6xHis-CnTps1 was purified as described above and concentrated in a buffer containing 20 mM Tris pH 8.0, 300 mM NaCl, 5% glycerol and 2 mM β-mercaptoethanol. For grids prepared with apo CnTps1, 3 μL of 0.75 mg/mL 6xHis-CnTps1 was applied to glow-discharged carbon Quantifoil grids. After a 15 s incubation, the grids were blotted for 2 s to remove excess protein and rapidly plunged into liquid ethane (−182 °C) using a Leica EM GP2 (Leica Microsystems) operated at 95% humidity and 22 °C. For determination of the structure of the CnTps1-UDP-G6P complex, 0.5 mg/mL 6xHis-CnTps1 was incubated with 10 mM uridine di-phosphate (UDP, Sigma) and 10 mM glucose-6-phosphate (G6P, Sigma) for 18 hours at 4 °C. 3 μL of the CnTps1-UDP-G6P mixture was deposited onto glow-discharged UltrAuFoil grids. After a 15 s incubation, the grids were blotted for 2 s to remove excess protein and rapidly plunged into liquid ethane (−182 °C) using a Leica EM GP2 (Leica Microsystems) operated at 95% humidity and 22 °C. Grids were transferred to liquid nitrogen for storage until data collection.
Em gp2
Manufactured by Leica
Sourced in Germany
The EM GP2 is a high-performance cryo-plunge freezer designed for rapid freezing of specimens for electron microscopy. The device uses a liquid ethane/propane mixture to achieve ultrafast cooling rates, enabling the preservation of delicate samples in their native state.
Automatically generated - may contain errors
Lab products found in correlation
Whatman, by Cytiva (12 mentions)
Vitrobot mark 4, by Thermo Fisher Scientific (5 mentions)
Pelco easiglow, by Ted Pella (4 mentions)
R1.2 1 3 cu 300, by Quantifoil (4 mentions)
Autogrid, by Thermo Fisher Scientific (3 mentions)
Mwco spin filter, by Merck Group (3 mentions)
10 kda mwco spin filter, by Merck Group (3 mentions)
Titan krios, by Thermo Fisher Scientific (2 mentions)
Ultraufoil r1.2 1 3 grids, by Quantifoil (2 mentions)
Jem 3200fsc tem, by JEOL (2 mentions)
Cu holey carbon grids, by Protochips (2 mentions)
Ultraufoil gold holey gold grids, by Quantifoil (2 mentions)
Superdex 200 increase column, by GE Healthcare (2 mentions)
Ultraufoil r1.2 1 3 300 mesh grid, by Quantifoil (2 mentions)
Qf 1.2 1 3 4au grid e1435q ycf1, by Electron Microscopy Sciences (2 mentions)
Tecnai arctica microscope, by Thermo Fisher Scientific (2 mentions)
Em ace600, by Leica (2 mentions)
Type 1 paper, by Cytiva (2 mentions)
Protein a gold fiducials, by Aurion (2 mentions)
Jem 3200fsc, by JEOL (2 mentions)
60 protocols using em gp2
1
Structural Determination of CnTps1 Enzyme
2
Cryo-EM Structural Analysis of MRS2 Protein
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3 μl purified MRS2 at approximately 0.5 mg/ml was applied to a glow-discharged 400-mesh R 1.2/1.3 Cu grid (Quantifoil). The cryo grids were blotted for 6 s at 4°C and 95%, and plunge-frozen into liquid ethane using a Leica EM GP2 (Leica) and stored in liquid nitrogen. The grids were screened using on an FEI Tecnai T20 TEM before data collection.
Cryo-EM datasets were acquired with SerialEM48 (link) using a Titan Krios (FEI, now ThermoFisher Scientific) operated at 300 keV and equipped with an energy filter and K3 camera (Gatan Inc.). Movies of 50 frames with a dose of 1 e−/Å2 per frame (50 e−/Å2 total dose) were recorded at a nominal magnification of 105,000x, corresponding to a physical pixel size of 0.83 Å/px (super-resolution pixel size 0.415 Å/px) in CDS mode at a dose rate of 10 e−/px/s and a defocus range of −0.7 to −2.0 µm. In total, 3,991 and 9,656 movies were collected for MRS2-Mg2+ and MRS2-EDTA, respectively (Supplementary Table 1 ).
Cryo-EM datasets were acquired with SerialEM48 (link) using a Titan Krios (FEI, now ThermoFisher Scientific) operated at 300 keV and equipped with an energy filter and K3 camera (Gatan Inc.). Movies of 50 frames with a dose of 1 e−/Å2 per frame (50 e−/Å2 total dose) were recorded at a nominal magnification of 105,000x, corresponding to a physical pixel size of 0.83 Å/px (super-resolution pixel size 0.415 Å/px) in CDS mode at a dose rate of 10 e−/px/s and a defocus range of −0.7 to −2.0 µm. In total, 3,991 and 9,656 movies were collected for MRS2-Mg2+ and MRS2-EDTA, respectively (
3
Cryo-EM Structural Analysis of Beta Spike and mACE2-Fc Complex
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Cryo-EM grids were prepared with a Leica EM GP2 (Leica) plunge-freezing device, using Quantifoil R2/1 copper 400 grids. 3.0 μL of a sample containing 0.4 μM Beta Spike and 0.7 μM mACE2-Fc was applied to the glow-discharged grids, and backblotted for 2 s with a 10 s wait time, 80% humidity and 10°C in the sample chamber, and the blotted grids were plunge-frozen in liquid nitrogen-cooled liquid ethane.
Grids were screened for particle presence and ice quality on a TFS Talos Arctica transmission electron microscope (TEM) operated at 200kV. Cryo-EM data was collected using the same microscope, equipped with a TFS Falcon 3 camera. Movies were recorded at a nominal magnification of 150kx, corresponding to a 0.9759Å pixel, with defocus values ranging from -0.8 to -2.5 μm. Exposures were adjusted automatically to 40 e-/Å2 total dose with automatic collection using EPU.
Grids were screened for particle presence and ice quality on a TFS Talos Arctica transmission electron microscope (TEM) operated at 200kV. Cryo-EM data was collected using the same microscope, equipped with a TFS Falcon 3 camera. Movies were recorded at a nominal magnification of 150kx, corresponding to a 0.9759Å pixel, with defocus values ranging from -0.8 to -2.5 μm. Exposures were adjusted automatically to 40 e-/Å2 total dose with automatic collection using EPU.
4
Vitrification and Analysis of SARS-CoV-2 Spike
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To increase hydrophilicity, grids were pretreated in a Tergeo EM Plasma Cleaner (PIE Scientific) under an argon atmosphere for 1 min, using the immersion mode at 25w. A 3 μL aliquot of 2.73 μM SARS CoV-2 S in Tris pH 8.0 50 mM and 160 mM NaCl buffer, mixed with each Nb was applied onto a clean UltrAufoil R1.2/1.3 300-mesh grid. Excess solution was removed by blotting with Whatman No. #1 prior to plunging into liquid ethane (-182°C) in a Leica EM GP 2 (Leica) vitrification robot (automated blot pressure, chamber at 25°C and 95% RH). The RBD-1-1G (1.5 mg/ml), RBD-1-2G (1.3 mg/ml), RBD-2-1F (1.8 mg/ml), RBD-1-3H (1.2 mg/ml), RBD-2-1B (1.7 mg/ml) and RBD-2-3A (4.2 mg/ml) were prepared in PBS (pH 7.4).
5
Cryo-EM Sample Preparation for Ribosome Complexes
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For RiboRad6IP and RiboRad6IP–XL, 3 μL of the samples at 1 μg/μL was applied to UltrAUfoil R1.2/1.3 grids (Quantifoil Micro Tools GmbH). Grids were glow-discharged for 30 s at 15 mA prior to sample application (PELCO easiGlow, Ted Pella, Inc.). Grids were blotted between 2.5 and 3 s and plunged frozen in liquid ethane using a Leica vitrification plunger (EM GP2, Leica Microsystems). For RiboRad6mix, cryo-grids were prepared by rapid immersion into liquid ethane with a Vitrobot Mark IV (Thermo Fisher Scientific) set to 22°C and 95% humidity. Quantifoil 300 mesh R1.2/1.3 grids were used and glow-discharged as above.
6
Cryo-EM analysis of extracellular vesicles from UM
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The EVs for cryoEM were isolated from pooled serum samples from healthy donors, primary UM patients and metastatic UM patients, resulting in 3 separate EV samples. The size and shape of extracellular vesicles were analyzed with cryogenic transmission electron microscopy (Cryo-EM). Briefly, 10 µL of each EV sample were snap frozen onto glow discharged 200 mesh copper microscopy grids (Jena Bioscience, Jena, Germany) using an automatic plunge freezer EM GP2 (Leica Microsystems, Wetzlar, Germany) with 30s pre-blotting incubation and 4s blotting. The images were taken using JEOL JEM 2100-Plus 200 kV transmission electron microscope (JEOL, Akishima, Tokyo, Japan) with 200 kV acceleration voltage protocol under 4×105 magnification. Around 20 pictures were taken for each sample and the shape and size of obtained EVs were analyzed in all of them.
7
Cryo-TEM Imaging of LNP Dispersions
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For cryo-TEM imaging, 3 μL
of the LNP dispersions (∼0.02 wt %) was applied on plasma-cleaned
(30 s oxygen plasma flash) copper grids with Lacey carbon support
films (300 mesh, Ted Pella, Inc.). The excess water was removed using
an automatic plunge freezer (EM GP2, Leica Microsystem) with a blotting
time of 3 s maintaining 90% humidity, followed by vitrification in
a 1:1 liquid propane/ethane mixture. The vitrified samples were cryo-transferred
to the microscope, and the cryo-TEM images were taken
using a JEOL JEM-3200FSC field emission cryo-TEM
(JEOL) operated at 300 kV in bright-field mode with an Omega-type
zero-loss filter. The images were acquired with Gatan Digital Micrograph
software, while the specimen temperature was maintained at −187
°C.
of the LNP dispersions (∼0.02 wt %) was applied on plasma-cleaned
(30 s oxygen plasma flash) copper grids with Lacey carbon support
films (300 mesh, Ted Pella, Inc.). The excess water was removed using
an automatic plunge freezer (EM GP2, Leica Microsystem) with a blotting
time of 3 s maintaining 90% humidity, followed by vitrification in
a 1:1 liquid propane/ethane mixture. The vitrified samples were cryo-transferred
to the microscope, and the cryo-TEM images were taken
using a JEOL JEM-3200FSC field emission cryo-TEM
(JEOL) operated at 300 kV in bright-field mode with an Omega-type
zero-loss filter. The images were acquired with Gatan Digital Micrograph
software, while the specimen temperature was maintained at −187
°C.
8
Cryo-EM Sample Preparation and Imaging
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5 µL of the sample was deposited on Quantifoil R 2/1 200 mesh holey carbon-coated copper grids. The excess solution was removed by blotting for 3 s in 80% relative humidity using an automatic plunge freezer (EM GP2, Leica Microsystem), followed by immediate vitrification using liquid ethane (-175 °C). Vitrified samples were cryo-transferred to the microscope and imaged using a JEOL JEM-3200 FSC TEM while maintaining specimen temperature at -187 °C.
9
Protein Sample Preparation for Cryo-EM
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3μl of protein (1mg/mL in 50 mM TRIS, 250 mM NaCl, pH 8.0) was applied to plasma-cleaned C-flat 1.2/1.3 400 mesh Cu holey carbon grids (Protochips, Raleigh, NC) or 1.2/1.3 300 mesh UltrAuFoil gold holey gold grids (Quantifoil Micro Tools GmbH, Großlöbichau, Germany), blotted for 2.5 s after a 30 s wait time, and then plunge frozen in liquid ethane, cooled by liquid nitrogen, using the EM GP2 (Leica Microsystems, Inc, Buffalo Grove, IL) or Vitrobot Mark IV (Thermo Fisher Scientific, Hillsboro, Oregon).
10
Cryo-EM Investigation of AVP-V2R-βarr1ΔCT-ScFv30
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For AVP-V2R-βarr1ΔCT-ScFv30 cryo-EM investigation, 3-μl samples were applied on glow-discharged Quantifoil R1.2/1.3 300-mesh UltrAufoil grids (Quantifoil Micro Tools GmbH, Germany), blotted for 3.5 s, and then flash-frozen in liquid ethane using the semiautomated EM GP2 (Leica Microsystems) plunge freezer (100% humidity and 4°C). Images were collected in one session at the European Molecular Biology Laboratory (EMBL) of Heidelberg (Germany) on a FEI Titan Krios (Thermo Fisher Scientific) at 300 keV through a Gatan Quantum 967 LS energy filter using a 20-eV slit width in zero-loss mode and equipped with a K3 Summit (Gatan Inc.) direct electron detector configured in counting mode. Movies were recorded at a nominal energy-filtered TEM magnification of ×130,000 corresponding to a 0.64-Å calibrated pixel size. The movies were collected in 40 frames in defocus range between −1 and −2 μm with a total dose of 52.63 e−/Å2. Data collection was fully automated using SerialEM, resulting in 14,080 movies.
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