The purified protein–nanodisc complex was applied onto C-Flat 1/1 grids (400 mesh) and plunge-frozen in a Vitrobot (ThermoFisher) with 7 s blotting. Data were automatically acquired at 300 kV with Leginon (Suloway et al., 2005 ▸ ) in a Polara electron microscope (FEI) equipped with an energy filter (slit width of 70 eV) and a K2 direct electron detector (Gatan) in counting mode. A total of 3467 micrographs were recorded, of which 2835 were selected manually for further processing. A typical micrograph is shown in Fig. 1 ▸ (a). Data-acquisition and processing parameters are summarized in Table 1 ▸ .
Polara electron microscope
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Polara electron microscope is a high-performance imaging tool designed for advanced materials research and analysis. It utilizes cutting-edge electron optics to provide high-resolution, high-contrast images of a wide range of samples. The Polara's core function is to enable detailed observation and characterization of materials at the nanometer scale.
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Lab products found in correlation
Vitrobot, by Thermo Fisher Scientific (4 mentions)
Ultracam, by Ametek (2 mentions)
R3.5 1 holey copper grid, by Quantifoil (2 mentions)
Gatan ultrascan usc 4000, by Ametek (2 mentions)
Holey carbon grid, by Quantifoil (2 mentions)
4k 4k ccd camera, by Ametek (1 mentions)
K2 summit direct electron detector, by Ametek (1 mentions)
Vitrobot mark 4, by Thermo Fisher Scientific (1 mentions)
10 protocols using polara electron microscope
1
Cryo-EM Imaging of Protein-Nanodisc Complex
2
Purification and Imaging of Yeast Spindle Pole Bodies
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Spindle pole bodies from Saccharomyces cerevisiae were purified following previous published procedures 34 ,35 . The purified SPB sample, initially in high concentration of sucrose, was first dialyzed at 4°C overnight in a buffer containing 10 mM Bis-Tris/Cl (pH=6.5), 0.1 mM MgCl2, 20% (v/v) DMSO. Next day, after mixing with 10 nm colloid gold, the sample was applied onto either a home-made holey carbon grid or a Quantifoil grid (PSI, Inc.) in a humidity chamber, then blotted and plunged into liquid ethane using a home-made plunger or a Vitrobot (FEI, Inc.). Frozen grids were stored in liquid nitrogen before use. Tomography data were collected on a Polara electron microscope (FEI, Inc.) running at 300kV. A post-column energy filter (GIF, Gatan, Inc.) was used and the slit width was set at 25 eV. Automatic data collection was carried out by UCSF Tomography software 36 . Single-axis tilt series were collected at a nominal magnification of 41,000. Images of dimension 2032x2032 were recorded on a CCD camera (UltraCam, Gatan, Inc.). The final pixel size on the images was 5.32 Å. The specimen was tilted from −60° to +60° in 1.5° step. The microscope defocus values were set in the range of 10 to 15 μm. The accumulated dose for each tilt series was ~60 e−/Å2.
3
Synaptosome Purification from Mouse Brain
4
Cryo-EM Analysis of 2D AQP0 Crystals
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2D crystals grown at molar sphingomyelin:cholesterol ratios of 1:2 and 2:1 were prepared by trehalose embedding as described above.
Electron diffraction patterns of AQP0 2D crystals were recorded with a Polara electron microscope (FEI Company, Hillsboro, OR) operated at an acceleration voltage of 300 kV and equipped with a 4k ⨉ 4k CCD camera (Gatan, Pleasanton, CA). The camera length was set to 1.9 m and a C2 aperture with a diameter of 30 μm was selected. The selected target areas on the grids were exposed for 30 seconds, corresponding to a total electron dose of approximately 10 electrons/Å2. Diffraction patterns were collected at tilt angles of 0°, 20°, 45°, 60°, 65°, and 70°, and details are provided in Table 3.
Electron diffraction patterns of AQP0 2D crystals were recorded with a Polara electron microscope (FEI Company, Hillsboro, OR) operated at an acceleration voltage of 300 kV and equipped with a 4k ⨉ 4k CCD camera (Gatan, Pleasanton, CA). The camera length was set to 1.9 m and a C2 aperture with a diameter of 30 μm was selected. The selected target areas on the grids were exposed for 30 seconds, corresponding to a total electron dose of approximately 10 electrons/Å2. Diffraction patterns were collected at tilt angles of 0°, 20°, 45°, 60°, 65°, and 70°, and details are provided in Table 3.
5
Cryo-ET of Mitochondria with Fiducial Markers
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Mitochondria were washed twice with 320 mM trehalose, 20 mM Tris pH 7.3, and 1 mM EGTA. Samples were mixed 1:1 with fiducial gold markers (10 nm gold particles conjugated to protein A, Aurion, The Netherlands) and immediately plunge-frozen in liquid ethane on Quantifoil holey carbon grids (Quantifoil Micro Tools, Germany). Single tilt series (typically ±60°, step size 1–1.5°) were collected at 300 kV with an FEI Polara electron microscope equipped with a post-column Quantum energy filter and an Ultrascan 4 × 4 k CCD camera (Gatan, USA), or with a post-column Tridiem energy filter and a 2 × 2 k CCD camera (Gatan). Alternatively, tilt series were recorded with an FEI Titan Krios electron microscope equipped with a Quantum energy filter and a K2 summit direct electron detector (Gatan). Underfocus was 8–9 µm and the magnification was chosen to give an object pixel size between 4.3 Å and 7.3 Å. The total electron dose per tilt series was 120–150 e-/Ų. Tilt series were aligned to gold fiducial markers and tomograms were reconstructed by back-projection with the IMOD software package (Kremer et al., 1996 (link)). A final filtering step applying non-linear anisotropic diffusion (Frangakis and Hegerl, 2001 (link)) was performed to increase contrast. Tomograms were manually segmented with the program AMIRA (FEI).
6
Cryo-EM Sample Preparation for Structural Biology
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The sample quality and filament concentration were checked by negative-stain EM prior to cryo-EM grid preparation. Grids for cryo-EM were coated with a thin layer of carbon as follows. A piece of homemade carbon-mica foil was floated for 5 min on a 30 µl sample on an ice-cooled home-made polyoxymethlene block. A Quantifoil R2.2 grid was glow-discharged for one minute at 25 mA (PELCO easy glow), applied on top of the floating carbon foil and quickly transferred to a Vitrobot Mark IV (FEI) for vitrification. Vitrobot filter papers (TED PELLA, INC.) were humidified prior to use and the Vitrobot was set to 4 °C and 100% humidity. A drop of 3 µl water was added to the specimen prior to blotting at a force of -5 for a total time of 45 s. The grid was finally flash-frozen in liquid ethane before storage.
Cryo-EM movies where automatically collected on an FEI Polara electron microscope operated at 300 KV. Micrographs were recorded on a K2 direct electron detector operated in counting mode using the software Latitude S, at a nominal magnification of 41270 × (corresponding to 1.21 Å/pixel at the specimen level), with a defocus range of -1.5 to -3μm. 720 movies of 40 frames were collected with a total dose of 50 electrons Å−2 (Supplementary Table3 ).
Cryo-EM movies where automatically collected on an FEI Polara electron microscope operated at 300 KV. Micrographs were recorded on a K2 direct electron detector operated in counting mode using the software Latitude S, at a nominal magnification of 41270 × (corresponding to 1.21 Å/pixel at the specimen level), with a defocus range of -1.5 to -3μm. 720 movies of 40 frames were collected with a total dose of 50 electrons Å−2 (Supplementary Table
7
Purification and Imaging of Yeast Spindle Pole Bodies
8
Cryo-EM of Purified Yeast F1Fo ATPase
9
Cryo-EM of Streptococcus pneumoniae
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For cryo-EM, 4 mL of S. pneumoniae R800 cells in exponential growth were loaded onto a Quantifoil R3.5/1 holey copper grid (Quantifoil Micro Tools GmbH, Germany). Vitrification of the sample was performed manually (blotting the sample for 1-2 s and plunging rapidly the grid into liquid ethane cooled with liquid nitrogen). The frozen grid was transferred into a 300 kV POLARA electron microscope (FEI). The images were taken under high dose conditions at 300 kV (80 e -3A ˚À2 ), with a magnification of 12,000 and a defocus between 4 and 5 mm using a CCD camera (GATAN ultrascan USC 4000).
10
Cryo-EM of Streptococcus pneumoniae
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For cryo-EM, 4 mL of S. pneumoniae R800 cells in exponential growth were loaded onto a Quantifoil R3.5/1 holey copper grid (Quantifoil Micro Tools GmbH, Germany). Vitrification of the sample was performed manually (blotting the sample for 1-2 s and plunging rapidly the grid into liquid ethane cooled with liquid nitrogen). The frozen grid was transferred into a 300 kV POLARA electron microscope (FEI). The images were taken under high dose conditions at 300 kV (80 e -3A ˚À2 ), with a magnification of 12,000 and a defocus between 4 and 5 mm using a CCD camera (GATAN ultrascan USC 4000).
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