Vitrified grids were imaged using an FEI Titan Krios operating at 300 keV and equipped with a Gatan K2 summit direct detector device. Micrographs were collected at 130,000 × , corresponding to a pixel size of 0.55 Å pixel−1 in super resolution mode. Each image received a dose rate of ~8 e− pixel−1 s−1 with 200 ms exposure per frame, and an estimated defocus ranging from 1.0 to 3.5 µm. Data were collected in three separate sessions, resulting in a total of 3454 images using EPU (908 micrographs) (FEI) and Leginon (2546 micrographs)61 (link) automated data collection softwares.
K2 summit direct
Manufactured by Ametek
Sourced in Germany
The K2 Summit direct is a lab equipment product designed for precise temperature control and monitoring. It provides accurate and reliable temperature measurements for a variety of applications in the scientific and research fields.
Automatically generated - may contain errors
Lab products found in correlation
Titan krios, by Thermo Fisher Scientific (3 mentions)
Vitrobot mark 4, by Thermo Fisher Scientific (2 mentions)
Vitrobot, by Thermo Fisher Scientific (2 mentions)
Titan krios electron microscope, by Thermo Fisher Scientific (2 mentions)
Gif quantum filter, by Ametek (2 mentions)
R1.2 1 3 400 mesh au holey carbon grids, by Quantifoil (1 mentions)
Titan krios microscope, by Thermo Fisher Scientific (1 mentions)
1.2 1 3 cu 300 mesh grids, by Quantifoil (1 mentions)
Titan krios 300 kv microscope, by Ametek (1 mentions)
Fei titan krios microscope, by Thermo Fisher Scientific (1 mentions)
9 protocols using k2 summit direct
1
Cryo-EM imaging of vitrified samples
2
Tomographic Tilt-Series of B. bacteriovorus
3
Cryo-EM of hSMO–Gi–Fab Complex
4
TORC1 Filaments Cryo-EM Imaging
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Cryo-EM images of TORC1 filaments were collected on a Titan Krios microscope (Thermo Scientific) at EMBL Heidelberg, Germany, operated at 300 kV and equipped with a K2 summit direct electron detector (Gatan) camera operated in counting mode. A total of 4,901 movies of 16 frames were collected, with a dose rate of 2.5 e−/Å2/s and a total exposure time of 8 s, corresponding to a total dose of 20 e−/Å2. Movies were collected at a magnification of 37,000×, corresponding to a calibrated pixel size of 1.35 Å per pixel at the specimen level.
5
Cryo-EM Sample Preparation Protocol
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For freezing, Quantifoil 1.2/1.3 Cu 300 mesh grids (Quantifoil Micro Tools) were glow-discharged in methanol vapor before sample application. 3 μl sample was applied to each grid, which was then blotted for 1.5 s and plunge-frozen into liquid ethane using a FEI Vitrobot Mark IV. Micrographs were collected on an FEI Titan Krios 300 kV microscope with a post energy filter Gatan K2-Summit direct detector camera. Movies were collected at nominal 165,000× magnification, equivalent to a pixel spacing of 0.82 Å. A total dose of 40.8 e−/Å2 was used to collect 40 frames over 6 s, using a nominal defocus range covering −2.0 to −3.8 μm. This defocus range was selected to optimize signal-noise ratio/contrast, which was relatively low because of preferential distribution of particles in regions of very thick ice.
6
Cryo-EM Sample Preparation for RNC Imaging
7
Automated Cryo-EM Data Collection
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A total of 6,878 raw micrographs were acquired across two datasets on the same 300 keV FEI Titan Krios microscope (LMB Krios3) at a calibrated pixel size of 1.049 Å/pixel (nominal magnification of 130,000 X). The K2 Summit direct electron detector (Gatan) was used in electron counting mode with a GIF Quantum energy filter slit width of 20 eV. EPU (ThermoFisher) was used for automated data collection, with a defocus range set at −1.4 to −2.6 μm and dose-fractionation into 20 fractions per movie, with a total exposure time of 7-8 s to achieve a dose of 37-38 e-/Å2 per micrograph.
8
Cryo-EM Data Collection Protocol
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Cryo-EM data for all samples were collected on a Titan Krios EM equipped with a Falcon III direct electron detector (FEI Thermo Fisher) or a K2 Summit direct electron detector (Gatan Inc.) at the Francis Crick Institute. See Supplementary Table 2 for image acquisition details.
9
Cryo-EM Sample Preparation for RNC Imaging
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Approximately 3 μL of freshly made RNC at a concentration of 40 to 60 nM were incubated for 1 min on plasma-cleaned 300-mesh holey carbon grids (C-flat R2/2), on which a home-made continuous carbon film was pre-deposited. Grids were blotted for 4 seconds with 100% humidity at 4 °C and plunge-frozen in liquid ethane using an FEI Vitrobot. Automated data collection was performed on a Titan Krios electron microscope (FEI) equipped with a K2 Summit direct detector and GIF Quantum filter (Gatan) at 300 kV (Tables 1 -4 ). The total exposure time was 9 seconds, with a total dose of 50 electrons per Å2 (frame dose 1.3 electrons per Å2) 39 (link).
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