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.
Epu v2
Manufactured by Thermo Fisher Scientific
EPU v2.4 is a data acquisition and processing software used for cryo-electron microscopy (cryo-EM) applications. It is designed to control the electron microscope, automate data collection, and perform preliminary image processing. The software provides an intuitive user interface and streamlines the cryo-EM workflow, enabling researchers to efficiently gather and analyze high-quality electron microscopy data.
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Lab products found in correlation
2 protocols using epu v2
1
Cryo-EM Structural Analysis of PSM Assemblies
2
Cryo-EM Structural Analysis of Archaeal Pili
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A 3μL aliquot of sample containing either Pyrobaculum calidifontis, Aeropyrum pernix, or Agrobacterium tumefaciens pili was applied to a plasma cleaned (Gatan Solarus) lacey carbon grid (Ted Pella, Inc.), blotted with automated blotting for 3 s at 90% humidity and flash frozen in liquid ethane using an EM GP Plunge Freezer (Leica). The dataset used for structure determination was collected at the Molecular Electron Microscopy Core at the University of Virginia 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 10 eV was used during data collection and was aligned automatically every hour. All 8127 P. calidifontis, 598 A. pernix, and 8363 A. tumefaciens movies were collected in counting mode using EPU v2.4 (Thermo Fisher) at a magnification of 81 K, pixel size of 1.08 Å, and a defocus range from −2.2 to −1.2 μm. Data were collected using a total dose of 50 e− Å−2 across 40 frames with an exposure time of 2.98 s.
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