The sample images included in the manuscript were collected on a JEOL 2200 microscope with a Gatan K2 camera. These images include a range of stained polymer nanoparticles, amyloid fibres and gold nanoparticles.
K2 camera
Manufactured by Ametek
Sourced in United States
The K2 camera is a high-performance scientific imaging device designed for a variety of laboratory applications. It features a large sensor, high resolution, and advanced image processing capabilities to capture detailed and accurate data.
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Lab products found in correlation
Titan krios microscope, by Thermo Fisher Scientific (7 mentions)
Titan krios, by Thermo Fisher Scientific (5 mentions)
Vitrobot mark 4, by Thermo Fisher Scientific (4 mentions)
Vitrobot, by Thermo Fisher Scientific (3 mentions)
Lacey grids, by Ted Pella (3 mentions)
Talos arctica microscope, by Thermo Fisher Scientific (3 mentions)
Talos arctica, by Thermo Fisher Scientific (3 mentions)
Tecnai f20 microscope, by Ametek (2 mentions)
Titan krios microscope, by Ametek (2 mentions)
Jem 2200f, by JEOL (2 mentions)
Epu software, by Thermo Fisher Scientific (2 mentions)
Titan krios cryo electron microscope, by Ametek (2 mentions)
4k x 4k ccd, by Ametek (2 mentions)
Krios microscope, by Ametek (2 mentions)
Vitrobot 4, by Thermo Fisher Scientific (2 mentions)
Talos f200c 200 kv, by Thermo Fisher Scientific (1 mentions)
Talos l120c 120 kv, by Thermo Fisher Scientific (1 mentions)
Titan krios electron microscope, by Thermo Fisher Scientific (1 mentions)
Ceta camera, by Thermo Fisher Scientific (1 mentions)
Titan krios cryo electron microscope, by Thermo Fisher Scientific (1 mentions)
36 protocols using k2 camera
1
Imaging Polymer Nanoparticles, Amyloid Fibers, and Gold Nanoparticles
2
Structural Determination of NDH Complexes
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Three microliter of NDH-1L (for solving the NDH-PQ structure) or NDH-Fd (for solving the NDH-Fd structure) samples at concentration of 12 mg mL−1 were applied to a glow-discharged holey carbon copper or gold grids (GIG, R1.2/1.3, 300 mesh). The grids were blotted for 2 s with blot force of level 2 at 95% humidity and 4 °C, and were plunge frozen into liquid ethane cooled by liquid nitrogen immediately using a semiautomatic plunge device (Thermo Fisher Scientific Vitrobot IV). Sample screen was performed using Talos F200C 200 kV or Talos L120C 120 kV electron microscopes equipped with a Ceta camera (Thermo Fisher Scientific). For structure determination, the micrographs of both NDH-PQ and NDH-Fd were collected using SerialEM software suite55 (link) on a 300 kV Titan Krios electron microscope (Thermo Fisher Scientific) equipped with a K2 camera (Gatan) and a GIF quantum energy filter (20 eV). Total of 1463 images for NDH-PQ and 2090 micrographs for NDH-Fd were acquired using a defocus range between 0.5 and 2.5 μm at 130,000 magnification. The micrographs were exposed for 10 s and dose fractionated into 32 frames, leading to a total dose of 60 e− Å−2.
3
Cryo-EM Structural Determination of Fab-PvCS Complexes
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2F2 Fab-PvCSPvk210 and 2E10.E9 Fab-PvCSPvk247 complexes were purified via Superose 6 Increase 10/300 GL chromatography (GE Healthcare) and concentrated to 0.5 mg/mL. 3 µL of the sample was deposited on homemade holey gold grids (Marr et al., 2014 (link)), which were glow-discharged in air for 15 s before use. Samples were blotted for 12.0 s and subsequently plunge-frozen in a mixture of liquid ethane and propane (Tivol et al., 2008 (link)) using a modified FEI Vitrobot (maintained at 4°C and 100% humidity). Data collection was performed with a FEI Tecnai F20 microscope operated at 200 kV with a K2 camera (Gatan Inc). A calibrated 34,483× magnification, resulting in a pixel size of 1.45 Å, and defocus range between 1.5 and 2.8 µm were used for data collection. Exposures were fractionated as movies of 30 frames with a total exposure of 35 electrons/Å2. A total of 269 movies were obtained for the 2E10.E9 Fab-PvCSPvk247 complex and 169 movies for the 2F2 Fab-PvCSPvk210 complex. Image processing was carried out in cryoSPARC v2 (Punjani et al., 2017 (link)). Initial specimen motion correction, exposure weighting, and CTF parameters estimation were done using patch-based algorithms. 100,133 and 87,287 particle images were extracted from micrographs of 2E10.E9 Fab-PvCSPvk247 and 2F2 Fab-PvCSPvk210 complex, respectively, and subjected to 4–5 rounds of 2D classification.
4
Cryo-ET Imaging of Protein Particles
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Cryo-ET tilt series were acquired using SerialEM software53 (link) from +60° to −60° with a step of 3°, on an FEI Titan Krios microscope (300 kV) equipped with a Gatan K2 camera. At each tilt angle, we collected movies containing 8 frames, with a sum dose of ~3 e− Å−2 s−1, and the total dose for every tilt series (+60° to −60°) was 120 e− Å−2. The pixel size was 1.25 Å. The movies were first motion-corrected by MotionCor247 (link) and then imported into Etomo54 (link) for alignment and reconstruction. The position of protein particles inside the ice was manually identified and plotted13 (link).
5
Cryogenic Electron Microscopy of Purified PfGCC
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To prepare cryoEM grids, we applied 2 μl of purified PfGCC sample to thin continuous carbon films on lacey grids (Ted Pella Inc.) for 1 min, blotted for 12 s under 100% humidity and plunged the grid into liquid ethane with an FEI Vitrobot (ambient temperature in the Vitrobot chamber was 20 °C). CryoEM images were collected at liquid nitrogen temperature in an FEI Titan Krios cryo electron microscope operated at 300 kV using parallel illumination. Before data collection, the microscope was carefully aligned, and beam tilt was minimized by coma-free alignment. Images were recorded on a Gatan K2 camera with the counting mode at a nominal magnification of × 29,000 on microscope. The magnification on the camera was calibrated as × 49,500 using a catalase crystal sample, giving a pixel size of 1.01 Å per pixel on specimen. The dose rate of the electron beam was set to ∼8 counts per pixel per s on the camera which corresponds to ∼10 e− Å−2 s−1 on specimen when including the electrons uncounted by the K2 camera. Image stacks were recorded at 4 frames per sec for 10 s. After drift correction with the UCSF software34 (link), the first 12 frames of each image stack (movie) were merged to generate a final image with a total dose of ∼30 e− Å−2 of the sample.
6
Structural Analysis of Fab-complexed AaRseP
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All purified samples were diluted to 1.0 µg ml−1. For negative-stain EM, 5 µl protein solution was applied onto glow-discharged, 600 mesh, carbon-coated grids. The grids were negatively stained with 2% ammonium molybdate, blotted with filter paper and air-dried. A JEM2200FS (JEOL) was operated at 200 kV to acquire EM images using a K2 camera (Gatan) in counting mode. Images were recorded at a magnification of 20 000× (1.98 Å per pixel) with a defocus of −0.5 to −2.0 µm. A movie of 50 frames was taken for each image, and motion correction was performed using MotionCor2 (Zheng et al., 2017 ▸ ). The contrast transfer function was estimated by Gctf (Zhang, 2016 ▸ ). All subsequent processing was carried out using RELION3 (Zivanov et al., 2018 ▸ ). Particles were selected using the Relion Autopicker and three rounds of 2D classification were performed to select particles for 3D reconstruction. The number of particles used for each reconstruction of the Fab-complexed AaRseP (181-PA14-184) and (235-PA14-236) mutants are indicated in Fig. 7 and Supplementary Fig. S6 , respectively. Fitting of the atomic coordinates of the Fab–PDZ complex into the 3D reconstruction model and figure preparation were performed with UCSF Chimera (Pettersen et al., 2004 ▸ ).
7
Cryo-EM Structural Analysis of N Protein
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The purified N protein was prepared on UltraAuFoil R1.2/1.3 gold support grids (Quantifoil). 3 μl of sample was applied to glow-discharged grids, blotted for 2 s with -10 force, and vitrified by plunging into liquid ethane using the FEI Vitrobot Mark IV at 4°C and 100% relative humidity. Micrographs were collected at the Diamond eBIC facility on a Titan Krios microscope (FEI) operating at 300 keV and equipped with K2 camera and an energy filter slit width of 20 eV (Gatan). Automated data collection was performed using FEI EPU software. 1879 movies with a total electron dose of ~41 e-/Å2 were recorded in counting mode over 11 s (40 frames) with a pixel size of 1.048 Å. The defocus range chosen for automatic collection was 0.5 to 2.1 μm.
8
Cryo-EM Structural Analysis of SARS-CoV-2 Spike Proteins
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Data were collected using EPU software (Thermo Scientific) on Titan Krios microscopes operating at 300 kV either with a Falcon 3 camera (Thermo Scientific) operating in electron-counting mode (60 s exposures, total dose of 35 e−/Å2, fractionated into 30 frames, 1.09 Å2 calibrated pixel size) or K2 camera (Gatan) with GIF Quantum LS energy filter (Gatan) with a slit width of 20 eV operating in the zero-loss mode (9.4 s exposures, 49 e−/Å2 total dose, fractionated into 32 frames, 1.08 Å2 calibrated pixel size). The following eight datasets were collected (Supplementary Table 1 ): FUR2P Beta (Falcon 3), ACE2 + FUR2P Beta (K2), FUR2P Alpha (Falcon 3), ACE2 + FUR2P D614 Alpha (K2), ACE2 + 2P Alpha furin-uninhibited (Falcon 3), ACE2 + 2P Alpha furin-inhibited (Falcon 3), FUR2P mink (K2), ACE2 + FUR2P D614 mink (K2). All micrographs were collected with defocus range between 1.5 and 3 µm.
9
Cryo-ET of Neuronal Organelles
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Tomograms were acquired using a Titan Krios microscope (Thermo Fisher Scientific) operated at 300 kV and equipped with an energy filter operated with slit width 20 eV. SerialEM (Mastronarde, 2005 (link)) was used to acquire tilt series between ±60° in 2° increments. At each tilt angle, a movie containing 10 frames with an exposure of ∼2 e−/Å2/tilt was acquired on a K2 camera (Gatan). The total dose for each tilt series was 110–120 e−/Å2. Two DRG datasets and one Dm dataset were used in this study. DRG dataset 1 was acquired on GFP-RFP-LC3 neurons at DIV 4 on MRC-LMB Krios 3 at nominal pixel size 3.44 Å/pix using a zero-centered tilt scheme from −30° to +60° then −30° to −60° (30 out of 32 acquired tomograms were reconstructed and analyzed). DRG dataset 2 was acquired on wild-type neurons at DIV 7 on eBIC Krios 1 at nominal pixel size 2.75 Å/pixel using a dose-symmetric tilt scheme (Hagen et al., 2017 (link); 39 out of 43 acquired tomograms were reconstructed and analyzed). The Dm dataset was acquired on MRC-LMB Krios 3 at nominal pixel size 3.44 Å/pixel using a zero-centered tilt scheme from −30° to +60° then −30 to −60° (37 out of 42 acquired tomograms were reconstructed and included in the analysis). The defocus for each tomogram was set to between −3 and −6 µm underfocus.
10
Cryo-EM Data Collection Protocol
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Data were collected on a Tecnai Polara (FEI) operated at 300 kV using a nominal underfocus of 1–3 μm. Data were recorded using a K2 camera (Gatan Inc., Pleasanton, CA, USA), collecting 40 frames with a length of 0.2 seconds each at a dose rate of 8 e−/pixel/s. Detailed collection strategies are found in Table 1 .
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