K3 summit direct electron detector

Manufactured by Ametek

Sourced in United States, United Kingdom

The K3 Summit direct electron detector is a high-performance imaging device designed for use in electron microscopy. It captures electron signals directly, providing high-resolution and low-noise data for scientific analysis and research.

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48 protocols using k3 summit direct electron detector

Structural Studies of PORCN-Ligand Complexes

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PORCN samples (~8 mg/ml) with 10 μM LGK974 and HHAT–SHH-N–Fab^3H02 complex (~10mg/ml) were applied to Quantifoil R1.2/1.3 400 mesh Au holey carbon grids (Quantifoil), respectively. For the preparation of palmitoleoyl-CoA-bound PORCN samples, palmitoleoyl-CoA was added into the apo-PORCN sample at the final concentration of 1 mM before vitrification. For the preparation of LGK974/WNT3Ap-bound PORCN samples, WNT3Ap (MHLKCKCHGLSGSCEVKTCWWS, C5-C19, C7-C14, Biomatik) were mixed with LGK974-bound PORCN at a final concentration of 1 mM. For the preparation of product-bound PORCN, pamWNT3Ap was added into the apo-PORCN at the final concentration of 1.2 mM. The above two peptide mixtures were incubated on ice for 30 min before vitrification. The grids were blotted and plunged into liquid ethane for flash freezing using a Vitrobot Mark IV (FEI). The grids were imaged in a 300 kV Titan Krios (FEI) with a Gatan K3 Summit direct electron detector (Gatan). Data were collected using SerialEM ^{45 (link)} at 0.83 Å/pixel or 0.842 Å/pixel. Images were recorded for 5-second exposures in 50 subframes with a total dose of ~60 electrons per Å².

Liu Y., Qi X., Donnelly L., Elghobashi-Meinhardt N., Long T., Zhou R.W., Sun Y., Wang B, & Li X. (2022). Mechanisms and Inhibition of Porcupine-Mediated Wnt Acylation. Nature, 607(7920), 816-822.

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Cryo-EM Imaging of AdeB-Et Protein Complex

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A 2 μM AdeB-nanodisc sample was incubated with 20 μM Et for 2 h to form the AdeB-Et complex. The sample was then applied to lysine-coated graphene-oxide grids (Quantifoil Cu R1.2/1.3, 300 mesh) prepared in-house, blotted for 3 s, and then plunge-frozen in liquid ethane using a Vitrobot (Thermo Fisher). The grids were then transferred into cartridges. The images were recorded at −1 to −2.0 μm defocus on a Titan Krios equipped with a K3 summit direct electron detector (Gatan) with counting mode at nominal ×81,000 magnification, corresponding to a sampling interval of 1.08 Å/pixel (superresolution, 0.54 Å/pixel). Each micrograph was exposed for 2.6 s with 18.02 e−/s/physical pixel dose rate (total specimen dose, 40 e−/Å²), and 40 frames were captured per specimen area using Latitude.

Morgan C.E., Glaza P., Leus I.V., Trinh A., Su C.C., Cui M., Zgurskaya H.I, & Yu E.W. (2021). Cryoelectron Microscopy Structures of AdeB Illuminate Mechanisms of Simultaneous Binding and Exporting of Substrates. mBio, 12(1), e03690-20.

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Cryo-EM Sample Preparation for Protein Complex

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Aliquots of NC22/17-HA 131E ectodomain (4 uL, 0.18 mg/mL, in 20 mM Tris pH 8.0, 150 mM NaCl.) were applied to graphene oxide (GO) grids (GO on Quantifoils R1.2/1.3 400 mesh copper grids, R1.2/1.3). In the Vitrobot Mark IV (Thermo Fisher Scientific), the humidity was set at 100%, the protein solution was applied to the grid and there was a wait of 1 s before blotting. A blot force of 0 and blot time of 2 s were applied to blot the grid after waiting. After blotting, the grid was plunged into precooled liquid ethane at a liquid nitrogen temperature (69 (link)).
Images for complex were recorded using FEI Titan Krios microscope (Thermo Fisher Scientific) operating at 300 kV with a Gatan K3 Summit direct electron detector (Gatan Inc.) at Shanxi Academy of Advanced Research and Innovation. The automated software (EPU)3 was used to collect 5,746 movies for complex in super-resolution mode at a nominal magnification of 105,000× with a calibrated pixel size of 0.85 Å and at a defocus range between −1.0 and −2.0 um. Each movie has a total accumulate exposure of 60 e-/Å2 fractionated in 32 frames of 1.38 s exposure (70 (link)).

Sun H., Deng G., Sun H., Song J., Zhang W., Li H., Wei X., Li F., Zhang X., Liu J., Pu J., Sun Y., Tong Q., Bi Y., Xie Y., Qi J., Chang K.C., Gao G.F, & Liu J. (2022). N-linked glycosylation enhances hemagglutinin stability in avian H5N6 influenza virus to promote adaptation in mammals. PNAS Nexus, 1(3), pgac085.

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Cryo-Electron Tomography of Cellular Structures

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Cryo-lamella grids were visualized in a Titan Krios electron microscope (Thermo Fisher Scientific) equipped with a field emission gun, a GIF Quantum LS postcolumn energy filter (Gatan), and a K3 Summit direct electron detector (Gatan). The electron microscope was operated at 300 kV in nanoprobe mode at magnification 26,000× (pixel size of 3.3 Å at the specimen level). Cryo-ET tilt series with a dose-symmetric acquisition scheme (43 (link)) were acquired at a target defocus of –6 μm without Volta phase plate (VPP) or –1 μm with VPP in SerialEM (44 , 45 (link)). The K3 detector was operated in counting mode, and 10 sequentially acquired 0.1-s frames were combined. The tilt range was typically between ±50° and ±60°, with increments of 3°, and the total electron dose was about 80 e/Å². Imaging details are shown in SI Appendix, Table S1.

Cai S., Wu Y., Guillén-Samander A., Hancock-Cerutti W., Liu J, & De Camilli P. (2022). In situ architecture of the lipid transport protein VPS13C at ER–lysosome membrane contacts. Proceedings of the National Academy of Sciences of the United States of America, 119(29), e2203769119.

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Cryo-EM structure determination of NAIP5

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The purified Flag-NAIP5 (0.3 mg ml⁻¹) was loaded onto a glow-discharged UltrAuFoil grid (R1.2/1.3 300-mesh, Electron Microscopy Sciences no. Q350AR13A). Vitrification was performed using Mark IV Vitrobot (Thermo Fisher) with 3–5 s blot time under 100% humidity at 4 °C. These vitrification conditions were optimized through screening on Arctica to check ice thickness and particle distribution, the best grids were used for data collection on a Titan Krios (Thermo Fisher) equipped with BioQuantum Imaging Filter (Gatan Inc., slit width 20 eV) and K3 Summit direct electron detector (Gatan Inc.). All videos were automatically collected using serialEM³⁹ at nominal magnification ×81,000 (super-resolution pixel size 0.5347 Å) with a total dose of 52 e⁻/Å², and were then fractionated into 52 frames. Defocus range was set from 1.0 to 2.5 μm for all datasets: 3,682 images were collected at 0° and 1,597 at 30° stage tilt.

Paidimuddala B., Cao J., Nash G., Xie Q., Wu H, & Zhang L. (2023). Mechanism of NAIP—NLRC4 inflammasome activation revealed by cryo-EM structure of unliganded NAIP5. Nature structural & molecular biology, 30(2), 159-166.

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Cryo-EM Data Collection Protocol

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Beam-shift single-particle cryo-EM data were collected by SerialEM^{54 (link)} on a Titan Krios 300 kV FEG electron microscope (Thermo Fisher) equipped with a K3 Summit direct electron detector (Gatan, Inc.) and the BioQuantum energy filter (Gatan, Inc.) (Table 1). The beam-shift pattern used to collect movies is four movies per hole and four holes per movement. The final pixel size is 1.370 Å/pixel. A total dose of 45 or 70 electrons per Å² was radiated to each movie with 40 frames. The defocus range was between −1.0 and −3.0 μm at an interval of 0.25 μm.

Ghanaeian A., Majhi S., McCafferty C.L., Nami B., Black C.S., Yang S.K., Legal T., Papoulas O., Janowska M., Valente-Paterno M., Marcotte E.M., Wloga D, & Bui K.H. (2023). Integrated modeling of the Nexin-dynein regulatory complex reveals its regulatory mechanism. Nature Communications, 14, 5741.

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Cryo-EM Sample Preparation and Data Collection

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Cryo grids were prepared on the Thermo Fisher Vitrobot Mark IV. Quantifoil R1.2/1.3 Cu grids were glow-discharged using the Pelco Easyglow. Concentrated xlCHPT1 (3.5 μL) was applied to glow-discharged grids. After blotting with filter paper (Ted Pella) for 3.5–4.5 s, the grids were plunged into liquid ethane cooled with liquid nitrogen. For cryo-EM data collection, movie stacks were collected using EPU (Thermo Fisher Scientific) on a Titan Krios at 300 kV with a Quantum energy filter (Gatan), at a nominal magnification of ×81,000 and with defocus values of −2.0 to −0.8 μm. A K3 Summit direct electron detector (Gatan) was paired with the microscope. Each stack was collected in the super-resolution mode with an exposing time of 0.175 s per frame for a total of 50 frames. The dose was about 50 e^- per Å² for each stack. The stacks were motion-corrected with Relion 3 and binned (2 × 2) so that the pixel size was 1.07 Å^{32 (link)}. Dose weighting was performed during motion correction, and the defocus values were estimated with Gctf^{33 (link)}.

Wang L, & Zhou M. (2023). Structure of a eukaryotic cholinephosphotransferase-1 reveals mechanisms of substrate recognition and catalysis. Nature Communications, 14, 2753.

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CBD and 2-APB Modulation of TRPV2 in Nanodiscs

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6.5 mg/mL TRPV2 in nanodiscs were incubated with 100 µM CBD and 1 mM 2-APB on ice for 30 min and then 3 µL aliquots were applied to glow-discharged Quantifoil grids (R 1.2/1.3 Cu 300 mesh). The grids were blotted for 4 s, with blot force of 4 and 100% humidity, at 16°C using an FEI Vitrobot Mark IV (Thermo Fisher Scientific), followed by plunging into liquid ethane cooled by liquid nitrogen. Images were acquired using an FEI Titan Krios equipped with a Gatan LS image energy filter (slit width, 20 eV) operating at 300 kV. A Gatan K3 Summit direct electron detector was used to record movies in superresolution mode with a nominal magnification of ×105,000,, resulting in a calibrated pixel size of 0.415 Å per pixel. The typical defocus values ranged from −0.5 to −1.5 μm. Exposures of 1.6 s were dose-fractionated into 32 frames, resulting in a total dose of 52 e⁻ Å⁻². Images were recorded using the automated acquisition program SerialEM (Mastronarde, 2005 (link)).

Gochman A., Tan X.F., Bae C., Chen H., Swartz K.J, & Jara-Oseguera A. (2023). Cannabidiol sensitizes TRPV2 channels to activation by 2-APB. eLife, 12, e86166.

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Cryo-EM of hXKR8–hBSGΔ-FLAG–Fab Complex

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In cryo-EM, fluorinated Fos-choline 8 was added at a final concentration of 0.075% to the hXKR8–hBSGΔ-FLAG–Fab complex in lipid nanodiscs. An aliquot (3 μl) of the sample was applied to a glow-discharged Quantifoil holey carbon grid (Au, R1.2/1.3, 300-mesh), blotted for 4 s (+10 blot force, 10 s wait time, and 100% humidity at 6 °C), and plunge-frozen by Vitrobot (Thermo Fisher Scientific). Movies were acquired using a Titan Krios G3i (Thermo Fisher Scientific) equipped with a K3 summit direct electron detector (Gatan) running at 300 kV operated in the counting mode with a nominal magnification of 105,000×, corresponding to a calibrated pixel size of 0.83 Å. Data were automatically acquired using EPU software (Thermo Fisher Scientific). The dose rate was 10.432 e⁻/Å² per second, and the total exposure time was 4.79 s, resulting in a total dose of 50 e⁻/Å² over 50 frames. A total of 6118 movies were collected.

Sakuragi T., Kanai R., Otani M., Kikkawa M., Toyoshima C, & Nagata S. (2024). The role of the C-terminal tail region as a plug to regulate XKR8 lipid scramblase. The Journal of Biological Chemistry, 300(3), 105755.

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Cryo-EM Imaging of Axoneme Structure

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Using a Titan Krios 300 kV FEG electron microscope (Thermo Fisher) with a K3 Summit direct electron detector (Gatan, Inc) and the BioQuantum energy filter (Gatan, Inc), movies of the axoneme were acquired at 64 kx nominal magnification (calculated pixel size of 1.370 Å/pixel) using SerialEM (Mastronarde, 2005 (link)). A total dose of 45 electrons per Å² over 40 frames for the WT and MEC17-KO datasets. A total dose of 73 electrons per Å² per frame over 30 frames for the K40R dataset. The defocus range was between −1.0 and −3.0 μm at an interval of 0.25 μm.

Yang S.K., Kubo S., Black C.S., Peri K., Dai D., Legal T., Valente-Paterno M., Gaertig J, & Bui K.H. (2024). Effect of α-tubulin acetylation on the doublet microtubule structure. eLife, 12, RP92219.

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