Holey carbon film-coated EM grids were nanofabricated with regular arrays of 500 to 800 nm holes36 (link) and coated with an additional layer of gold. Cryo-EM specimens were prepared with a FEI Vitrobot grid preparation robot at 4 °C and 100 % humidity by applying 3 μl of sample (3 mg/ml) to glow-discharged grids, allowing the grids to equilibrate for 1 s, and blotting for 12 s before freezing in a liquid ethane/propane mixture (1:1 v/v)37 (link). Grids were subsequently stored in liquid nitrogen before shipping to the New York Structural Biology Center for imaging with a FEI Titan Krios electron microscope equipped with a Gatan K2 Summit camera and automated with Leginon38 (link).
K2 summit camera
Manufactured by Ametek
Sourced in United States
The K2 Summit camera is a high-performance imaging device designed for scientific and industrial applications. It features a large sensor and advanced optics, providing high-resolution, low-noise images. The camera's core function is to capture and digitize visual data for various analytical and research purposes.
Automatically generated - may contain errors
Lab products found in correlation
Vitrobot mark 4, by Thermo Fisher Scientific (26 mentions)
Gif quantum energy filter, by Ametek (6 mentions)
Titan krios, by Thermo Fisher Scientific (5 mentions)
Titan krios electron microscope, by Ametek (4 mentions)
Titan krios, by Ametek (4 mentions)
Vitrobot, by Thermo Fisher Scientific (4 mentions)
Titan krios microscope, by Thermo Fisher Scientific (4 mentions)
Tecnai f20, by Thermo Fisher Scientific (4 mentions)
K3 summit camera, by Ametek (3 mentions)
Em gp, by Leica (3 mentions)
Polara microscope, by Thermo Fisher Scientific (3 mentions)
Bioquantum, by Ametek (3 mentions)
Titan krios transmission electron microscope, by Thermo Fisher Scientific (3 mentions)
Titan krios g3 electron microscope, by Thermo Fisher Scientific (3 mentions)
Titan krios electron microscope, by Thermo Fisher Scientific (3 mentions)
Vitrobot grid preparation robot, by Thermo Fisher Scientific (2 mentions)
Trpa1 to 1.2 1 3 holey carbon grids, by Quantifoil (2 mentions)
Holey carbon grid, by Quantifoil (2 mentions)
Polara and titan krios microscopes, by Thermo Fisher Scientific (2 mentions)
R1.2 1 3 holey carbon grid, by Quantifoil (2 mentions)
66 protocols using k2 summit camera
1
Cryo-EM Specimen Preparation via Nanofabricated Grids
2
Cryo-EM Specimen Preparation via Nanofabricated Grids
3
Cryo-EM of Coxsackievirus A24 Variant
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Cryo-EM grids were prepared by applying 3 μL of purified CV-A24v (9.6 mg/mL) to 400 mesh lacey grids coated in a 3 nm carbon film (Agar Scientific, Essex, UK). The sample was left to adsorb for 30 s, and then the excess sample was removed by manual blotting. On-grid binding of pentavalent Neu5Ac conjugate was then performed by applying 3 μL of a 5 mM solution of compound 14 in PBS to the pre-blotted, CV-A24v-coated grid, and leaving for 30 s before blotting and freezing using a Leica EM GP plunge freeze device (Leica Microsystems, Wetzlar, Germany). The Leica EM chamber temperature was set to 8 °C with 80% relative humidity and liquid ethane was used for sample vitrification. Grids were glow discharged at 20 mA for 30 s prior to application of the sample, using a PELCO easiGlow™ (Ted Pella, Redding, CA, USA). All data sets were collected on a Titan Krios (Thermo Fisher Scientific, Waltham, MA, USA) transmission electron microscope operating at 300 kV, at a nominal magnification of 165,000×. A total of 1443 exposures were recorded using EPU automated acquisition software on a Gatan K2 summit camera operating in super-resolution mode, using a total electron dose of 48.9 e−/Å2. Each movie had a total exposure of 10 s and contained 50 fractions. The final calibrated physical pixel size was 0.82 Å. Detailed information on data collection was shown in Table 2 .
4
Cryo-EM imaging at high resolution
5
Cryo-EM Imaging of TRPA1 Protein
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Samples for cryo-EM were prepared by applying 4 μl of purified TRPA1 to 1.2/1.3 holey carbon grids (Quantifoil) and blotting for 8-12 sec. in a Vitrobot Mark IV (Thermo Fisher) prior to plunge freezing in liquid ethane. For multi-shot imaging, samples were prepared on 2/2 holey carbon grids (Quantifoil) and blotted for 6-8 s. Cryo-EM samples were imaged on Polara and Titan Krios microscopes (Thermo Fisher, see Extended Data Table 1 for details) equipped with the K2 Summit camera (Gatan). Movies were drift-corrected using MotionCor234 (link) and CTF parameters estimated with gctf35 (link). Particle images were selected from micrographs using gautomatch (MRC-LMB, https://www.mrc-lmb.cam.ac.uk/kzhang/ ) and extracted in Relion36 (link). 2- and 3-D classification of particle images was performed in cryoSPARC37 (link) and 3D maps refined in cryoSPARC and cisTEM38 (link). Conversion of data from cryoSPARC to Relion and generation of orientation distribution plots were performed using pyem39 (link). Directional Fourier shell correlations of cryo-EM maps were performed as previously described40 (link).
6
Structural Analysis of Primosome Assembly
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To alleviate any preferred orientation of the particles, 0.2% n-Octyl-β-D-glucoside was quickly mixed with the various primosome assembly samples before preparing the grids. Aliquots (4 μL) of the primosome assembly samples were applied to glow-discharged holey carbon grids (Quantifoil R2/1 Copper, 300 mesh) in the climate-controlled chamber of an FEI Vitrobot Mark IV. The EM grids were blotted for 3 s with filter paper and then plunged into liquid ethane and stored in liquid nitrogen. Pilot datasets of around 300 micrographs were collected on a 200-kV FEI Arctica electron microscope equipped with a K2 summit camera (Gatan) for screening purposes. The 3D reconstruction and refinement led to preliminary 3D maps with resolutions around 6 Å to confirm the quality of the grids. Two individual datasets for each primosome assembly sample were then collected using SerialEM50 (link) on a TFS Titan Krios electron microscope operated at 300 kV and at a nominal magnification of 130,000× equipped with a K3 summit camera (Gatan) using the objective lens defocus range of –1.0 to –2.0 μm. All the EM images were recorded in the super-resolution counting and movie mode with a dose rate of 0.88 electrons per Å2 per frame; a total of 75 frames were recorded in each movie micrograph.
7
Cryo-EM Analysis of Primosome Assembly
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To alleviate any preferred orientation of the particles, 0.2% n-Octyl-β-D-glucoside was quickly mixed with the various primosome assembly samples before preparing the grids. Aliquots (4 μL) of the primosome assembly samples were applied to glow-discharged holey carbon grids (Quantifoil R2/1 Copper, 300 mesh) in the climate-controlled chamber of a FEI Vitrobot Mark IV. The EM grids were blotted for 3 s with filter paper and then plunged into liquid ethane and stored in liquid nitrogen. Pilot datasets of around 300 micrographs were collected on a 200-kV FEI Arctica electron microscope equipped with a K2 summit camera (Gatan) for screening purposes. The 3D reconstruction and refinement led to preliminary 3D maps with resolutions around 6 Å to confirm the quality of the grids. Two individual datasets for each primosome assembly sample were then collected using SerialEM50 (link) on a TFS Titan Krios electron microscope operated at 300 kV and at a nominal magnification of 130,000× equipped with a K3 summit camera (Gatan) using the objective lens defocus range of −1.0 to −2.0 μm. All the EM images were recorded in the super-resolution counting and movie mode with a dose rate of 0.88 electrons per Å2 per frame; a total of 75 frames were recorded in each movie micrograph.
8
Cryo-EM Structure Determination of GluK2
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Vitrified samples of GluK2 solublized in DDM-CHS were prepared with protein concentrated to 4.2 mg/mL. A volume of 3 μL was added to R1.2/1.3 holey carbon grids (Quantifoil) rendered hydrophilic with self-assembled monolayer functionalization27 (link), and grids were frozen with a Vitrobot Mk IV robot (FEI Company, Hillsboro, OR, USA) or a Leica EM GP (Leica Microsystems Inc., Buffalo Grove, IL, USA).
Data was collected using a Titan Krios, operated at 300 kV, aligned for parallel illumination, and equipped with a GIF Quantum Energy Filter (Gatan, Inc.) operated in zero-energy-loss mode with a slit width of 20 eV (Extended Data Table 2 ). Images were acquired manually on a K2 Summit camera (Gatan, Inc.), at 105,000 X nominal magnification corresponding to a 1.324 Å physical pixel size. Each exposure was recorded in super-resolution mode as a 38-frame movie, with dose rate and exposure time of 3 e Å−1 s−1 and 15 s, respectively.
Data was collected using a Titan Krios, operated at 300 kV, aligned for parallel illumination, and equipped with a GIF Quantum Energy Filter (Gatan, Inc.) operated in zero-energy-loss mode with a slit width of 20 eV (
9
Cryo-EM Data Acquisition Protocol
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Cryo‐micrographs were acquired on a 300 kV Polara microscope (FEI) with a K2 Summit camera (Gatan) operated in counting mode after a Quantum energy filter (Gatan) with a 20 eV slit. The magnified pixel size was 1.39 Å. The dose rate was 2.6–2.8 e‐/Å2/s during 9‐s exposures, resulting in the total dose of 23–25 e‐/Å2 on the specimen. These exposures were collected manually and fractionated into 36 movie frames (0.25 s/frame) with SerialEM (http://bio3d.colorado.edu/SerialEM/ ), at defocus ranging from −0.4 to −2.5 μm defocus.
10
Cryo-EM Imaging of Biological Samples
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Cryo-EM imaging was performed on an FEI Titan Krios microscope (FEI Company) operated at 300 kV, aligned for parallel illumination. Data collection images (Figure 2A ) were acquired with a K2 Summit camera operated in super-resolution counting mode with a calibrated physical pixel size of 0.858 Å and a super-resolution pixel size of 0.429 Å with a defocus range between −1.0 and −2.5 µm using the Latitude S software version 3.21.1253 (Gatan Inc., Pleasanton, CA). Focusing improved by using the edge of the previously imaged hole on the gold foil grid. No energy filter or Cs corrector was installed on the microscope. The dose rate used was ∼4.5 e-/pixel·s to ensure operation in the linear range of the detector. The total exposure time was 15.2 s, and intermediate frames were recorded every 0.4 s giving an accumulated dose of ∼40 e-/Å2 and a total of 38 frames per image. A total of 3,085 images were collected over six days. 2,928 and 1,852 images were collected using the same setup on Cu carbon grids and Cu carbon grids with a 2 nm carbon layer, respectively.
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