Cryo‐micrographs were acquired on a 300 kV Polara microscope (FEI) with a K2 Summit camera (Gatan) operated in counting mode after a Quantum energy filter (Gatan) with a 20 eV slit. The magnified pixel size was 1.39 Å. The dose rate was 2.6–2.8 e‐/Å2/s during 9‐s exposures, resulting in the total dose of 23–25 e‐/Å2 on the specimen. These exposures were collected manually and fractionated into 36 movie frames (0.25 s/frame) with SerialEM (http://bio3d.colorado.edu/SerialEM/ ), at defocus ranging from −0.4 to −2.5 μm defocus.
Polara microscope
Manufactured by Thermo Fisher Scientific
The Polara microscope is a high-performance electron microscope designed for advanced imaging and analysis. It features a powerful electron beam, high-resolution optics, and sophisticated control systems to enable detailed examination of samples at the nanoscale level.
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Lab products found in correlation
K2 summit camera, by Ametek (3 mentions)
Quantum energy filter, by Ametek (1 mentions)
Tecnai f20, by Thermo Fisher Scientific (1 mentions)
Cryoplunge 3 instrument, by Ametek (1 mentions)
K2 summit electron detector, by Ametek (1 mentions)
Vitrobot mark 3, by Thermo Fisher Scientific (1 mentions)
Gif quantum energy filter, by Ametek (1 mentions)
K2 summit, by Ametek (1 mentions)
Vitrobot, by Thermo Fisher Scientific (1 mentions)
7 protocols using polara microscope
1
Cryo-EM Data Acquisition Protocol
2
Cryo-EM sample preparation and data collection
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Ctf19c samples were applied to glow-discharged C-flat grids (CF-1.2/1.3–3C; Electron Microscopy Sciences). In all cases, 3.5 μL of protein solution were applied, and grids were blotted from both sides for 4 s before vitrification in liquid ethane using a Cryoplunge 3 instrument (Gatan) operating at 80–90% humidity. For screening of sample preparations and generation of initial maps, we used a Tecnai F20 (FEI) microscope operating at 200 kV. Images, collected using the UCSF Image4 software package (Li et al., 2015 (link)), were recorded on a K2 Summit electron detector (Gatan) operating in super-resolution movie mode (50 frames, 0.2 s/frame,~60 electrons per Å2 total dose, 0.64 Å/super-resolution pixel).
For collection of high-resolution data, we used an FEI Polara microscope (FEI) operating at 300 kV. Images, collected using the SerialEM software package (Mastronarde, 2005 (link)), were recorded on a K2 Summit electron detector operating in super-resolution movie mode (40 frames, 0.2 s/frame, 52 electrons per Å2 total dose, 0.615 Å/super-resolution pixel). In total, we collected 15,439 movies over three sessions.
For collection of high-resolution data, we used an FEI Polara microscope (FEI) operating at 300 kV. Images, collected using the SerialEM software package (Mastronarde, 2005 (link)), were recorded on a K2 Summit electron detector operating in super-resolution movie mode (40 frames, 0.2 s/frame, 52 electrons per Å2 total dose, 0.615 Å/super-resolution pixel). In total, we collected 15,439 movies over three sessions.
3
High-Resolution Cryo-Electron Microscopy
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Micrographs were acquired on a 300 kV Polara microscope (FEI) combined with a K2 Summit camera (Gatan) operated in counting mode after energy filter with a 20 eV slit. The magnification at the specimen plane was 35,971x resulting in a pixel size of 1.39 Å. The dose rate was ~5 e-/pixel/sec, corresponding to ~2.6 e-/Å2/sec. The total dose on the specimen was ~23.4 e- collected over 9 sec exposures fractionated into 36 movie frames (0.25 sec/frame). We used SerialEM software (http://bio3d.colorado.edu/SerialEM/ ) to manually collect exposures with -0.4 to -2.5 μm defocus range.
4
Cryo-EM Imaging of Bacterial Cells
5
High-Resolution Cryo-Electron Microscopy
6
Cryo-EM Data Collection Protocol
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Data collection was carried out using a Tecnai “Polara” microscope (FEI) equipped with a Gatan GIF Quantum energy filter with an energy selecting slit width of 30 eV, operated at 300 kV. Data images were recorded on a Gatan K2 Summit direct electron detector operating in counting mode, as movies each containing 32 frames with a total electron dose of 35 e− Å−2, using serial EM. The defocus range was 0.5–2.5 μm. The calibrated magnification was ×37,037, corresponding to a pixel size of 1.35 Å. The dose rate was ~4 e− Å−2 s−1.
7
Cryo-EM Imaging of PB1, PB1-ZZ, and p62
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A total of 3.5 ml of PB1, PB1-ZZ, and p62 sample were applied to glow-discharged C-flat 1.2/1.3 and 200 mesh Quantifoil multi-A grids and plungefrozen in a reservoir of liquid ethane using a custom-made cryo-plunger (PB1 [1-102], PB1 [1-122], PB1-ZZ) or an FEI Vitrobot (p62). Frozen grids were transferred to the FEI Titan Krios loading cassette for EM observation. Vitrified samples of PB1 type T/F, p62, and PB1-ZZ were imaged using a FEI Titan Krios at 120, 200, and 300 kV at a nominal magnification of 60,000 and 28,000. Micrographs were taken at an underfocus between 1.6 and 3.5 mm onto a Gatan US4000 4k 3 4k CCD camera (PB1 type T/F, p62) or on a direct electron detector FEI Falcon II (PB1 [1-102], PB1-ZZ). We estimated the dose deposited on PB1 (1-102), PB1 (1-122), p62, and PB1-ZZ between 10 and 20 e À /A ˚2, respectively. Tomograms of negatively stained samples were acquired on a FEI Polara microscope using Serial EM software (Mastronarde, 2005) and by tilting the stage ±60 with a sampling step of 2 followed by smaller increments for higher angles using a Saxton acquisition scheme at a pixel size of 0.6 nm.
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