For the initial preparation, electron microscopy grids (Quantifoil, 200-mesh copper R1.2/1.3) were glow-discharged for 90 s using PELCO easiGlow. Four microliters of sample was applied on the grid in Vitrobot Mark IV chamber (Thermo Fisher Scientific). The chamber of Vitrobot was set to 100% humidity at 4 °C. The sample was blotted for 5.5 s with a blot force of 25, and then plunged into ethane. For the second preparation, acetone prewashed electron microscopy grids (Ultrafoil R1.2/1.3 Au 300 mesh) were glow-discharged and 3 μL of sample was applied to the grid in a Vitrobot Mark IV chamber (Thermo Fisher Scientific), set to 100% humidity at 4 °C. The sample was blotted for 10 s with a blot force of 19, and then flash frozen in liquid ethane.
Vitrobot mark 4 chamber
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Vitrobot Mark IV chamber is a piece of laboratory equipment designed for preparing samples for cryo-electron microscopy. It provides a controlled environment for vitrifying (rapid freezing) samples in a thin layer of ice, which is a crucial step in the cryo-EM sample preparation process.
Automatically generated - may contain errors
Lab products found in correlation
K3 direct electron detector, by Ametek (3 mentions)
Jec 3000fc sputter coater, by JEOL (2 mentions)
Cryoarm300, by JEOL (1 mentions)
R1.2 1 3 cu 200 mesh grid, by Quantifoil (1 mentions)
R1.2 1 3 au 300 mesh, by Thermo Fisher Scientific (1 mentions)
Falcon 3ec, by Thermo Fisher Scientific (1 mentions)
Krios g4 cryo transmission electron microscope, by Thermo Fisher Scientific (1 mentions)
R1.2 1 3 200 mesh copper grids, by Quantifoil (1 mentions)
Bioquantum gif k3, by Ametek (1 mentions)
Titan krios g3 microscope, by Thermo Fisher Scientific (1 mentions)
9 protocols using vitrobot mark 4 chamber
1
Cryo-EM Grid Preparation Protocols
2
Cryo-EM Structural Analysis of S Protein Trimer
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S protein trimer with D614G mutation was prepared as described previously26 (link) and mixed with 1:1 molar ratio of NIBIC-71 and 7G7/κ solutions at a final concentration of 0.5 mg/mL of the S protein trimer. Quantifoil grids (R1.2/1.3 Cu 200 mesh) were glow-discharged using a JEC-3000FC sputter coater (JEOL) at 20 mA for 20 s. After 3 mL of the complex solutions were applied, the grids were blotted with a force of –10 and a time of 2 s in a Vitrobot Mark IV chamber (Thermo) equilibrated at 4 °C and 100% humidity, and then immediately plunged into liquid ethane. The grids were stored in liquid nitrogen. All cryo-EM image datasets were acquired using SeriaIEM27 (link), yoneoLocr28 (link), and a JEM-3300 (CRYO ARM™ 300 II, JEOL) operated at 300 kV with a K3 direct electron detector (Gatan, Inc.) in CDS mode. The W-type in-column energy filter was operated with a slit width of 20 eV for zero-loss imaging. The nominal magnification was 60,000×, corresponding to 0.86 Å per pixel. Defocus varied between − 0.5 and − 2.0 mm. Each movie was fractionated into 60 frames with a total dose of 60 e−/Å2.
3
Structural Characterization of Bp109-92 and TNFR2-MBP Complex
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The Bp109-92 and TNFR2-MBP complex was prepared as a 2:3 mixture (5.6 μM Bp109-92 and 8.4 μM TNFR2-MBP) at room temperature for 5 min, and purified in a Superose 6 Increase 10/300 GL column. Three microliters of the complex solution (0.2 mg/mL) were applied to a Quantifoil R1.2/1.3 Cu 200 mesh grid (Quantifoil Micro Tools GmbH, Jena, Germany) that was glow-discharged for 20 s at 20 mA using a JEC-3000FC sputter coater (JEOL). The grid was blotted with a blot force of 0 and a blot time of 3 s in a Vitrobot Mark IV chamber (Thermo Fisher Scientific) equilibrated at 4 °C and 100% humidity, and then immediately plunged into liquid ethane. Excess ethane was removed with filter paper, and the grids were stored in liquid nitrogen. The image dataset was collected using SerialEM45 (link), yoneoLocr46 (link), and JEM-Z300FSC (CRYO ARM 300: JEOL) operated at 300 kV with a K3 direct electron detector (Gatan, Pleasanton, CA, USA) in CDS mode. The Ω-type in-column energy filter was operated with a slit width of 20 eV for zero-loss imaging. The nominal magnification was 60,000×. Defocus varied between −0.5 and −2.0 μm. Each movie was fractionated into 60 frames (0.081 s each, total exposure: 4.87 s), with a total dose of 60 e−/Å2.
4
Cryo-EM Structure of SARS-CoV-2 Spike with Nanobodies
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An epoxidized graphene grid (EG-grid)89 was used to increase the number of protein particles. The trimer complex of the SARS-CoV-2 spike (D614G) with the furin-resistant mutation (“GSAS”) at a concentration of 0.1 mg ml–1 was mixed with a 5-time molar excess of P86 or P17 monomer and incubated on ice for 10 min, and 3 μl of the spike-nanobody complex solution was applied to the EG-grid. After incubation at room temperature for 5 min, the grids were blotted with a force of –3 and a time of 2 s in a Vitrobot Mark IV chamber (Thermo) equilibrated at 4 °C and 100% humidity and then immediately plunged into liquid ethane. Excessive ethane was removed with filter paper, and the grids were stored in liquid nitrogen. All cryo-EM image datasets were acquired using a JEM-Z300FSC (CRYO ARM 300: JEOL, Tokyo, Japan) operated at 300 kV with a K3 direct electron detector (Gatan, Inc., Pleasanton, CA) in CDS mode90 (link). The Ω-type in-column energy filter was operated with a slit width of 20 eV for zero-loss imaging. The nominal magnification was ×60,000, corresponding to 0.870 Å per pixel. Defocus varied between –0.5 and –2.0 μm. Each movie was fractionated into 60 frames (0.0505 s each, total exposure: 3.04 s) with a total dose of 60 e−/Å2.
5
Cryogenic Electron Microscopy Grid Preparation
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Acetone pre-washed electron microscopy grids (Ultrafoil R1.2/1.3 Au 300 mesh) were glow-discharged and 3 μL of the sample was applied to the grid in a Vitrobot Mark IV chamber (Thermo Fisher Scientific), set to 100% humidity at 4 °C. The sample was blotted for 10 s with a blot force of 19 and then flash frozen in liquid ethane.
6
Reconstitution of RNA Polymerase III Initiation Complex
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τA was incubated with equimolar amounts of double-stranded His_upstream_Abox DNA, followed by the addition of the Brf1–TBP fusion protein and finally full-length Bdp1. The sample was incubated for 10 min before adding the next complex component, respectively. The complex was subsequently diluted to 1.3 mg/mL in dilution buffer (20 mM HEPES pH 7.5, 5 mM DDT, and 2 mM MgCl2) and thereby adjusted to 75 mM NaCl. Directly before grid freezing 4 mM CHAPSO (3-([3-cholamidopropyl]dimethylammonio)-2-hydroxy-1-propanesulfonate in water) was added to the protein sample. A total of 2.5 µL of protein sample were applied to Quantifoil Cu 2/1 which had been previously glow discharged with a Pelco EasyGlow instrument. Excess liquid was removed with a Vitrobot Mark IV chamber (Thermo Fischer Scientific) at 4 °C, and 100% humidity for 4 s and at a blot force of 2.
7
Cryo-EM of GATA3-nucleosome complex
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For the cryo-EM specimen of the GATA3znf–nucleosome complex, 2 µl portions of the sample (0.8 µg DNA/µl) were applied to glow-discharged Quantifoil R1.2/1.3 200-mesh grids. The grids were blotted for 8 s under 100% humidity at 4 °C in a Vitrobot Mark IV chamber (Thermo Fisher Scientific, USA), and were immediately plunged into liquid ethane.
Images of the GATA3znf–nucleosome complex in the vitrified ice were collected using the EPU (Thermo Fisher Scientific, USA) auto acquisition software on a Krios G3i cryo-electron microscope (Thermo Fisher Scientific, USA), operated at 300 kV at a pixel size of 1.05 Å. Images were recorded with 64 s exposure times on a Falcon 3EC (Thermo Fisher Scientific, USA) direct electron detector in the electron counting mode, retaining a total of 51 frames with a total dose of ~50 electron/Å2. The two data collections produced 3,139 images and 2,471 images of the GATA3znf–nucleosome complex.
Images of the GATA3znf–nucleosome complex in the vitrified ice were collected using the EPU (Thermo Fisher Scientific, USA) auto acquisition software on a Krios G3i cryo-electron microscope (Thermo Fisher Scientific, USA), operated at 300 kV at a pixel size of 1.05 Å. Images were recorded with 64 s exposure times on a Falcon 3EC (Thermo Fisher Scientific, USA) direct electron detector in the electron counting mode, retaining a total of 51 frames with a total dose of ~50 electron/Å2. The two data collections produced 3,139 images and 2,471 images of the GATA3znf–nucleosome complex.
8
Cryo-EM Sample Preparation for p53-Nucleosome
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For the cryo-EM specimen preparation, R1.2/1.3 200 mesh copper grids (Quantifoil) were washed with ethyl acetate and glow-discharged by soft plasma ion bombardment (PIB-10, Vacuum Device Inc.). Aliquots (2.5 µL) of the p53-nucleosome complex or the p53 DBD-nucleosome complex were applied to the Quantifoil grids in the Vitrobot Mark IV chamber (Thermo Fisher Scientific) at 100% humidity and 16°C, and then blotted and plunged into liquid ethane. Data collections were performed on a Krios G4 cryo-transmission electron microscope (Thermo Fisher Scientific) operated at 300 kV, using the EPU software. Digital micrographs of the p53-nucleosome complex and the p53 DBD-nucleosome complex were recorded on a K3 BioQuantum (Gatan) direct electron detector calibrated at a pixel size of 1.06 and 1.1 Å in the electron counting mode, using a slit width of 25 eV, and retaining 40 frames with total doses of 56.8 and 46 electron/Å2, respectively (Table S1 ).
9
Cryo-EM structure determination of KCTD complexes
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For the KCTD5-Gγ2/Gβ1 fusion complex, a glow-charged 300-mesh holey carbon grid (Quantifoil R1.2/1.3 Au) was loaded into a Vitrobot Mark IV chamber (Thermo Fisher Scientific) at 8°C and 100% humidity. A total of 3.0 μl of the concentrated complex sample was applied onto the grid and blotted for 4.0 s before plunge-freezing in liquid ethane. Cryo-EM images were collected using EPU on a Titan Krios G4 microscope (Thermal Fisher Scientific) equipped with a Falcon 4 direct electron detector and operated at 300 kV at a magnification of ×96,000 with a pixel size of 0.86 Å. Each movie stack was dose-fractionated to 32 frames with a total dose of 49.71 e−Å−2 for 5.59 s.
For the KCTD7/Cul3 complex, 3.0 μl of the sample was loaded onto a glow-charged 400-mesh holey grid (Quantifoil R1.2/1.3 Au) coated with graphene oxide and blotted for 2 s with a blot force of 2 followed by plunge-freezing in liquid ethane. The images were collected using EPU on a Titan Krios G3 microscope (Thermal Fisher Scientific) equipped with a BioQuantum GIF/K3 (Gatan) direct electron detector in a super-resolution mode and operated at 300 kV at a magnification of ×64,000 with a pixel size of 1.087 Å. Each movie stack was dose-fractionated to 32 frames with a total dose of 50 e−Å−2 for 2.56 s.
For the KCTD7/Cul3 complex, 3.0 μl of the sample was loaded onto a glow-charged 400-mesh holey grid (Quantifoil R1.2/1.3 Au) coated with graphene oxide and blotted for 2 s with a blot force of 2 followed by plunge-freezing in liquid ethane. The images were collected using EPU on a Titan Krios G3 microscope (Thermal Fisher Scientific) equipped with a BioQuantum GIF/K3 (Gatan) direct electron detector in a super-resolution mode and operated at 300 kV at a magnification of ×64,000 with a pixel size of 1.087 Å. Each movie stack was dose-fractionated to 32 frames with a total dose of 50 e−Å−2 for 2.56 s.
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