MBS was purified using detergent DDM and was concentrated to ~5 mg/ml. Cryo-EM grids were prepared under the aerobic conditions and the processes took 20–30 min (MBS in air has a half-life of 19 h19 (link)). In brief, three-microliter aliquots of the sample were applied to glow-discharged Quantfoil R 1.2/1.3 gold grids (300 mesh). The grids were blotted for 3 s at 10 °C with 100% humidity and were flash-frozen in liquid ethane using an FEI Vitrobot Mark IV device. Cryo-EM data were recorded on a K2 camera positioned post a GIF quantum energy filter in a 300 kV FEI Titan Krios electron microscopy. Two datasets were automatically collected with Serial EM 3.7beta and FEI EPU software package, respectively, containing 8352 and 6355 movies respectively. Micrographs were recorded in counting mode at a nominal magnification of 130,000× with a pixel size of 1.03 Å on sample. Defocus values varied from −1.1 to −3 μm. The dose rate was 10 electrons per pixel per second. A total exposure of 6 s was dose-fractionated into 30 sub-frames, resulting in a total accumulated dose of 52 electrons per Å2.
Mark 4 vitrobot device
Manufactured by Thermo Fisher Scientific
The Mark IV Vitrobot device is a self-contained instrument designed for the preparation of vitrified samples for cryo-electron microscopy (cryo-EM). It automates the process of plunge-freezing samples in liquid ethane or propane, ensuring consistent and reproducible sample preparation.
Automatically generated - may contain errors
Lab products found in correlation
Titan krios electron microscopy, by Thermo Fisher Scientific (1 mentions)
Lacey carbon grid, by Ted Pella (1 mentions)
4k 4k ccd camera, by Thermo Fisher Scientific (1 mentions)
Tecnai g2 spirit biotwin, by Thermo Fisher Scientific (1 mentions)
Gatan 626 cryo holder, by Ametek (1 mentions)
Vitrobot mark 4, by Thermo Fisher Scientific (1 mentions)
F20 electron microscope, by Thermo Fisher Scientific (1 mentions)
K2 summit, by Ametek (1 mentions)
Epu software, by Thermo Fisher Scientific (1 mentions)
6 protocols using mark 4 vitrobot device
1
Cryo-EM Structural Analysis of MBS
2
Cryo-EM Imaging of xPCD Samples
3
Cryo-EM Sample Preparation Protocol
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Typically, 3.5 μl of T5 tail sample (with or without FhuA nanodisc) was deposited on a freshly glow discharged (25 mA, 30 s) Cu/Rh 300 mesh Quantifoil R 2/1 EM grids and plunge-frozen in nitrogen-cooled liquid ethane using a Thermo Fisher Scientific Mark IV Vitrobot device (100% humidity, 20°C, 5-s blotting time, blot force 0).
4
Cryo-EM Sample Preparation of PI3KC2α
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Purified PI3KC2αΔN (0.8 mg ml−1 in 20 mM Tris-HCl, 100 mM NaCl at pH 7.4) were used for plunge-freezing. Double-application of 3 μl of diluted PI3KC2αΔN were applied onto freshly plasma-cleaned (NanoClean, model 1070, Fischione Instruments) QUANTIFOIL Holey Au-carbon-R2/2 specimen grids, and vitrified by plunge-freezing into liquid ethane using a Mark IV Vitrobot device (Thermo Fisher Scientific). Cryo-EM specimen prepared with different blotting conditions (blot force, blot time) were screened. Final datasets were collected from the cryo-EM grids with thinner uniform ice thickness and good particle orientation distribution.
5
Cryo-EM Grid Preparation for Protein Samples
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A 4 µl protein sample was placed on an Au 300 mesh Quantifoil R 1.2/1.3 EM grid that had been glow-discharged (Med, 30 s). The grid was then rapidly frozen in liquid ethane that had been chilled with liquid nitrogen, using the Mark IV Vitrobot device from Thermo Fisher Scientific. The grid was blotted for 3 s after a 5 s waiting period, and then flash frozen in liquid ethane cooled by liquid nitrogen with a Vitrobot Mark IV (FEI) at 100% humidity and 8 °C.
6
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.
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