope (FEI, Hillsboro, USA) operated in EFTEM mode at 300 kV and equipped with a K2 Summit direct detector (Gatan, Pleasanton, USA). Automated data acquisition was carried out using the FEI EPU software package at a nominal magnification of 105,000x (1.37 Å/pix). A total of 14,000 image stacks were collected at a defocus range from -0.5 µM to -5.0 µM. Each stack contained 40 frames that were acquired over a 10 seconds exposure time window in the counting mode of the camera. A dose rate of 4.2 e^-/Å²s was applied, resulting in a total dose of 42 e^-/Å².

Schilbach S., Hantsche M., Tegunov D., Dienemann C., Wigge C., Urlaub H, & Cramer P. (2017). Structures of transcription pre-initiation complex with TFIIH and Mediator. Nature, 551(7679), 204-209.

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Cryo-EM Imaging of SARS-CoV-2 S-Protein

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Holey gold film (Russo and Passmore, 2014 (link)) coated EM grids with 2 µm holes were prepared following a previously described procedure (Marr et al., 2014 (link)). The S-protein solution contained 0.4 to 0.8 mg/mL protein in 10 mM Tris, pH 8.0 and 150 mM NaCl. Vitrification of the cryo-EM specimen was performed using a Gatan Cryoplunge 3 device. For each cryo-EM specimen, 3 µL of S-protein solution was applied to a glow-discharged (2 min, 15 mA) holey grid. The grids were blotted for 13 s at 100% humidity and 295 K and then plunge-frozen in a 50:50 ethane:propane mixture (Tivol et al., 2008 (link)) at 77 K. Specimen screening and optimization was done with an FEI Tecnai TF20 electron microscope equipped with a Gatan K2 summit direct detector. The final high-resolution data collection was performed with a Thermo Fisher Scientific Titan Krios G3 300 kV microscope equipped with a Falcon 3EC direct detector. The final high-resolution data were collected at 75000x nominal magnification, resulting in a calibrated pixel size of 1.06 Å. Each movie was recorded in counting mode with a 60 s exposure and saved in 30 fractions. The exposure rate was adjusted to 0.8 electrons per pixel per second, resulting in a total exposure of 42.7 electrons per Å². The data were collected with a 1.8 to 2.2 µm defocus. A total of ~4000 movies were collected for the final high-resolution dataset.

Li Z., Tomlinson A.C., Wong A.H., Zhou D., Desforges M., Talbot P.J., Benlekbir S., Rubinstein J.L, & Rini J.M. (2019). The human coronavirus HCoV-229E S-protein structure and receptor binding. eLife, 8, e51230.

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Graphene-Coated Grids for Cryo-EM of hSGLT1 and hSGLT2

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The surface of Quantifoil Au 300 mesh R 0.6/1.0 grids were coated with graphene oxide²³. Aliquots of 2.5 µl of the hSGLT1-MAP17 complex or the hSGLT2-MAP17 complex in presence of 0.5 mM fluorinated octyl-maltoside (FOM, Anatrace) were applied to the grids. After incubation at 4 °C under 100% humidity for 60 s, the grids were blotted for 4 s using a blot force of 4, and then plunge-frozen into liquid ethane using a Vitrobot Mark IV (Thermo Fisher Scientific). The grids were transferred to a Titan Krios electron microscope (Thermo Fisher) operating at 300 kV. SerialEM-3.6.11 was used for automated data collection. Movies from the dataset of the hSGLT1-MAP17 were recorded on a K3 Summit direct detector (Gatan) mounted post a quantum energy filter (slit width 20 eV) in super-resolution mode with a defocus range of −1.4 to −1.8 μm and a magnification of 105,000×, resulting in a calibrated pixel size of 0.417 Å. Movies from datasets of the hSGLT2-MAP17 in the apo state and the AMG binding state were collected on a K2 Summit direct detector (Gatan) at a magnification of 165,000× with a calibrated pixel size of 0.4105 Å. Each stack of 32 frames was exposed for 8 s, with an exposing time of 0.25 s per frame at a dose rate of 4.7 electrons per Å⁻² per second.

Cui W., Niu Y., Sun Z., Liu R, & Chen L. (2023). Structures of human SGLT in the occluded state reveal conformational changes during sugar transport. Nature Communications, 14, 2920.

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Cryo-EM Imaging of Phage Tail-Receptor Complexes

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pb6 tubes and purified T5 tails incubated with or without FhuA were prepared following the same procedure. An aliquot of 3 μL of sample was applied to glow discharged 2:1 Quantifoil holey carbon grid (Quantifoil Micro Tools GmbH, Germany) and the grids were plunge-frozen in liquid ethane with a Vitrobot Mark II (FEI/Thermo Fisher Scientific). Samples were observed with an FEI/Thermo Fisher Scientific Polara at 300 kV. Images were recorded on a K2 Summit direct detector (Gatan Inc., USA) in super resolution counting mode. Movies were recorded for a total exposure of 4 s and 100 ms per frame, resulting in 40-frame movies with a total dose of ~40 e⁻ Å⁻². For tails incubated with FhuA and pb6 fibres, the magnification was ×23,000 (0.82 Å per pixel at the sample level) while for T5 tails, the nominal magnification was ×15,500 (1.24 Å per pixel at the sample level).

Arnaud C.A., Effantin G., Vivès C., Engilberge S., Bacia M., Boulanger P., Girard E., Schoehn G, & Breyton C. (2017). Bacteriophage T5 tail tube structure suggests a trigger mechanism for Siphoviridae DNA ejection. Nature Communications, 8, 1953.

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Cryo-EM analysis of MMTV STC

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Purified MMTV STC sample (0.5 mg/ml) was applied to R1.2/1.3 gold UltrAufoil grids, Au 400 mesh (Quantifoil), and cryo-EM grids were prepared by freezing using a manual plunger at 4°C. The grids were clipped and subsequently stored in liquid nitrogen for future data acquisition. Data were collected at the Scripps Research Institute Cryo-EM facility, La Jolla using a FEI Titan Krios (300 kV) microscope equipped with a Gatan K2 summit direct detector. The complex was imaged at 22 500× magnification and the pixel size was 1.31 Å. Movies were collected in counting mode with an electron dose rate of 3.3 electron per pixel per second. The defocus range was −1.3 to −3.0 μm. A total of 1578 movies of 100 frames/movie were collected. The data collection parameters are presented in Supplementary Table S1.

Jóźwik I.K., Li W., Zhang D.W., Wong D., Grawenhoff J., Ballandras-Colas A., Aiyer S., Cherepanov P., Engelman A.N, & Lyumkis D. (2022). B-to-A transition in target DNA during retroviral integration. Nucleic Acids Research, 50(15), 8898-8918.

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Cryo-EM Imaging of Vitrified Samples

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Sample preparation was as described (Monroe et al., 2017 (link)). Vitrified grids were loaded onto a Titan Krios (FEI) operating at 300 kV. Images were acquired using a defocus range between −1.0 to −2.2 µm. A total of 2,349 cryo-EM movies were recorded using a K2 Summit direct detector (Gatan) in counting mode with a pixel size of 1.10 Å and at a dose rate of ~7.4 e^-/pixel/sec. Each movie was recorded as a stack of 40 frames accumulated over 10 s, totaling ~62e^-/Å².

Han H., Monroe N., Sundquist W.I., Shen P.S, & Hill C.P. (2017). The AAA ATPase Vps4 binds ESCRT-III substrates through a repeating array of dipeptide-binding pockets. eLife, 6, e31324.

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Cryo-EM Structure Determination with Volta Phase Plate

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Automated data collection was performed on a Titan Krios electron microscope (FEI) operated at 300 kV and equipped with a K2 Summit direct detector, a Quantum energy filter (Gatan, Pleasanton, CA) and an FEI Volta phase plate (FEI) using SerialEM20 (link) software. Movies comprising 40 frames, 2 s exposure time and a total dose of 40 e⁻ Å⁻² were recorded on a K2 Summit direct detection camera (Gatan) in counting mode, at a calibrated magnification of 95,200 corresponding to a magnified pixel size of 0.525 Å. The small pixel size was selected to get better signal-to-noise ratio in the high-resolution region by placing it below the half-Nyquist frequency of the detector where the detective quantum efficiency is higher. For comparison, in our first attempt at solving the structure of Hgb we used a pixel size of 1.35 Å with in-focus VPP data collection and a set of ∼10,300 particles from 233 micrographs yielded a 6 Å density map (Fig. 6). The second attempt presented here comprised 2,261 micrographs acquired in one 89 h microscope session.

Khoshouei M., Radjainia M., Baumeister W, & Danev R. (2017). Cryo-EM structure of haemoglobin at 3.2 Å determined with the Volta phase plate. Nature Communications, 8, 16099.

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High-Resolution Cryo-EM Data Collection

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Images were recorded on a Titan Krios electron microscope (FEI/Thermo Fisher) equipped with a K2 summit direct detector (Gatan) at 0.394 Å per pixel in super-resolution counting mode (0.788 Å for the physical pixel size) using the Leginon software package^{18 (link)}. Data collection was performed using a dose of ~22.5 e⁻ Å⁻² across 70 frames (50 m frame⁻¹) at a dose rate of ~4 e⁻ pix⁻¹ s, using a set nominal defocus range of −0.6 to −2 μm. On our microscope, the 100 μm objective aperture allows for transmission of information up to ~1.4 Å, but could not be aligned to produce a coma-free diffractogram. In contrast, the 70 μm aperture would truncate information at the ~2 Å limit. For this reason, the objective aperture was removed to prevent physical truncation of the most widely scattering electrons—and thus the highest resolution information. A total of 1317 micrographs was recorded over a single 3.5-day collection.

Tan Y.Z., Aiyer S., Mietzsch M., Hull J.A., McKenna R., Grieger J., Samulski R.J., Baker T.S., Agbandje-McKenna M, & Lyumkis D. (2018). Sub-2 Å Ewald curvature corrected structure of an AAV2 capsid variant. Nature Communications, 9, 3628.

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Cryo-EM Imaging of γTuRC

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Freeze-thawed human γTuRC (purified as described above) was briefly spun to remove aggregates. Lacey grids (400 mesh) with a layer of ultra-thin carbon (Agar Scientific) were glow-discharged at 45 mA for 1 min using a K100X Glow Discharge Unit (EMS). A 4 μl-droplet was then applied directly onto the carbon-side of the grid loaded into the humidity chamber of a Vitrobot Mark IV (Thermo Fisher) set to room temperature and 90% humidity. After an incubation time of 60 seconds, the grid was blotted for 3s and plunged into liquid ethane. The ice quality was assessed on a 200 kV Talos Arctica (Thermo Fisher) and a small dataset was collected to evaluate the sample quality.
The highest-quality grid was imaged using a 300kV Titan Krios electron microscope (Thermo Fisher) using a GIF Quantum energy filter (Gatan) and a K2 Summit direct detector (Gatan), operated in counting mode. A total of 2,4000 movies were collected over two sessions at a pixel size of 1.08 Å/px with a total dose of ∼50 e−/A2 and a defocus range of -1.0 - -3.5 μm.

Consolati T., Locke J., Roostalu J., Chen Z.A., Gannon J., Asthana J., Lim W.M., Martino F., Cvetkovic M.A., Rappsilber J., Costa A, & Surrey T. (2020). Microtubule Nucleation Properties of Single Human γTuRCs Explained by Their Cryo-EM Structure. Developmental Cell, 53(5), 603-617.e8.

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