To prepare for freezing cryo-EM grids, the fresh samples were obtained and concentrated. The p3dsRNA:RIG-I, p2dsRNA:RIG-I, p1dsRNA:RIG-I, OHdsRNA:RIG-I, p3SLR30:RIG-I and OHSLR30:RIG-I complexes were concentrated to 0.3, 1.5, 0.4, 1.5, 1.5 and 1.5 mg/ml. The Quantifoil holey carbon R1.2/1.3 300 mesh Cu grids (Ted Pella) were glow discharged using the PELCO easiGlow™ Glow Discharge Cleaning System (Ted Pella) for 35 s at 25 mA. With purified RNA:RIG-I complexes (3 µl) applied onto the grids, the grids were blotted and plunged to the liquid ethane for flash freezing using a Vitrobot Mark IV (ThermoFisher). The blotting conditions for all grids were similar, under conditions of 22 °C and 100% humidity with force -4. Blotting times were 4 s, 5 s, 4 s, 4 s, 3 s and 3 s for p3dsRNA:RIG-I, p2dsRNA:RIG-I, p1dsRNA:RIG-I, OHdsRNA:RIG-I, p3SLR30:RIG-I and OHSLR30:RIG-I complexes, respectively. In the case of p3SLR30:RIG-I complex with ATP in solution, OHSLR30:RIG-I complex with ATP in solution and p3SLR30:RIG-I with AMPPNP in solution, 3, 20, 20 µM (0.3, 2.3, 2.3 mg/ml) of complex was incubated with 2.5 mM ATP/AMPPNP and 5 mM MgCl2 on ice for 30 min before being applied to the grids, and the blotting conditions are the same as p3SLR30:RIG-I complex in the absence of ATP described above. The frozen grids were all transferred and kept in liquid nitrogen prior to use in data collection.
Pelco easiglow glow discharge cleaning system
Manufactured by Ted Pella
Sourced in United States
The PELCO easiGlow glow discharge cleaning system is a laboratory equipment designed for the cleaning and treatment of various surfaces. It utilizes a glow discharge process to modify the surface properties of samples, preparing them for subsequent applications.
Automatically generated - may contain errors
Lab products found in correlation
Vitrobot mark 4, by Thermo Fisher Scientific (18 mentions)
Filter paper, by Cytiva (5 mentions)
Titan krios transmission electron microscope, by Thermo Fisher Scientific (4 mentions)
K2 summit detector, by Ametek (3 mentions)
Grade 595 filter paper, by Ted Pella (3 mentions)
Formvar and carbon coated 200 mesh containing copper em grids, by Electron Microscopy Sciences (3 mentions)
Tkr 010, by Oerlikon Balzers (3 mentions)
Titan krios microscope, by Thermo Fisher Scientific (3 mentions)
C flat holey carbon grids cf 1.2 1 3 2 c, by Electron Microscopy Sciences (2 mentions)
Titan krios, by Thermo Fisher Scientific (2 mentions)
Gold grid, by Quantifoil (2 mentions)
Vitrobot mark 3, by Thermo Fisher Scientific (2 mentions)
Temcam f216, by TVIPS (2 mentions)
Formvar and carbon coated 200 mesh copper em grids, by Electron Microscopy Sciences (2 mentions)
Whatman grade 595 filter paper, by GE Healthcare (2 mentions)
Jem 1400 transmission electron microscope, by JEOL (2 mentions)
F216 camera, by TVIPS (2 mentions)
Jem 1400 tem, by JEOL (2 mentions)
Gatan k3 summit detector, by Thermo Fisher Scientific (2 mentions)
Titan krios electron microscope, by Thermo Fisher Scientific (2 mentions)
36 protocols using pelco easiglow glow discharge cleaning system
1
Cryo-EM sample preparation for RIG-I complexes
2
Cryo-EM Analysis of Fibril Morphology
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C-flat holey carbon grids (CF 1.2/1.3-2 C, Electron Microscopy Sciences) were glow-discharged for 40 s at 20 mA using a PELCO easiGlow glow discharge cleaning system (TED PELLA). Four microliters of the fibril extract was applied on the glow discharged grid for 30 s, followed by both side blotting and plunging into liquid ethane. Blotting and plunging was done using a Gatan Cryoplunge 3 (Gatan) operated at 20 °C and >90% relative humidity. To optimize the specimen quality regarding e.g. fibril distribution and ice thickness, the cryo-EM specimens were initially analyzed using a JEM-2100F TEM (Jeol) that was equipped with a DE12 direct electron detector (Direct Electron) and operated at an accelerating voltage of 200 kV. High-resolution data sets for reconstruction of the fibrils were recorded with a Titan Krios (Thermo Fisher Scientific) microscope that was equipped with a K2-Summit detector (Gatan) and operated at an acceleration voltage of 300 kV (see Supplementary Table 2 for further details). The width and crossover distance of the three major morphologies were determined for 30 fibrils each from cryo-TEM images using ImageJ software.
3
Cryo-EM Analysis of NPR1–TGA3–LS7-DNA Complex
4
Cryo-EM Imaging of IGF1/IGFBP3/ALS Complex
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Quantifoil R1.2/1.3 300 mesh gold holey carbon grids (Cat#Q3100AR1.3, Electron Microscopy Sciences) was glow-discharged with a PELCO easiGlow Glow Discharge Cleaning system (Ted Pella) for 90 s at 15 mA, and then 2.5 μl of the purified IGF1/IGFBP3/ALS complex was applied to the grid in 100% humidity at 4 °C. After 2 s of blotting, the grid was plunged into liquid ethane using a Vitrobot MkIV (Thermo Fisher Scientific). Micrographs were acquired on a Titan Krios G4 TEM operated at 300 keV with a K3 direct electron detector (Gatan) at the Institute for Basic Science (IBS), using a lit width of 20 eV on a GIF-quantum energy filter. EPU software was used for automated data collection at a calibrated magnification of ×105,000 under the single-electron counting mode and correlated-double sampling (CDS) mode49 (link), yielding a pixel size of 0.883 Å/pixel. A total of 14,754 movies were collected with a defocus range from –0.9 to –2.7 μm. Each micrograph was dose-fractionated to 42 frames under a dose rate of 14.5 e–/pixel/s with total exposure time of 3.4 s, resulting in a total dose of about 63 e–/Å2. Detailed image acquisition parameters are summarized in Supplementary Table 1 .
5
Cryo-EM Sample Preparation Protocol
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Samples were diluted to 3.0 mg/mL from their respective stocks, and 3.0 µL was applied onto glow discharge for 60 s on a PELCO easiGlow™ Glow Discharge Cleaning System (Ted Pella, Inc., Redding, CA, USA); UltrAuFoil 300 mesh holey gold UltrAuFoil R 1.2/1.3 grids; and R 1/1 grids for TehA purified in GDN (Quantifoil Micro Tools GmbH, Jena, Germany). Grid preparation was performed using a Vitrobot Mark IV (Thermo Fisher Scientific, Waltham, MA, USA), with the environmental chamber set to 95% humidity, and a temperature of 4 °C. The blotting condition parameters are described in Table 1 .
Samples were imaged on a Glacios Cryo-Transmission Electron Microscope (Thermo Fisher Scientific, Waltham, MA, USA) equipped with a Falcon 4 camera (Thermo Fisher Scientific, Waltham, MA, USA) utilizing a 50 µm C2 aperture and 100 µm objective lens. Movies were acquired using the Leginon software version 3.6 at a nominal magnification of 240,000× with a calibrated pixel size of 0.566 Å and a dose rate of 10.41 e−/Å2/s with a total exposure of 3.50 s, for an accumulated dose of 36.43 e−/Å2 [22 (link),23 (link)]. Movies were collected at a nominal defocus range of −0.5 µm to −1.5 µm.
Samples were imaged on a Glacios Cryo-Transmission Electron Microscope (Thermo Fisher Scientific, Waltham, MA, USA) equipped with a Falcon 4 camera (Thermo Fisher Scientific, Waltham, MA, USA) utilizing a 50 µm C2 aperture and 100 µm objective lens. Movies were acquired using the Leginon software version 3.6 at a nominal magnification of 240,000× with a calibrated pixel size of 0.566 Å and a dose rate of 10.41 e−/Å2/s with a total exposure of 3.50 s, for an accumulated dose of 36.43 e−/Å2 [22 (link),23 (link)]. Movies were collected at a nominal defocus range of −0.5 µm to −1.5 µm.
6
Cryo-EM Imaging of Omicron S Trimer
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To prepare cryo EM grids, 4.0 μL of the freshly purified full-length Omicron S trimer from the peak fraction in DDM, concentrated to ∼3.5 mg/mL was applied to a 1.2/1.3 Quantifoil gold grid (Quantifoil Micro Tools GmbH), which were glow discharged with a PELCO easiGlow™ Glow Discharge Cleaning system (Ted Pella, Inc.) for 60 s at 15 mA in advance. Grids were immediately plunge-frozen in liquid ethane using a Vitrobot Mark IV (ThermoFisher Scientific), and excess protein was blotted away by using grade 595 filter paper (Ted Pella, Inc.) with a blotting time of 4 s, a blotting force of −12 at 4°C with 100% humidity. The grids were first screened for ice thickness and particle distribution. Selected grids were used to acquire images by a Titan Krios transmission electron microscope (ThermoFisher Scientific) operated at 300 keV and equipped with a BioQuantum GIF/K3 direct electron detector. Automated data collection was carried out using SerialEM version 3.8.6 (Mastronarde, 2005 (link)) at a nominal magnification of 105,000× and the K3 detector in counting mode (calibrated pixel size, 0.83 Å) at an exposure rate of 13.362 electrons per pixel per second. Each movie add a total accumulated electron exposure of ∼53.592 e−/Å2, fractionated in 50 frames. Data sets were acquired using a defocus range of 0.5-2.2 μm.
7
Cryo-EM sample preparation for RIG-I complexes
8
Negative Staining for Electron Microscopy
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Each sample was diluted 100 × using pure water. We used 2% uranyl acetate as the staining solution. Samples were mounted on a glow-discharge carbon-coated grid, using the PELCO easiGlow Glow Discharge Cleaning System (Ted Pella, Redding, CA, USA). The current used was 15 mA and grids were glow-discharged for 30 s. The glow-discharged grid was held using reverse-force anti-capillary forceps with the carbon-coated side facing upwards, then 2.5 μl of each sample was applied to the glow-discharge grid and incubated for 60 s. The sample was blotted off using filter paper and then the grid was briefly contacted twice with the stain, followed by additional blotting with filter paper. This was procedure was repeated a second time. The grid was then left to dry for 5 min.
9
Cryo-EM Characterization of Protein Fibrils
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C-flat 1.2/1.3 400-mesh holey carbon-coated grids (Science Services) were glow-discharged at 40 mA for 40 s using a PELCO easiGlow glow-discharge cleaning system (Ted Pella). Conditions of grid preparation were optimized with the help of a Vitrobot Mark 3 (Thermo Fisher Scientific) and checked in a 200-kV JEM 2100 F transmission electron microscope (JEOL) that was equipped with a DE12 detector (Direct Electron). The grids for data collection were prepared by application of 3.5 μL of fibril solution to a grid, incubation for 30 s at >95% humidity, both-side blotting using filter paper (Whatman), and plunging into liquid ethane (~103 K). The data set was recorded with a Titan Krios transmission electron microscope (Thermo Fisher Scientific) at 300 kV and applying a Gatan imaging filter with a 20-eV slit. The images were recorded with a K2-Summit detector (Gatan) in counting mode. The software SerialEM v3.7 was used for data collection. In total, 3033 micrographs were collected from a single grid. See Supplementary Table 1 for further details. Global fibril parameters, such as width and crossover distance, were measured using Fiji v1.5257 (link). The proportion of fibrils showing the reconstructed morphology was determined by analyzing all fibrils (length at least 200 nm) in 100 micrographs.
10
TDP-43 Oligomer Visualization Protocol
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Prior to sample
loading, Formvar and carbon-coated 200 mesh containing copper EM grids
(Electron Microscopy Sciences) were glow-discharged for 30 s at 20
mA using a PELCO easiGlow glow discharge cleaning system (TED PELLA,
Inc.). Subsequently, 5 μL of samples were spotted onto the EM
grids and waited for 5 min. TDP-43 oligomer samples were then carefully
blotted out using the edge of filter papers and air-dried for 1 min.
After that, the grids were washed 3 times with ultrapure water, followed
by staining with 0.7% (w/v) uranyl formate solution. The grids were
subsequently examined using a Tecnai Spirit BioTWIN electron microscope.
The microscope was equipped with a LaB6 gun operated at an acceleration
voltage of 80 kV, and images were captured using a 4K × 4K charge-coupled
device camera (FEI Eagle).
loading, Formvar and carbon-coated 200 mesh containing copper EM grids
(Electron Microscopy Sciences) were glow-discharged for 30 s at 20
mA using a PELCO easiGlow glow discharge cleaning system (TED PELLA,
Inc.). Subsequently, 5 μL of samples were spotted onto the EM
grids and waited for 5 min. TDP-43 oligomer samples were then carefully
blotted out using the edge of filter papers and air-dried for 1 min.
After that, the grids were washed 3 times with ultrapure water, followed
by staining with 0.7% (w/v) uranyl formate solution. The grids were
subsequently examined using a Tecnai Spirit BioTWIN electron microscope.
The microscope was equipped with a LaB6 gun operated at an acceleration
voltage of 80 kV, and images were captured using a 4K × 4K charge-coupled
device camera (FEI Eagle).
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