To break the preferential particle orientation, 0.0125% CHAPSO (final concentration) was added into the sample before grid preparation. 2.7 µl of freshly purified sample was applied onto glow-discharged (30 s, 5 mA) grids (Quantifoil R 0.6/1 300 mesh Cu or Ultrafoil 0.6/1 300 mesh Au) and plunge-frozen into liquid ethane using a Vitrobot Mark IV (FEI, Thermo Fisher Scientific) with the following parameters: 4 °C, 100% humidity, 7 s wait time, 4–4.5 s blot time, and a blot force of 25. Movies were collected using the semi-automated acquisition program EPU (FEI, Thermo Fisher Scientific) on a Titan Krios G2 microscope operated at 300 keV paired with a Falcon 3EC direct electron detector (FEI, Thermo Fisher Scientific). Images were recorded in an electron counting mode, at 96,000x magnification with a calibrated pixel size of 0.832 Å and defocus range of 0.8–3 µM. For the VaPomAB sample purified in LMNG, 6467 micrographs were collected, with each micrograph containing 40 frames and a total exposure dose of 37.98 (e/Å2). For the VaPomAB sample reconstituted into saposin nanodiscs, 3927 micrographs were collected, with each micrograph containing 40 frames and a total exposure dose of 37 (e/Å2 (link)). For the VaPomAB MSP1D1 sample, 5450 micrographs were collected, with each micrograph containing 40 frames and a total exposure dose of 40 (e/Å2 (link)).
Titan krios g2 microscope
Manufactured by Thermo Fisher Scientific
The Titan Krios G2 microscope is a high-performance cryo-electron microscope designed for advanced structural biology research. It features a stable and powerful electron beam, a versatile sample stage, and state-of-the-art optics to enable high-resolution imaging of biological specimens in their native state.
Automatically generated - may contain errors
Lab products found in correlation
Vitrobot mark 4, by Thermo Fisher Scientific (4 mentions)
Falcon 3ec direct electron detector, by Thermo Fisher Scientific (1 mentions)
K2 summit direct electron detector, by Ametek (1 mentions)
Tecnai arctica microscope, by Thermo Fisher Scientific (1 mentions)
Bioquantum energy filter, by Ametek (1 mentions)
Gloqube, by Quorum Technologies (1 mentions)
K3 summit direct electron camera, by Ametek (1 mentions)
Vitrobot mark 4 system, by Thermo Fisher Scientific (1 mentions)
K2 summit, by Ametek (1 mentions)
6 protocols using titan krios g2 microscope
1
Cryo-EM Sample Preparation for VaPomAB
2
Cryo-EM Imaging of Purified Carboxysomes
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Purified carboxysomes were applied to a glow-discharged Quantifoil R1.2/1.3 300mesh copper holey carbon grid (Quantifoil, Micro Tools), blotted under 100% humidity at 4°C and plunged into liquid ethane using a Mark IV Vitrobot (FEI). Micrographs were acquired on a Titan Krios G2 microscope (FEI) operated at 300 kV with a K2 Summit direct electron detector (Gatan). A calibrated magnification of 130k times was used for imaging, yielding a pixel size of 1.04 Å on images. The defocus was set at 1.5 to 2.0 μm. Each micrograph was dose-fractionated to 32 frames under a dose rate of 8 e -/pixel/s with a total exposure time of 10.4 s.
3
Time-resolved Cryo-EM of Molecular Complexes
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The cryo-grids were initially screened in a 200 kV Tecnai Arctica microscope (Thermo Fisher). Good-quality grids were then transferred to a 300 kV Titan Krios G2 microscope (Thermo Fisher) equipped with the post-column BioQuantum energy filter (Gatan) connected to a K2 Summit direct electron detector (Gatan). Coma-free alignment and parallel illumination were manually optimized prior to each data collection session. Cryo-EM data were acquired automatically using SerialEM software45 (link) in a super-resolution counting mode with 20 eV energy slit, with the nominal defocus set in the range of −0.8 to −2.0 μm. A total exposure time of 10 s with 250 ms per frame resulted in a 40-frame movie per exposure with an accumulated dose of ~50 electrons per Å2. The calibrated physical pixel size and the super-resolution pixel size were 1.37 Å and 0.685 Å, respectively. For time-resolved sample conditions, 1,781, 2,298, 15,841, 2,073 and 2,071 movies were collected for cryo-grids made with the reaction time of 0, 0.5, 1, 5, and 10 min, respectively. For the condition of exchanging ATP to ATPγS at 1 min after substrate addition, 21,129 movies were collected.
4
Cryo-EM structure determination of Nf1-23a
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Quantifoil R2/1 holey carbon grids (Au 300 mesh, Electron Microscopy Sciences) were glow-discharged for 60 s at 20 mA using a GloQube (Quorum) instrument. Purified Nf1-23a was thawed, centrifuged (14,000g for 5 min at 4 ºC) and diluted to ~0.5 mg ml−1 with gel filtration buffer. Protein was loaded into the freshly glow-discharged grids and plunge-frozen in LN2-cooled liquid ethane using a Vitrobot Mark IV (Thermo Fisher Scientific) with a blot force of −2 for 2.5 s. Temperature and relative humidity were maintained at 4 ºC and 100%, respectively. Grids were clipped and loaded into a 300-kV Titan Krios G2 microscope (Thermo Fisher Scientific, EPU 2.8.1 software) equipped with a Gatan BioQuantum energy filter and a K3 Summit direct electron detector (AMETEK). Grids were screened for quality control based on particle distribution and density, and images from the best grid were recorded. Micrographs were recorded at a nominal magnification of ×105,000, corresponding to a calibrated pixel size of 0.86 Å. The dose rate was 12 electron physical pixels per second, and images were recorded for 3.3 s divided into 40 frames, corresponding to a total dose of 40 electrons per Å2. Defocus range was set between −0.5 μm and −4 μm. Gain-corrected image data were acquired.
5
Cryo-EM Sample Preparation and Data Collection
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The purified samples were applied to Quantifoil R 1.2/1.3300 Mesh Gold grids. The grids were glow discharged for 90 s at 30 mA with a PELCO easiGlow™ Glow Discharge Cleaning System to obtain a hydrophilic surface. The glow‐discharged grids were used to prepare vitrified samples with the Thermo Scientific Vitrobot Mark IV System. We applied 3 μL of purified protein to the glow‐discharged surface of the grid at 4°C at 100% of humidity and blotted the solution for 5.0–5.5 s, with blot force of either 18 or 19.
The data were acquired with a 300 kV Titan Krios G2 microscope (Thermo Fisher) equipped with a K3 Summit direct electron camera (Gatan) run in super‐resolution mode at a nominal magnification of 105,000×, with a physical pixel of 0.834 Å. A phase plate was not used and the objective aperture was not inserted. SerialEM was used for automated data collection in beam‐image shift mode with nine images collected per stage movement, with a defocus range from −1.0 to −3.0 μm and beam‐image shift compensation (Mastronarde, 2005 (link)). The slit width of the GIF Quantum Energy Filter was set to 25 eV. Movies were dose‐fractionated into 100 frames with a total dose of ~80 e−/Å2. Two batches of data were collected. The first batch consisted of 2398 movies, and the second batch consisted of 4509 movies.
The data were acquired with a 300 kV Titan Krios G2 microscope (Thermo Fisher) equipped with a K3 Summit direct electron camera (Gatan) run in super‐resolution mode at a nominal magnification of 105,000×, with a physical pixel of 0.834 Å. A phase plate was not used and the objective aperture was not inserted. SerialEM was used for automated data collection in beam‐image shift mode with nine images collected per stage movement, with a defocus range from −1.0 to −3.0 μm and beam‐image shift compensation (Mastronarde, 2005 (link)). The slit width of the GIF Quantum Energy Filter was set to 25 eV. Movies were dose‐fractionated into 100 frames with a total dose of ~80 e−/Å2. Two batches of data were collected. The first batch consisted of 2398 movies, and the second batch consisted of 4509 movies.
6
Cryo-EM Structural Analysis of Purified Virus
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Three µL of purified virus particles were loaded on a glow-charged lacey carbon copper grid and flash frozen in liquid ethane after 2 s blotting in 100% humidity at 4 o C using a Vitrobot Mark IV (Thermo Fisher Scientific). The particles images were collected using a 300 kV Titan Krios G2 microscope (Thermo Fisher Scientific) equipped with a K2 Summit direct electron detector using counting mode and a GIF energy filter of 20 eV slit width (Gatan Inc). Two datasets were collected, dataset 1 with 20 frames per micrograph movie, and dataset 2 with 40 frames per micrograph movie. These datasets were taken at a nominal magnification of 64,000× (pixel size on specimen is 2.21 Å/pixel), at 0.8 -3.0 µm under focus, and at 26.4 e -/Å 2 total dose.
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