L. pneumophila Lp02 cells were grown till early stationary stage (OD600 ~2.8) and harvested. Cells were mixed with 10-nm colloidal gold beads (Sigma-Aldrich, St. Louis, MO) precoated with bovine serum albumin. Four μl of this mixture was applied onto freshly glow-discharged copper R2/2 Quantifoil holey carbon grids (Quantifoil Micro Tools GmbH, Jena, Germany). Using an FEI Vitrobot Mark IV, grids were then blotted (under 100% humidity conditions) and plunge-frozen in a liquid ethane/propane mixture.
R2 2 holey carbon grids
Manufactured by Quantifoil
Sourced in Germany
The Quantifoil R2/2 holey carbon grids are a type of sample support film used in transmission electron microscopy (TEM). The grids consist of a uniform array of circular holes with a diameter of 2 microns, spaced 2 microns apart, and supported by a thin carbon film. These grids provide a stable and consistent platform for mounting and imaging samples in the TEM.
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Lab products found in correlation
Vitrobot mark 4, by Thermo Fisher Scientific (6 mentions)
Titan krios, by Thermo Fisher Scientific (6 mentions)
10 nm colloidal gold beads, by Merck Group (2 mentions)
Fei vitrobot mk 4, by Thermo Fisher Scientific (2 mentions)
Fei titan krios microscope, by Thermo Fisher Scientific (2 mentions)
Titan krios cryo electron microscope, by Thermo Fisher Scientific (2 mentions)
Vitrobot, by Thermo Fisher Scientific (2 mentions)
Mark 4, by Thermo Fisher Scientific (1 mentions)
Falcon 2 direct electron detector, by Thermo Fisher Scientific (1 mentions)
Vitrobot mk 4, by Thermo Fisher Scientific (1 mentions)
Vitrobot plunge freezer, by Thermo Fisher Scientific (1 mentions)
K2 camera, by Thermo Fisher Scientific (1 mentions)
E coli total lipid extract, by Avanti Polar Lipids (1 mentions)
Imaging filter quantum, by Ametek (1 mentions)
K2 summit, by Ametek (1 mentions)
Tecnai polara, by Thermo Fisher Scientific (1 mentions)
19 protocols using r2 2 holey carbon grids
1
Plunge-freezing of L. pneumophila for cryo-EM
2
Plunge-freezing of L. pneumophila for cryo-EM
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L. pneumophila Lp02 cells were grown till early stationary stage (OD600 ~2.8) and harvested. Cells were mixed with 10-nm colloidal gold beads (Sigma-Aldrich, St. Louis, MO) precoated with bovine serum albumin. Four μl of this mixture was applied onto freshly glow-discharged copper R2/2 Quantifoil holey carbon grids (Quantifoil Micro Tools GmbH, Jena, Germany). Using an FEI Vitrobot Mark IV, grids were then blotted (under 100% humidity conditions) and plunge-frozen in a liquid ethane/propane mixture.
3
Cryo-EM Imaging of Liganded GluA2 and GluK2
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Vitrified specimens were prepared by adding 2.5 μL of liganded GluA2 or GluK2 at 1.8 mg/mL to R2/2 holey carbon grids (Quantifoil, Jena, Germany) rendered hydrophilic by chemical treatment (J. R. M, P. R., J. K., S. C., J. P., M. L. M. and S. S., manuscript in preparation). Grids were blotted for 2 seconds, then plunge-frozen in liquid ethane using an FEI Vitrobot Mk IV (FEI Company, Hillsboro, OR), with the chamber maintained at 22°C and 100% humidity. Following vitrification, grids were post-mounted into autoloader cartridges and transferred to the microscope. Cryo-EM imaging was done on an FEI Titan Krios microscope (FEI Company, Hillsboro, OR) operated at 300 kV, aligned for parallel illumination, and equipped with a high-brightness XFEG and Cs corrector. Projection images were acquired as seven-frame movies with a 4,096×4096 back-thinned Falcon II CMOS detector at a nominal magnification of 47,000×, corresponding to a pixel size of 1.406 Å at the specimen plane. Imaging was carried out using FEI EPU automated data-acquisition software to collect approximately equal numbers of images at nominal focus values of −2.0, −2.5, −3.0, and −3.5 μm, and with a dose rate and exposure time of 20 e/Å2/sec and 3.5 sec, respectively.
4
Cryo-EM Imaging of Liganded GluA2 and GluK2
5
Structural Studies of RNAP-Ocr Complex
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For structural studies using cryoEM, the His-tag of Ocr was cleaved and the purification procedure was as previously reported (Sturrock et al., 2001 (link)). The complex was formed as described above. The RNAP-Ocr complex sample was concentrated to 0.6 mg/ml and 3.5 µl of the complex was applied to R2/2 holey carbon grids (Quantifoil). The vitrification process was performed using a Vitrobot Mark IV (FEI) at 4°C and 95% humidity, blotted for 1.5 s with blotting force −6. The grid was then flash frozen in liquid ethane and stored in the liquid nitrogen before data collection.
6
Structural Determination of Hoxa9 IRES-80S Complex
7
Cryo-EM Sample Preparation and Data Collection
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3 μL of samples at a concentration of 0.5 mg/ml were applied to R2/2 holey carbon grids (Quantifoil). Vitrification was carried out using a Vitrobot Mark IV (FEI) at 4°C and 95% humidity for 0.5 s with a blotting force of −5.
All data were collected on a Titan Krios using EPU (FEI) operated at 300 KeV and a K2 Summit direct electron detector (Gatan) using a defocus range of −1.2 μm to −3.2 μm. The RPo dataset was collected at eBIC (Diamond Light Source, UK) with a pixel size of 1.06 Å/pixel and a total dose of 45 e−/Å2 fractionated into 25 frames (1.8 e-/Å2/frame). The RPitc dataset was collected at the Francis Crick Institute with a pixel size of 1.08 Å/pixel and a dose of 48 e-/Å2 over 30 frames (1.6 e-/Å2/frame). A total of 916 micrographs were collected for the RPo and 4205 for the RPitc.
All data were collected on a Titan Krios using EPU (FEI) operated at 300 KeV and a K2 Summit direct electron detector (Gatan) using a defocus range of −1.2 μm to −3.2 μm. The RPo dataset was collected at eBIC (Diamond Light Source, UK) with a pixel size of 1.06 Å/pixel and a total dose of 45 e−/Å2 fractionated into 25 frames (1.8 e-/Å2/frame). The RPitc dataset was collected at the Francis Crick Institute with a pixel size of 1.08 Å/pixel and a dose of 48 e-/Å2 over 30 frames (1.6 e-/Å2/frame). A total of 916 micrographs were collected for the RPo and 4205 for the RPitc.
8
Cryo-EM Sample Preparation and Data Collection
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Quantifoil R2/2 holey carbon grids, covered with homemade amorphous carbon (~50 Å thick) was used with prior glow discharging. Using the Vitrobot Mk IV, 3 μl of our sample was applied to the grids, blotted for 4–6 s, vitrified by plunging into liquid ethane, and stored in liquid nitrogen. Data was collected over seven separate sessions in integrating mode at a magnification of 75,000, pixel size 1.04 Å on the FEI Titan Krios 300 kV electron microscope using the FEI Falcon III detector and EPU software. A total of 46,109 movies were collected using a defocus range from −1.1 to −3.2 at a dose of 1.5 e− per frame per Å2 at 1 s exposure (39 frames).
9
Cryo-EM of E. coli 30S-KsgA Complex
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70 nM submethylated E. coli 30S were mixed with 700 nM KsgA and incubated 10 min at 37°C. The sample was spun 5 min at 16 000 rcf and 4°C to remove aggregates. R2/2 holey carbon grids (Quantifoil) washed with ethylacetate were glow discharged for 30 s at negative 15 mA. For each grid 3 μl of 0.2 mg/ml grapheneoxide suspension was applied on the carbon side of the grid and excess liquid was blotted after 4 min incubation at room temperature. Grids were washed once with 20 μl of MilliQ, excess liquid was blot and grids were dried. The grid was transferred to a Vitrobot (FEI Company) at 4°C and 100% relative humidity and 5 μl sample were incubated for 30 s on the graphene oxide coated grids. Grids were blotted for 6 and 9 s and frozen in a 1:2 mixture of liquid ethane and propane. The grids were transferred to a Titan Krios cryo-electron microscope (FEI Company) operated at 300 kV and a magnification of 100720 × corresponding to a physical pixel size of 1.39 Å. Micrographs were recorded from two grids on a Falcon III direct electron detector in integration mode using dose fractionation with 25 frames and a total average dose of 35 e−/Ų. Defocus values were in the range from −0.7 μm to −3 μm (Supplementary Table S1 ).
10
Cryo-EM Sample Preparation Protocol
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The electron microscopy grids were cleaned by argon oxygen for 45 s. A droplet of 4 µL of freshly purified samples containing class I complexes were applied at a concentration of 0.5 mg mL‐1 to R2/2 holey carbon grids (Quantifoil) whereas the class II complex at a concentration of 0.5 mg mL‐1 were applied to R1.2/1.3 holey grids (Quantifoil), blotted 0.5 s with the blotting force ‐9, and vitrified using a Vitrobot Mark IV (FEI) at 4 °C and 100% humidity. The grids were then flash frozen in liquid ethane and stored in the liquid nitrogen before data collection.
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