K2 summit

Manufactured by Ametek

Sourced in United States, France, United Kingdom

The K2 Summit is a high-performance laboratory equipment designed for precise analysis and measurement. It features advanced capabilities for accurate data collection and analysis, enabling researchers and scientists to conduct their work efficiently and effectively.

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K2 Summit direct electron detector (187 citations) K2 Summit detector (73 citations) K2 Summit camera (66 citations) K2 Summit direct detector (31 citations) K2 Summit direct detection camera (12 citations) K2 Summit direct (9 citations) K2 Summit direct electron camera (8 citations) Gatan K2 Summit (7 citations) K2 Summit direct electron detection camera (6 citations) K2 Summit direct detector camera (5 citations)

297 protocols using k2 summit

Cryo-EM Sample Preparation for FAS

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To prepare samples for imaging via electron microscope, freshly purified FAS complexes were concentrated to 4 mg/mL before applying 3 μl onto the nanofabricated holey sputtered gold grid^{29 (link)} with a hole size of ~2 μm. Grid freezing was done in Vitrobot Mark IV (FEI) with 3 sec blotting at 4 °C, 100% humidity and using liquid ethane at liquid nitrogen temperature. For KS-inhibited state, purified FAS at 0.5 mg/ml was incubated with 25 μM of cerulenin for 1 h at room temperature. The sample was then concentrated to 10 mg/ml followed by addition of acetyl-CoA, malonyl-CoA, and NADPH pH-adjusted mixture. The final concentrations were 4 mg/ml FAS, 1 mM acetyl-CoA, 3 mM NADPH, and 1.4 mM malonyl-CoA. Freezing was done as with the Apo sample.
Cryo-grids were screened for ice-thickness, particle distribution, and sample behaviour with a FEI Tecnai F20 field emission electron microscope equipped with a Gatan K2 summit direct detector device (DDD) camera. Images were acquired in counting mode with 1.45 Å/pixel, 2 frames/s for 15 s, and an exposure rate of 1.2 e⁻/Å²/frame. Data collection was done with Titan Krios G3 electron microscope. See Table S1 for details on data collection from Titan Krios electron microscope.

Lou J.W., Iyer K.R., Hasan S.M., Cowen L.E, & Mazhab-Jafari M.T. (2019). Electron cryomicroscopy observation of acyl carrier protein translocation in type I fungal fatty acid synthase. Scientific Reports, 9, 12987.

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Cryo-EM Structure Determination of Bacterial RNAP

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The grids were imaged using a 300 kV Titan Krios (FEI, Inc.) equipped with a K2 Summit direct electron detector (Gatan, Inc.). Images were recorded with Serial EM in counting mode with a physical pixel size of 1.307 Å and a defocus range of 1.5–2.5 μm. Data were collected with a dose of 8 e/pixel/s. Images were recorded with a 12 s exposure and 0.25 s subframes to give a total dose of 59 e/Å². Subframes were aligned and summed using MotionCor2 (22 (link)). The contrast transfer function was estimated for each summed image using CTFFIND4 (23 (link)). From the summed images, ∼10 000 particles were manually picked and subjected to 2D classification in RELION (24 (link)). 2D averages of the best classes were used as templates for auto-picking in RELION. Auto-picked particles were manually inspected, then subjected to 2D classification in RELION. Poorly populated classes were removed, resulting in a dataset of 610 066 particles. These particles were 3D classified in RELION using a map of E. coli RNAP-promoter open complex low-pass filtered to 40 Å resolution as a reference (25 (link)). 3D classification resulted in four classes. Particles in Class 1 (RPo) were 3D auto-refined and post-processed in RELION. Particles in Class 3 were subjected to an additional 3D classification and the class with density for SspA was 3D auto-refined and post-processed.

Wang F., Shi J., He D., Tong B., Zhang C., Wen A., Zhang Y., Feng Y, & Lin W. (2020). Structural basis for transcription inhibition by E. coli SspA. Nucleic Acids Research, 48(17), 9931-9942.

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Cryo-EM Data Acquisition using FEI Titan Krios

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Cryo-EM data was collected using a FEI Titan Krios transmission electron microscope equipped with a Gatan K2 Summit direct electron detector. Automated data acquisition was facilitated by SerialEM software in super-resolution counting mode²³. Each raw movie stack consists of 40 frames with a total dose of 49.6 e^–/Å² for 8 s. Nominal defocus values were set to range from –1.2 to –1.9 μm.

Ruan Z., Osei-Owusu J., Du J., Qiu Z, & Lü W. (2020). Structures and pH sensing mechanism of proton-activated chloride channel. Nature, 588(7837), 350-354.

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Cryo-EM Structure Determination of MotA Protein Complex

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The grids were imaged using a 300 kV Titan Krios (FEI, Inc.) equipped with a K2 Summit direct electron detector (Gatan, Inc.). Images were recorded with Serial EM (31 (link)) in counting mode with a physical pixel size of 1.307 Å and a defocus range of 1.5–2.5 μm. Data were collected with a dose of 8 e/pixel/s. Images were recorded with a 12 s exposure and 0.25 s subframes to give a total dose of 56 e/Å². Subframes were aligned and summed using MotionCor2 (32 (link)). The contrast transfer function was estimated for each summed image using CTFFIND4 (33 (link)). From the summed images, ∼10 000 particles were manually picked and subjected to 2D classification in RELION (34 (link)). 2D averages of the best classes were used as templates for auto-picking in RELION. Auto-picked particles were manually inspected, then subjected to 2D classification in RELION. Poorly populated classes were removed, resulting in a dataset of 654 794 particles. These particles were 3D classified in RELION using a map of E. coli elongation complex (EMD-8585) (35 (link)) low-pass filtered to 40 Å resolution as a reference. 3D classification resulted in 4 classes. Particles in Class 3 and Class 4 were 3D auto-refined, then subjected to 3D classification focused on the MotA box. From this classification, the best-resolved class containing 105 108 particles was 3D auto-refined and post-processed in RELION.

Shi J., Wen A., Zhao M., You L., Zhang Y, & Feng Y. (2019). Structural basis of σ appropriation. Nucleic Acids Research, 47(17), 9423-9432.

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Cryo-EM Tilt Series Imaging of IDL

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Tilt image series of IDL cryo-EM samples were collected from −60° to +60° in 3° increments using a Zeiss Libra 120 Plus TEM (Carl Zeiss NTS) equipped with an in-column energy filter and a Gatan UltraScan 4 K × 4 K CCD. During data acquisition, the Gatan tomography module (Gatan Inc., Pleasanton, CA, USA) operated in Advanced Tomography mode was used to track the specimen and maintain defocus at ~2.0 μm. The acquired tilt image series at magnification of 50 k× (each pixel corresponds to 0.24 nm) represents a total dose of ~60 e⁻/Å².
Tilt image series of IDL-antibody mixture cryo-EM samples were collected from −60° to +60° at 1.5° increments on an FEI Tecnai TF20 TEM equipped with a Gatan K2 Summit direct electron detection camera. During data acquisition, the UCSF Tomography software package was used to automatically track the specimen and maintain defocus at ~7 μm. The acquired tilt image series at magnification of 19 k× (each pixel corresponds to 0.19 nm) represents a total dose of ~90 e⁻/Å².

Lei D., Yu Y., Kuang Y.L., Liu J., Krauss R.M, & Ren G. (2018). Single-molecule 3D imaging of human plasma intermediate-density lipoproteins reveals a polyhedral structure. Biochimica et biophysica acta. Molecular and cell biology of lipids, 1864(3), 260-270.

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Cryo-EM Sample Preparation and Imaging

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Gold grids were a generous gift from Dr. Bridget Carragher and were prepared as in [42 (link)]. Sample was diluted to ~225 nM in gradient buffer and applied to a plasma cleaned (6s, Gatan Solarus) gold grid in humidified CP3 chamber (FEI). Sample was blotted automatically for 2.5s and plunged into liquid ethane, then stored in liquid nitrogen until imaged.
Data were collected on an Thermo Fisher Scientific (formerly FEI) Titan Krios electron microscope operating at 300 kV equipped with a Gatan K2 Summit detector using the Leginon software [43 (link)] with an estimated underfocus ranging from 1.0 μm to 3.5 μm (distributed in an approximately Gaussian manner). The total dose was 45 e-/ Å², fractionated over 50 raw frames collected over a 10 second exposure time (200 ms per frame), with each frame receiving a dose of ~7 e-/ Å². 1958 movies were recorded at a calibrated magnification for the position of the detector of 38,167 (nominal magnification of 22,500), corresponding to a pixel size 1.31 Å. To overcome problems of preferred orientation on the grid, particles were imaged at different tilt angles (0, 10, 20, 30, 40, 50 degrees) to obtain different views [44 (link)].

Rabuck-Gibbons J.N., Popova A.M., Greene E.M., Cervantes C.F., Lyumkis D, & Williamson J.R. (2019). SrmB Rescues Trapped Ribosome Assembly Intermediates. Journal of molecular biology, 432(4), 978-990.

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Cryo-EM Imaging of Protein Complexes

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Cryo-EM un-tilted images were acquired by using a Zeiss Libra 120 Plus TEM (for IDL and IDL-antibody mixture) and a FEI Tecnai TF20 TEM (IDL-antibody mixture). On the Zeiss Libra 120 Plus TEM which was operated at 120 kV high tension, the images were acquired at a magnification of 50 k× (each pixel corresponds to 0.24 nm) using a Gatan UltraScan 4 K × 4 K CCD and an in-column energy filter. A 75-μm-diameter condenser aperture was used to select only the central portion of the electron source, and a 50-μm-diameter objective aperture was used to increase micrograph contrast. On the FEI Tecnai TF20 TEM which was operated at 200 kV high tension, the images were acquired at a magnification of 19 k× (each pixel corresponds to 0.19 nm) using a Gatan K2 Summit direct electron detection camera.

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Cryo-EM Structural Analysis of ClpC and ClpC-MecA

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S. aureus ClpC WT (6 µM) was incubated for 15 min at room temperature in 25 mM Tris-HCl (pH 7.5), 25 mM KCl, 10 mM MgCl₂, 1 mM DTT and 2 mM ATPγS. For ClpC-MecA complex formation, S. aureus MecA was incubated with ClpC in a 3:1 molar ratio. Samples were vitrified with liquid ethane on Quantifoil R2/2 grids using a Vitrobot Mark IV (FEI) at 100% humidity, 24°C temperature and blotting time of 3 s.
Images of ClpC were collected using the EPU software on a Titan Krios TEM (FEI) operating at 300kV, using a Falcon two direct electron detector (FEI). Images of ClpC in complex with MecA were collected using the EPU software on a Titan Krios TEM (FEI) operating at 300kV equipped with a Gatan K2 Summit direct electron detector and bioquantum energy filter with 20 eV slit. The defocus range was set between −1 and −3 µm with a total dose of 30 electrons/Å (Doyle et al., 2013 (link)) in 17 frames for ClpC and 50 electrons/Å (Doyle et al., 2013 (link)) in 40 frames for ClpC-MecA. Pixel size was 1.34 Å/pixel for ClpC and 1.37 Å/pixel for ClpC-MecA. The dose rate on the K2 camera was 4.125 e/pixel/sec and the exposure time 23 s.

Carroni M., Franke K.B., Maurer M., Jäger J., Hantke I., Gloge F., Linder D., Gremer S., Turgay K., Bukau B, & Mogk A. (2017). Regulatory coiled-coil domains promote head-to-head assemblies of AAA+ chaperones essential for tunable activity control. eLife, 6, e30120.

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Cryo-Electron Tomography of Viral Envelope

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The frozen-hydrated grids were imaged at −170 °C on a cryo-electron microscope (Titan Krios, Thermo Fisher Scientific), equipped with a field emission gun, an energy filter, Volta phase plate, and a direct detection device (K2 Summit, Gatan). Volta phase plate enhances the contrast of the tomogram, helping a better visualization of the Env on virus surface (Fig. 1a and b). The microscope was operated at 300 keV with a magnification of 64,000×, resulting in 2.245 Å/pixel at the specimen level. The tomographic package SerialEM^{57 (link)} was utilized to collect 35 image stacks at a range of tilt angles from −51° to +51° with increments of 3° for each tilt series. The raw images were collected from single-axis tilt series at focus with accumulative dose of ~50 e⁻/Å². For every single tilt series collection, the does-fractionated mode was used to generate 8–10 frames per projection image.

Li Z., Li W., Lu M., Bess J J.r., Chao C.W., Gorman J., Terry D.S., Zhang B., Zhou T., Blanchard S.C., Kwong P.D., Lifson J.D., Mothes W, & Liu J. (2020). Sub-Nanometer Structures of HIV-1 Envelope Trimers on Aldrithiol-2 Inactivated Virus Particles. Nature structural & molecular biology, 27(8), 726-734.

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High-Resolution Electron Microscopy Protocols

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The
electron dose is reported
for each HRTEM image. The images were acquired with two types of detectors:
a CCD camera with 2048 × 2048 pixels (UltraScan 1000, Gatan Inc.),
and a direct electron detection camera (K2 Summit, Gatan Inc.), with
7420 × 7676 pixels in super-resolution mode. The K2 camera is
able to capture images on a large field of view with atomic resolution,
while minimizing electron exposure. The HRTEM images were acquired
with the K2 camera unless specified.

Dang Z., Shamsi J., Palazon F., Imran M., Akkerman Q.A., Park S., Bertoni G., Prato M., Brescia R, & Manna L. (2017). In Situ Transmission Electron Microscopy Study of Electron Beam-Induced Transformations in Colloidal Cesium Lead Halide Perovskite Nanocrystals. ACS Nano, 11(2), 2124-2132.

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