For cryo-EM/ET infection studies, HeLa cells were grown on gold Quantifoil R 2/1 grids (Quantifoil, Großlöbichau, Germany) and inoculated with RSV A2-mK+ at a M.O.I. of 10. The infected cells were rocked for 1 h at room temperature to facilitate virus binding and were then washed twice with PBS to remove the unbound viral particles. New medium containing 600 nM fusion inhibitor (BMS-433771) [51 (link),52 (link)] was added to prevent viral entry and fusion events, and the infected cells with fusion inhibitor were allowed to incubate for 24 h. The samples were processed for cryo-EM/ET imaging as described below.
R2 1 grid
Manufactured by Quantifoil
Sourced in Germany
The Quantifoil R2/1 grid is a laboratory equipment product designed for use in electron microscopy. It features a regular array of circular holes in a carbon film supported by a metal mesh. The core function of this product is to provide a stable and consistent substrate for the preparation and analysis of samples using transmission electron microscopy (TEM) techniques.
Automatically generated - may contain errors
Lab products found in correlation
Vitrobot mark 4, by Thermo Fisher Scientific (5 mentions)
Vitrobot, by Thermo Fisher Scientific (5 mentions)
Titan krios electron microscope, by Thermo Fisher Scientific (3 mentions)
K2 summit, by Ametek (2 mentions)
Titan krios microscope, by Thermo Fisher Scientific (2 mentions)
K2 summit direct electron detector, by Ametek (2 mentions)
Titan krios g3, by Thermo Fisher Scientific (2 mentions)
Titan krios, by Thermo Fisher Scientific (2 mentions)
Vitrobot mk 4, by Thermo Fisher Scientific (2 mentions)
Tecnai g2 transmission electron microscope, by Thermo Fisher Scientific (1 mentions)
4k fei eagle ccd camera, by Thermo Fisher Scientific (1 mentions)
K2 direct electron detector, by Ametek (1 mentions)
Fei titan krios electron microscope, by Thermo Fisher Scientific (1 mentions)
Quantum 964 post column energy filter, by Ametek (1 mentions)
Falcon 2 detector, by Thermo Fisher Scientific (1 mentions)
Krios 300 kv microscope, by Ametek (1 mentions)
Epu software, by Thermo Fisher Scientific (1 mentions)
So163 films, by Kodak (1 mentions)
Super coolscan 9000 ed scanner, by Nikon (1 mentions)
Mk 3 vitrobot, by Thermo Fisher Scientific (1 mentions)
22 protocols using r2 1 grid
1
Cryo-EM Imaging of RSV Infection
2
Cryo-EM Imaging of Purified TRPV2
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Purified rat TRPV2 was frozen using conditions as previously described16 (link). Briefly, a 3.0 μl aliquot of purified protein at a concentration of 2.5 mg ml−1 was blotted onto Quantifoil R2/1 grids (Quantifoil Micro Tools) and manually plunged into liquid ethane. The frozen grid samples were imaged on the FEI Titan Krios Microscope operated at 300 kV at the nominal magnification of × 31,000 with a Gatan K2 Summit direct detector camera. Movies (32 frames per movie) were recorded under super-resolution counting mode at a calibrated pixel size of 0.645 Å and a dose rate of ∼8 electrons per physical pixel per s and underfocus values ranging from −1.5 to −3.0 μm with the automated collection software Leginon49 (link).
3
Cryogenic Electron Microscopy of Extracellular Vesicles
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Samples for cryo-EM were vitrified using an FEI Vitrobot Mark IV on Quantifoil R2/1 grids with the following settings: 4 µl sample; wait time 10 s; blot time 2 s; blot force −2. EVs were examined with Tecnai G2 transmission electron microscope (FEI, Brno, CZ) equipped with 4k FEI Eagle CCD camera, at 200 kV. The micrographs were acquired using EPU (FEI, Brno, CZ) acquisition software at defocus varying between 3 and 5 μm at 29,000 × or 80,000 × magnification (dose of 20 e− Å−2).
4
Vitrification and Cryogenic Imaging of Reovirus
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Aliquots of 3.5 μL of purified reovirus virion and the uncoated core particles generated by digestion were applied to glow-discharged Quantifoil R2/1 grids (Quantifoil Micro Tools GmbH, Jena, Germany), blotted for 3 s in a 100% humidity chamber, and plunged into liquid ethane (cooled by liquid nitrogen) in an FEI Vitrobot Mark IV vitrification robot.
For T-core and nT-core particles, samples were prepared as a transcription reaction in vitro, except for [α-32P]-UTP, which was replaced by UTP. After the reaction, samples were applied to grid and were plunge-frozen as above.
For all particles in different states, we collected dose-fractionated super-resolution movies using a 300-kV FEI Titan Krios electron microscope (FEI Company) with a K2 direct electron detector (Gatan). The magnification was 105,000×, yielding a physical pixel size of 1.35 Å, corresponding to a super-resolution pixel size of 0.68 Å. For virion, each exposure of 7.6 s was dose-fractionated into 32 movie frames, leading to a total dose of ~40 e−/Å2. For the core particles, including the uncoated core, nT-core and T-core, the exposure time was 5.44 s, which was dose-fractionated into 32 movie frames, leading to a total dose of ~30 e−/Å2.
For T-core and nT-core particles, samples were prepared as a transcription reaction in vitro, except for [α-32P]-UTP, which was replaced by UTP. After the reaction, samples were applied to grid and were plunge-frozen as above.
For all particles in different states, we collected dose-fractionated super-resolution movies using a 300-kV FEI Titan Krios electron microscope (FEI Company) with a K2 direct electron detector (Gatan). The magnification was 105,000×, yielding a physical pixel size of 1.35 Å, corresponding to a super-resolution pixel size of 0.68 Å. For virion, each exposure of 7.6 s was dose-fractionated into 32 movie frames, leading to a total dose of ~40 e−/Å2. For the core particles, including the uncoated core, nT-core and T-core, the exposure time was 5.44 s, which was dose-fractionated into 32 movie frames, leading to a total dose of ~30 e−/Å2.
5
Cryo-EM Grid Preparation for NDH-1MS
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To prepare cryo-EM grids, four microliter of NDH-1MS at 5 mg mL−1 were applied to R2/1 grids (Quantifoil) that were glow-discharged for 20 s immediately before use. The sample was incubated 30 s at 100% humidity and 4 °C before blotting for 3.5 s with blotforce 5 and then plunge-frozen into liquid ethane/propane mix cooled by liquid nitrogen using a Vitrobot Mark IV (FEI). Data was acquired on a Titan Krios electron microscope (ThermoFisher, FEI) operated at 300 kV, equipped with a K2 Summit direct electron detector (Gatan) and a GIF quantum energy filter (20 eV) (Gatan). Movies were recorded in counting mode at a pixel size of 1.35 Å per pixel using a cumulative dose of 40.20 e−/Å2 and 50 frames. Data acquisition was performed using SerialEM34 (link) with four exposures per hole with a target defocus range of 0.5 to 3.0 μm.
6
Cryo-EM Imaging of Protein Vesicles
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For grid preparation, 3.5 μl of vesicles were mixed with 10-nm gold fiducials on Quantifoil R2/1 grids and plunge-frozen in a propane/ethane mixture using a manual plunge freezer. Microscopy was performed using a Tecnai F30 “Polara” microscope (FEI Thermo Fisher Scientific) at 300 kV equipped with a Quantum 964 post-column energy filter (Gatan) operated in zero-loss imaging mode. Images were recorded on a 4k × 4k K2 Summit electron detector with a calibrated pixel size of 0.14 nm at the specimen level. Transmission images were recorded using SerialEM (49 (link)) at a −3-μm defocus. Vesicle diameters were measured in 3dmod.
7
Cryo-EM Imaging of MxB Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Three microliters of the MxB tubes (0.5 mg/ml) was applied on the carbon side of glow-discharged holey R2/1 Quantifoil grids (Quantifoil Micro Tools GmbH), manually blotted from the backside, and then plunge-frozen in liquid ethane using a homemade manual plunger. Images were collected under low-dose conditions (~40 e−/Å2 total) using a Polara 300-kV microscope with a field-emission gun and an FEI Falcon II detector. Movies (~1000, each with seven frames) were manually collected using SerialEM (39 (link)) at a nominal magnification of ×98,000 (1.147 Å/pixel), with under-focus values ranging from 1.5 to 3.5 μm.
8
Cryo-EM Imaging of Protein Complexes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
3 µl of 0.5 mg/ml tubes were applied on the carbon side of glow-discharged holey R2/1 Quantifoil grids (Quantifoil Micro Tools GmbH), manually blotted from the back side, and then plunge frozen in liquid ethane using a homemade manual plunger. Images were collected at the Electron Bio-Imaging Centre at Diamond Light Source under low-dose conditions (∼40 e−/Å2 total) using a Titan Krios 300-kV microscope equipped with a Schottky X field emission gun and a Gatan Quantum energy filter with K2 Summit detector. Videos (6,426; each with 40 frames) were collected using EPU software (FEI) at a nominal magnification of 98,000 (1.06 Å/pixel) with under focus values ranging from 1.5 to 3.5 µm.
9
Cryo-EM Imaging of Dynamin Tubes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Three microliters of the dynamin tubes (0.5 mg/ml) was applied on the carbon side of glow-discharged holey R2/1 Quantifoil grids (Quantifoil Micro Tools GmbH), manually blotted from the backside, and then plunge-frozen in liquid ethane using a homemade manual plunger. The grids were imaged on a Titan Krios microscope (Thermofisher Scientific, USA) operated at 300 KeV, with a nominal magnification of 130,000 × in EFTEM mode on a post-column Quantum-K2 detector (Gatan, USA) operated in counting mode with an energy selecting slit of 20 eV. Detailed parameters for data collection are listed in Table 1 . All data were collected using EPU software (Thermofisher Scientific, USA).
10
Cryo-EM Data Collection of MmuPV1
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The MmuPV1 sample was assessed for purity and concentration before vitrification for cryo-EM data collection on the Penn State Titan Krios (https://www.huck.psu.edu/core-facilities/cryo-electron-microscopy-facility/instrumentation/fei-titan-krios ). Three microliters of the purified virus sample was pipetted onto glow-discharged R2/1 Quantifoil grids (Quantifoil Micro Tools GmbH, Jena, Germany), blotted for 2.5 s, and plunge-frozen in liquid ethane using a Vitrobot Mark IV (Thermo Fisher, Waltham, MA, USA). Vitrified grids were imaged using a Titan Krios G3 (Thermo Fisher, Waltham, MA, USA) under automated control of the EPU software. An atlas image was taken at 165× magnification, and suitable areas were selected for imaging on the Falcon 3EC direct electron detector. The microscope was operated at 300 kV with a 70 μm condenser aperture and a 100 μm objective aperture. Magnification was set at 59,000× yielding a calibrated pixel size of 1.1 Å. Four, nonoverlapping exposures were acquired per each 2-um-diameter hole of the grid with the beam in parallel mode, for an overall collection of 2859 micrographs. The total dose per exposure was set to 45 e−/Å2 (Supplementary Table S1 ).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!