About 3 μl aliquots of the emptied mature T4 capsids prepared as above were frozen onto Lacey carbon EM grids using a Gatan CP3 freezer with a blotting time of 6 s. The EM grids were then loaded on an FEI Titan Krios EM operated at 300 kV and equipped with a Gatan K2 Summit detector. The data collection was performed using the Leginon program46 (link) with a magnification of 14,000 (physical pixel size: 2.08 Å) in the “super-resolution” mode, which resulted in a pixel size of 1.04 Å per pixel. The dose rate was ~10 e−/(pixel·s). A total of 4377 movies, each composed of 40 frames, were collected. Each frame has an exposure time of 250 ms.
Titan krios em
Manufactured by Ametek
The Titan Krios EM is a transmission electron microscope designed for high-resolution structural biology applications. It features a stable and high-performance electron optical system, enabling the acquisition of high-quality images for advanced research and analysis.
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5 protocols using titan krios em
1
Cryo-EM Imaging of T4 Capsids
2
Cryo-EM Structural Analysis of PSM Assemblies
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An aliquot (3 μL) of sample containing either PSMα3 or PSMβ2 assemblies was applied to a plasma-cleaned (Gatan Solarus) lacey carbon grid (Sigma-Aldrich), blotted with force of 3 to 6 for 4.0 s at 100% humidity, and flash frozen in liquid ethane using a Vitribot Mark IV (FEI). The datasets used for structure determination were collected at the National Cryo-Electron Microscopy Facility at the Frederick National Laboratory for Cancer Research on a Titan Krios EM operated at 300 keV, equipped with an energy filter and K3 direct electron detector (Gatan). An energy filter slit width of 20 eV was used during data collection and was aligned automatically every hour. A total of 18,751 (PSMα3) and 8,700 (PSMβ2) movies were collected in counting mode using EPU v2.4 (Thermo Fisher Scientific) at a magnification of 81K, a pixel size of 1.08 Å, and a defocus range from −2.2 to −1.2 μm. Data collection was performed using a total dose of 50 e− Å−2 across 40 frames with an exposure time of 2.98 s.
3
Cryo-EM Data Collection of Lamellae P. purpureum
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The lamellas of P. purpureum data were collected on a FEI Titan Krios EM operated at 300 kV equipped with a Gatan K3 Summit detector and an energy filter (20 eV slit width). For each lamella, the grid was tilted to either −15° or +15° before imaging, which making the lamella plane perpendicular to the incident beam. Data collection was performed with SerialEM41 (link) using a nominal magnification of 53,000 with a total dose of ~35 e− at super-resolution mode, resulting in a pixel size of 0.834 Å/pixel. A total of 1437 such movies were collected. The defocus value was set from 2.0 to 4.0 μm. Each micrograph was dose-fractionated to 80 frames. Motion correction was done by MotionCorr242 and micrographs were dose-weighted and binned with a factor of 2.
4
Bunyavirus Cryo-EM Imaging Protocol
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Bunyavirus data were collected on an FEI Titan Krios EM operated at 300 kV equipped with a Gatan K2 Summit detector. Data collection was performed with Serial EM using a nominal magnification of 22,500 with a total dose of ~50 e -resulting in a pixel size of 1.3 Å/pixel. Each micrograph was recorded as a movie composed of 36 frames. A total of 1,849 such movies were collected and 10,950 viral particles were extracted from motion-corrected micrographs.
5
Bunyavirus Cryo-EM Imaging Protocol
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Bunyavirus data were collected on an FEI Titan Krios EM operated at 300 kV equipped with a Gatan K2 Summit detector. Data collection was performed with Serial EM using a nominal magnification of 22,500 with a total dose of ~50 e -resulting in a pixel size of 1.3 Å/pixel. Each micrograph was recorded as a movie composed of 36 frames. A total of 1,849 such movies were collected and 10,950 viral particles were extracted from motion-corrected micrographs.
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