The grids were plasma-cleaned and protein samples were prepared as described above. Blotting was performed from the side using Whatman No. 1 filter paper, as normally performed for negative staining. Plunging was performed by hand, simply submerging the grids completely in liquid nitrogen. These frozen-hydrated samples were imaged at liquid-nitrogen temperature on a Tietz 4k × 4k CCD camera with a defocus of −2.5 µm using the Leginon automated electron-microscopy package with an FEI F20 electron microscope operating at 200 keV at a nominal magnification of 62 000× (a pixel size of 1.774 Å).
F20 electron microscope
Manufactured by Thermo Fisher Scientific
Sourced in United States
The F20 electron microscope is a high-performance imaging tool designed for materials characterization. It utilizes an electron beam to produce high-resolution images of samples at the nanoscale level. The F20 provides detailed information about the structure and composition of materials, enabling researchers to gain a deeper understanding of their properties and behavior.
Automatically generated - may contain errors
Lab products found in correlation
Epu software, by Thermo Fisher Scientific (3 mentions)
Falcon 3, by Thermo Fisher Scientific (3 mentions)
No 1 filter paper, by Cytiva (1 mentions)
Cryoplunge 3 instrument, by Ametek (1 mentions)
Superdex 200 column, by GE Healthcare (1 mentions)
Ccd camera, by Thermo Fisher Scientific (1 mentions)
Mark 4 vitrobot device, by Thermo Fisher Scientific (1 mentions)
K2 summit, by Ametek (1 mentions)
Superose 6 column, by GE Healthcare (1 mentions)
10 protocols using f20 electron microscope
1
Cryo-EM Imaging of Protein Samples
2
In Vitro Reconstitution of Ctf3c-Cnn1-Ulp2 Complex
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Purified Ctf3c (120 pmol) was incubated with an equimolar amount of Cnn1-Wip1 and a threefold molar excess of the FITC-Ulp2-KIM peptide (FITC-AHA-DDSDVNLIGSS; Tufts University Core Facility). After incubation on ice for 1 h, the mixture was subjected to gel filtration on a Superdex 200 column (S200 increase 5/150 GL; GE) equilibrated with GF150 supplemented with 0.05% sodium azide (wt:vol). Absorbance at 488 nm confirmed the presence of the FITC-Ulp2 peptide. Equivalent gel filtration experiments using a biotin-labeled Ulp2-KIM peptide coupled with analysis of the eluted material by dot blot using streptavidin-HRP further confirmed the retention of the peptide during gel filtration. For all gel filtration experiments, 50-µl fractions were collected manually and analyzed by SDS-PAGE.
Protein concentration after gel filtration was measured by absorbance at 280 nm, and a fraction containing ∼0.5 µg/ml protein was selected for analysis by cryo-EM. 3.5 µl of the eluate was applied to a cryo-EM grid (C-flat 2/1, glow discharged for 30 s at 15 mA). The grid was plunged into liquid ethane after blotting from both sides for 4 s using a Cryoplunge 3 instrument (Gatan). Vitrified samples were screened for ice thickness and sample distribution using an F20 electron microscope (FEI) operating at 200 kV.
Protein concentration after gel filtration was measured by absorbance at 280 nm, and a fraction containing ∼0.5 µg/ml protein was selected for analysis by cryo-EM. 3.5 µl of the eluate was applied to a cryo-EM grid (C-flat 2/1, glow discharged for 30 s at 15 mA). The grid was plunged into liquid ethane after blotting from both sides for 4 s using a Cryoplunge 3 instrument (Gatan). Vitrified samples were screened for ice thickness and sample distribution using an F20 electron microscope (FEI) operating at 200 kV.
3
Negative Stain Microscopy of Outer Dynein Arms
4
Electron Microscopy Sample Preparation
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A PELCO easiGlowTM glow discharger was used to apply a negative charged to a Cu grid at 30 mA for 20 s. Then, 3 µl of 0.05 to 0.1 mg/ml fractionated cell lysate was applied to the glow-discharged carbon-coated Cu grid. The fractionated sample was incubated on the grid for 1 min, blotted, washed twice with distilled water, washed once with 1.5% (w/v) uranyl acetate and incubated on a 1.5% uranyl acetate drop for 1 min. The stained grid was blotted and air-dried before observation. The prepared grid was analyzed with a Tecnai F20 electron microscope (FEI) with a Gatan CCD camera at the KAIST Analysis Center for Research Advancement (KARA). A total of 46, 43, 11, and 16 micrographs were collected for Group 1, 2, 3, and 4 from the E. coli lysate. The collected micrographs were processed using EMAN 2.0. Particles were auto-picked in different box sizes, 200 Å, 300 Å, and 420 Å. Selected particles were subjected to 2D classification.
5
Cryo-EM Structure Determination of FhuA-RBP pb5
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Typically, 3.5 μL of the FhuA-RBP pb5 complex was deposited on a freshly glow-discharged (25 mA, 30 s) Cu/Rh 400-mesh Quantifoil R 2/1 EM grid and flash-frozen in nitrogen-cooled liquid ethane using a ThermoFisher Mark IV Vitrobot device (100% humidity, 20°C, 2 s blotting time, blot force 1). Preliminary screening of freezing conditions was performed on an FEI F20 electron microscope.
Two different data sets were acquired on the same grid. For both data sets, 60-frame movies with a total dose of 60 e -/Å 2 were acquired on a ThermoFisher Scientific Titan Krios G3 transmission electron microscope (European Synchrotron Radiation Facility, Grenoble, France) (53) operated at 300 kV and equipped with a Gatan Quantum LS/967 energy filter (slit width of 20 eV used) coupled to a Gatan K2 summit direct electron detector. Automated data collection was performed using ThermoFisher Scientific EPU software. A nominal magnification of ×130,000 was used, resulting in a calibrated pixel size at the specimen level of 1.052 Å/pixel. For the first data set, 777 movies were acquired with a phase plate, close to focus (between -0.5 and -1.0 μm), while for the second data set, 8,752 movies were acquired without a phase plate and with a defocus ranging between -1.2 and -2.6 μm.
Two different data sets were acquired on the same grid. For both data sets, 60-frame movies with a total dose of 60 e -/Å 2 were acquired on a ThermoFisher Scientific Titan Krios G3 transmission electron microscope (European Synchrotron Radiation Facility, Grenoble, France) (53) operated at 300 kV and equipped with a Gatan Quantum LS/967 energy filter (slit width of 20 eV used) coupled to a Gatan K2 summit direct electron detector. Automated data collection was performed using ThermoFisher Scientific EPU software. A nominal magnification of ×130,000 was used, resulting in a calibrated pixel size at the specimen level of 1.052 Å/pixel. For the first data set, 777 movies were acquired with a phase plate, close to focus (between -0.5 and -1.0 μm), while for the second data set, 8,752 movies were acquired without a phase plate and with a defocus ranging between -1.2 and -2.6 μm.
6
Nanoring Structural Determination by Cryo-EM
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NRM was size excluded in PBS on a Superose 6 column (GE Healthcare) and diluted to a concentration of 0.015 mg/ml. The sample was adsorbed to a glow-discharged carbon-coated copper grid (EMS, Hatfield, PA, United States) washed with deionized water and stained with a solution of uranyl formate 0.75%. Observation was made using an F20 electron microscope (Thermo Fisher, Hillsboro, OR, USA) operated at 200 kV. Digital images were collected using a direct detector camera Falcon III (Thermo Fisher, Hillsboro, OR, USA) 4,098 × 4,098 pixels. Automatic data collection was performed using EPU software (Thermo Fisher, Hillsboro, OR, USA) at a nominal magnification of 50,000×, corresponding to a pixel size of 2 Å, and defocus range of −1 μm to −2 μm.
Contrast transfer function for each image was estimated using CTFFIND4 [68 (link)]. One thousand particles of nanorings were picked using XMIPP manual-picking utility within SCIPION framework [69 (link)]. Manually picked particles were used as input into XMIPP auto-picking utility, resulting in 13,861 particles. Particles were extracted and binned to have the box size of 100 pixels, corresponding to the pixel size of 4 Å; phase-flipped; and subjected for three rounds of reference-free 2D classification without contrast transfer function correction in RELION-3.0 Beta [70 (link)].
Contrast transfer function for each image was estimated using CTFFIND4 [68 (link)]. One thousand particles of nanorings were picked using XMIPP manual-picking utility within SCIPION framework [69 (link)]. Manually picked particles were used as input into XMIPP auto-picking utility, resulting in 13,861 particles. Particles were extracted and binned to have the box size of 100 pixels, corresponding to the pixel size of 4 Å; phase-flipped; and subjected for three rounds of reference-free 2D classification without contrast transfer function correction in RELION-3.0 Beta [70 (link)].
7
Non-cross-linked IkCON3 complex visualization
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Negative-stain EM grids of the non–cross-linked IKC0N3 sample were prepared using 0.1 mg/ml of the complex stained with 2% uranyl acetate. A total of 776 images were collected on an F20 electron microscope (Thermo Fisher Scientific) equipped with a Falcon III detector. Data were processed with RELION 4.0.
8
Structural Analysis of KAP1 Protein
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KAP1 Fl was diluted to 0.05 mg/ml in 20 mM Hepes, pH 7.5, 300 mM NaCl, and 2 mM TCEP and cross-linked with 0.1% glutaraldehyde for 2 h at 23°C. The reaction was stopped by addition of 100 mM Tris–HCl, pH 7.5. The sample was diluted 20 times, and adsorbed to a glow-discharged carbon-coated copper grid (EMS, Hatfield) washed with deionized water and stained with a solution of 2% uranyl acetate. The grids were observed using an F20 electron microscope (Thermo Fisher Scientific) operated at 200 kV. Digital images were collected using a direct detector camera Falcon III (Thermo Fisher Scientific) with 4,098 × 4,098 pixels. The magnification of work was 29,000× (px = 0.35 nm), using a defocus range of −1.5 to −2.5 μm. After manual picking of 400 particles, Relion (105 (link)) was used to sort them into 2D class averages.
9
Electron Microscopy Protocol for Structural Analysis
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Digital images were collected using a direct detector camera Falcon III (Thermo Fisher) under an F20 electron microscope (Thermo Fisher) operated at 200 kV with the set-up of 4098 x 4098 pixels. The homogeneity and coverage of staining samples on the grid was first visualized at low magnification mode before automatic data collection. Automatic data collection was performed using EPU software (Thermo Fisher) at a nominal magnification of 50,000x, resulting in a pixel size of 2 Å, and defocus range from -1 µm to -2 µm.
10
Visualizing VLP in Biofilms Using TEM
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A first confirmation of the presence of VLP in biofilms was obtained from TEM. For this, 5 µL of unprocessed sample was adsorbed to a glow-discharged carbon-coated copper grid (Canemco & Marivac, Gore, QC, Canada), washed with deionized water and stained with 5 µL of 2% uranyl acetate. TEM observations were made on an F20 electron microscope (ThermoFisher, Hillsboro, OR, USA) operated at 200 kV and equipped with a 4,098 × 4,098 pixel camera (CETA, ThermoFisher, Waltham, MA, USA). Magnification ranged from 10,000 to 29,000×, using a defocus range of −1.5 to −2.5 µm.
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