A 2 μM AdeB-nanodisc sample was incubated with 20 μM Et for 2 h to form the AdeB-Et complex. The sample was then applied to lysine-coated graphene-oxide grids (Quantifoil Cu R1.2/1.3, 300 mesh) prepared in-house, blotted for 3 s, and then plunge-frozen in liquid ethane using a Vitrobot (Thermo Fisher). The grids were then transferred into cartridges. The images were recorded at −1 to −2.0 μm defocus on a Titan Krios equipped with a K3 summit direct electron detector (Gatan) with counting mode at nominal ×81,000 magnification, corresponding to a sampling interval of 1.08 Å/pixel (superresolution, 0.54 Å/pixel). Each micrograph was exposed for 2.6 s with 18.02 e−/s/physical pixel dose rate (total specimen dose, 40 e−/Å2), and 40 frames were captured per specimen area using Latitude.
Cu r1 2 1
Manufactured by Quantifoil
Quantifoil Cu R1.2/1.3 is a copper grid designed for electron microscopy applications. It features a perforated carbon film with a uniform hole size and distribution. The grid provides a stable support for sample preparation and imaging.
Automatically generated - may contain errors
Lab products found in correlation
Vitrobot mark 4, by Thermo Fisher Scientific (11 mentions)
Vitrobot, by Thermo Fisher Scientific (6 mentions)
Pelco easiglow, by Ted Pella (3 mentions)
Fei vitrobot mark 4, by Thermo Fisher Scientific (3 mentions)
Fei titan krios microscope, by Thermo Fisher Scientific (3 mentions)
Fei ceta camera, by Thermo Fisher Scientific (3 mentions)
Titan krios cryo electron transmission microscope, by Thermo Fisher Scientific (2 mentions)
K3 direct electron detector, by Ametek (2 mentions)
Mark 4, by Thermo Fisher Scientific (2 mentions)
Krios electron microscope, by Thermo Fisher Scientific (2 mentions)
R1.2 1 3 grid, by Quantifoil (2 mentions)
Talos f200c microscope, by Thermo Fisher Scientific (2 mentions)
K2 summit camera, by Thermo Fisher Scientific (2 mentions)
K3 camera, by Ametek (2 mentions)
Titan krios electron microscope, by Ametek (2 mentions)
Titan krios, by Ametek (1 mentions)
K3 summit direct electron detector, by Ametek (1 mentions)
Chapso, by Merck Group (1 mentions)
Titan krios electron microscope, by Thermo Fisher Scientific (1 mentions)
Vitrobot, by Quantifoil (1 mentions)
25 protocols using cu r1 2 1
1
Cryo-EM Imaging of AdeB-Et Protein Complex
2
Cryo-EM of E. coli Membrane and Lysate
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The BpHpnN, E. coli K12 membrane and E. coli K12 cell lysate samples were concentrated to 0.7 mg/ml, 1 mg/ml and 1 mg/ml, respectively. These samples were applied to glow-discharged holey carbon grids (Quantifoil Cu R1.2/1.3, 300 mesh), blotted for 5 s, 7 s and 7 s, respectively, and then plunge-frozen in liquid ethane using a Vitrobot (Thermo Fisher). The grids were transferred into cartridges. A Titan Krios cryo-electron transmission microscope (Thermo Fisher) was used to collect cryo-EM images. The images were recorded at 1-2.5 μm defocus on a K3 direct electron detector (Gatan) with super-resolution mode at a physical pixel size of 1.08 Å/phys. pixel (super resolution 0.54 Å/pixel). For the BpHpnN sample, each micrograph was exposed for 3.2 s with 18.2 e−/sec/phys. pixel dose rate (total specimen dose, 50 e−/A2). 40 frames were captured per specimen area using Latitude (Gatan, Pleasanton, CA). A total of 13,093 images were recorded and processed. For the E. coli K12 cell membrane and cell lysate samples, 7,643 and 5,067 movies were collected over 42 frames (4.2 s exposure time; 11.97 e−/sec/phys. pixel dose rate; 40 e−/A2 total dose) in SerialEM33 (link), respectively.
3
Cryo-EM Structural Analysis of NHEJ Complex
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Proteins were concentrated using a centricon (Amicon) with a 30-kDa cutoff and buffer exchanged into 20 mM Hepes (pH 7.6), 200 mM NaCl, 0.5 mM EDTA, 2 mM MgCl2, and 5 mM DTT. Purified Ku70/80 full length was then first mixed with Y-shaped 42- to 55-bp DNA before being mixed with purified DNA-PKcs, LX4, and PAXX in a 2:2:2:2 ratio respectively.
Aliquots of 3 μl of the NHEJ super complex (~2.5 mg/ml) were mixed with 8 mM CHAPSO to eliminate particle orientation bias (final concentration; Sigma-Aldrich) before being applied to Holey carbon grids (Quantifoil Cu R1.2/1.3, 300 mesh), glow-discharged for 60 s at a current of 25 mA in PELCO easiGlow (Ted Pella Inc.). The grids were then blotted with filter paper once to remove any excess sample and plunge-frozen in liquid ethane using a FEI Vitrobot Mark IV (Thermo Fisher Scientific) at 4°C and 95% humidity.
Aliquots of 3 μl of the NHEJ super complex (~2.5 mg/ml) were mixed with 8 mM CHAPSO to eliminate particle orientation bias (final concentration; Sigma-Aldrich) before being applied to Holey carbon grids (Quantifoil Cu R1.2/1.3, 300 mesh), glow-discharged for 60 s at a current of 25 mA in PELCO easiGlow (Ted Pella Inc.). The grids were then blotted with filter paper once to remove any excess sample and plunge-frozen in liquid ethane using a FEI Vitrobot Mark IV (Thermo Fisher Scientific) at 4°C and 95% humidity.
4
Cryo-EM Structure Determination of SLC26A9
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The purified SLC26A9 protein was concentrated to ~10 mg/mL. Aliquots (3 μL) of the protein were placed on glow-discharged holey carbon grids (Quantifoil Cu R1.2/1.3). The grids were blotted for 3.5 s and flash-frozen in liquid ethane cooled by liquid nitrogen with Vitrobot (Mark IV, Thermo Fisher Scientific). The prepared grids were transferred to a Titan Krios operating at 300 kV equipped with Cs corrector, Gatan K2 Summit detector and GIF Quantum energy filter. A total of 5281 movie stacks were automatically collected using AutoEMation36 (link) with a slit width of 20 eV on the energy filter and a preset defocus range from –1.8 to –1.5 µm in super-resolution mode at a nominal magnification of ×105,000. Each stack was exposed for 5.6 s with an exposure time of 0.175 s/frame, resulting in a total of 32 frames/stack. The total dose rate was ~48 e–/Å2 for each stack. The stacks were motion corrected with MotionCor237 (link) and binned 2-fold, resulting in a pixel size of 1.091 Å. Meanwhile, dose weighting38 (link) was performed. The defocus values were estimated with Gctf39 (link).
5
Cryo-EM Structural Analysis of PchE Protein
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Four-microliter aliquots of specimens at ~4 mg/mL were applied to glow-discharged holey carbon grids (Quantifoil Cu, R1.2/1.3, 200 mesh) for 6 s of incubation, blotted for 2.5 s and plunge-frozen into liquid ethane precooled by liquid nitrogen using a Vitrobot Mark IV (FEI) operated at approximately 100% humidity and 4.5 °C. Cryo-EM images were collected with a Titan Krios electron microscope (FEI) operated at 300 kV and equipped with a K2 Summit direct electron detector (Gatan). Thirty-eight frames were recorded for each movie stack at a nominal magnification of 22500-fold in super-resolution mode with a pixel size of 1.0 Å within the defocus range of 1.5 to 2.5 µm. A total of 3749 movie stacks of PchE were automatically collected using SerialEM29 with an exposure time of 7.6 s (0.2 s per frame) and a total dose of 60.8 e-/Å2.
6
Cryo-EM Sample Preparation for Ku-PAXX Complex
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Proteins were concentrated using a centricon (Amicon) with a 10-kDa cutoff and buffer exchanged in 20 mM Hepes (pH 7.6), 200 mM NaCl, 0.5 mM EDTA, 2 mM MgCl2, and 5 mM dithiothreitol (DTT). Ku was mixed with a 15-bp duplex DNA containing a 5′ 15-bp overhang and PAXX in a 1:1.2:2 ratio. The complex was then incubated with methyl–polyethylene glycol (PEG4)–N-hydroxysuccinimide (Thermo Fisher Scientific) at a final concentration of 2 mM to reduce the impact of particle orientation bias and incubated for 2 hours at room temperature. The reaction was quenched by adding 0.1 (v/v) of 1 M tris-HCl (pH 8.0).
Aliquots of 3 μl of ~1.7 mg/ml were applied to Holey carbon grids (Quantifoil Cu R1.2/1.3, 300 mesh), glow-discharged for 60 s at a current of 25 mA in PELCO easiGlow (Ted Pella Inc.). The grids were then blotted with filter paper once to remove any excess sample and plunge-frozen, (blotting force of −5 and blotting time of 3 s) in liquid ethane using a FEI Vitrobot Mark IV (Thermo Fisher Scientific) at 4°C and 95% humidity.
Aliquots of 3 μl of ~1.7 mg/ml were applied to Holey carbon grids (Quantifoil Cu R1.2/1.3, 300 mesh), glow-discharged for 60 s at a current of 25 mA in PELCO easiGlow (Ted Pella Inc.). The grids were then blotted with filter paper once to remove any excess sample and plunge-frozen, (blotting force of −5 and blotting time of 3 s) in liquid ethane using a FEI Vitrobot Mark IV (Thermo Fisher Scientific) at 4°C and 95% humidity.
7
AdeJ-Era Complex Formation and Ribosome Interaction
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For the AdeJ-Era sample, a 10-μM AdeJ-nanodisc sample was incubated with 20 μM Era (eravacycline dihydrochloride; MedChemExpress) for 1 h to form the AdeJ-Era complex. The sample was applied to glow-discharged holey carbon grids (Quantifoil Cu R1.2/1.3, 300 mesh), blotted for 5 s, and then plunge-frozen in liquid ethane using a Vitrobot (Thermo Fisher). The grids were then transferred into cartridges. The ribosome sample was prepared by incubation of 100 nM A. baumannii ribosome with 50 μM Era for 1 h prior to plunge freezing (15 s blot) on graphene oxide-coated Quantifoil R1.2/1.3 grids. The grids were then transferred into cartridges prior to data collection.
8
Cryo-EM of E. coli Membrane and Lysate
9
Budsome Morphology via TEM and Cryo-EM
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Transmission electron microscopy (TEM) was used to observe the morphology of budsomes. Budsome solution (0.1 mg/mL, budesonide-equivalent) was dropped onto a 300-mesh copper grid coated with carbon. After 2 min of deposition, the surface liquid was removed with filter paper and the deposit was subsequently stained with 2 wt% aqueous uranyl acetate solution for 1 min. Budsome morphology was observed using a Tecnai G2 spirit (Thermo FEI) at an acceleration voltage of 120 kV (Huang et al., 2021 ).
Budsome morphology was also characterized using cryo-electron microscopy (Cryo-EM) at the Center of Cryo-Electron Microscopy, Zhejiang University School of Medicine. An aliquot of 2.5 μL of the budsome solution (1.5 mg/mL, budesonide-equivalent) was deposited on a glow-discharged holey carbon grid (Quantifoil Cu R1.2/1.3, 300 mesh), blotted for 5 s, and plunge-frozen in liquid ethane using a Vitrobot Mark IV (ThermoFisher Scientific)(Grassucci et al., 2007 (link)). Images were recorded with a Talos F200C equipped with a Ceta 4k × 4k camera.
Budsome morphology was also characterized using cryo-electron microscopy (Cryo-EM) at the Center of Cryo-Electron Microscopy, Zhejiang University School of Medicine. An aliquot of 2.5 μL of the budsome solution (1.5 mg/mL, budesonide-equivalent) was deposited on a glow-discharged holey carbon grid (Quantifoil Cu R1.2/1.3, 300 mesh), blotted for 5 s, and plunge-frozen in liquid ethane using a Vitrobot Mark IV (ThermoFisher Scientific)(Grassucci et al., 2007 (link)). Images were recorded with a Talos F200C equipped with a Ceta 4k × 4k camera.
10
Cryo-EM Analysis of LovBC Complex
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Immediately prior to specimen preparation, Tween 20 (10%) was added to the freshly purified LovBC complex to a final concentration of 0.1%, which improved the quality of vitreous ice in the specimen. Four microliter aliquots of specimen at ~8 mg/ml were applied to glow-discharged holey carbon grids (Quantifoil Cu, R1.2/1.3, 200 mesh) for 60 s of incubation and then blotted for 2.5 s and plunge-frozen into liquid ethane precooled by liquid nitrogen using a Vitrobot Mark IV (FEI) operated at approximately 100% humidity and 22 °C. Cryo-EM images were collected with a Titan Krios electron microscope (FEI) operated at 300 kV and equipped with a K2 Summit direct electron detector (Gatan). Thirty-eight frames were recorded for each movie stack at a nominal magnification of 22,500-fold in super resolution mode with a pixel size of 1.0 Å in the defocus range of 1.5–2.5 µm. A total of 8136 movie stacks of the LovBC complex were automatically collected using SerialEM20 (link) with an exposure time of 7.6 s (0.2 s per frame) and a total dose of 60.8 e−/Å2.
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