9 μM purified PFKL was combined with 1 mM ATP, 2 mM F6P, and 50 μM NA-11 in 20 mM HEPES pH 7.5, 1 mM DTT, 500 μM ammonium sulfate, 5% glycerol, and 100 μM EDTA. The PFKL sample was applied to glow-discharged CFLAT 2/2 holey-carbon grids (Protochips) and blotted away 4 times successively, before being plunged into liquid ethane using a Vitrobot (ThermoFisher). Data collection was performed on a Titan Krios (ThermoFisher) equipped with a Quantum GIF energy filter (Gatan Inc.) operating in zero-loss mode with a 20 eV slit width. Movies were acquired on a K-2 Summit Direct Detect camera (Gatan Inc.), operating in super-resolution mode with a pixel size of 0.525 Å/pixel, with 50 frames and a total dose of 90 electrons/Å2. Images were collected at both 0° and 30° tilt. Data collection was automated using Leginon software (Suloway et al., 2005 (link)). Data collection and refinement statistics are listed in Table S2 .
K 2 summit direct detect camera
Manufactured by Ametek
Sourced in United States
The K-2 Summit Direct Detect camera is a lab equipment product designed for scientific and industrial applications. It provides direct detection of various samples and materials. The camera's core function is to capture high-quality images and data for analysis and research purposes.
Automatically generated - may contain errors
Lab products found in correlation
Vitrobot, by Thermo Fisher Scientific (4 mentions)
Titan krios, by Thermo Fisher Scientific (3 mentions)
Cflat 2 2 holey carbon grids, by Protochips (3 mentions)
Gif quantum energy filter, by Ametek (2 mentions)
Fei tecnai g2 f20, by Thermo Fisher Scientific (1 mentions)
Glacios, by Thermo Fisher Scientific (1 mentions)
4 protocols using k 2 summit direct detect camera
1
Cryo-EM Structural Analysis of PFKL
2
Cryo-EM Structural Analysis of PFKL
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9 μM purified PFKL was combined with 1 mM ATP, 2 mM F6P, and 50 μM NA-11 in 20 mM HEPES pH 7.5, 1 mM DTT, 500 μM ammonium sulfate, 5% glycerol, and 100 μM EDTA. The PFKL sample was applied to glow-discharged CFLAT 2/2 holey-carbon grids (Protochips) and blotted away 4 times successively, before being plunged into liquid ethane using a Vitrobot (ThermoFisher). Data collection was performed on a Titan Krios (ThermoFisher) equipped with a Quantum GIF energy filter (Gatan Inc.) operating in zero-loss mode with a 20 eV slit width. Movies were acquired on a K-2 Summit Direct Detect camera (Gatan Inc.), operating in super-resolution mode with a pixel size of 0.525 Å/pixel, with 50 frames and a total dose of 90 electrons/Å2. Images were collected at both 0° and 30° tilt. Data collection was automated using Leginon software (Suloway et al., 2005 (link)). Data collection and refinement statistics are listed in Table S2 .
3
Cryo-EM Imaging of Red Blood Cell-Derived Extracellular Vesicles
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RBC-EV sample preparation was conducted based on a previously described protocol [65 (link)]. Briefly, RBC-EV samples were pre-incubated with antibodies against CD63 (556,019, BD Biosciences, USA) overnight at 4 °C, followed by incubation with 10-nm gold conjugated goat anti-mouse IgG antibody (abcam) for 60 min at room temperature. Then, samples were applied to glow-discharged holey grids, following with loading and blotting of 10 μl RBC-EV samples for 5 min (repeated 3 times). Grids with EV samples were plunged in liquid ethane using a Vitrobot (FEI) and maintained in liquid nitrogen until imaging. Images were acquired and collected on a FEI Tecnai G2 F20 (FEI Co, USA) at 200 kV Cryo-S/TEM equipped with a Gatan K-2 Summit Direct Detect camera. Leginon software was used to automate data collection [90 (link)].
4
Cryo-EM Sample Preparation for CTPS
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To prepare samples for cryo-EM, CTPS was applied to glow-discharged CFLAT 2/2 holey-carbon grids (Protochips) and blotted away four times successively, before being plunged into liquid ethane using a Vitrobot (ThermoFisher). Sample conditions for CTP-bound CTPS2 were as described previously (10 (link)). Conditions for CTP-bound CTPS1 were 5 mM CTP and 10 mM MgCl2 in 20 mM Tris ⋅ HCl pH 7.9, 100 mM NaCl, and 0.5 mM DTT. Conditions for human and mouse CTPS bound to small molecule inhibitors were 5 μM CTPS, 2 mM UTP, 7.5 mM glutamine, 10 mM MgCl2, and 50 μM R80 or T35 in 20 mM Tris ⋅ HCl pH 7.9, 100 mM NaCl, and 0.5 mM DTT. Data for CTP-bound CTPS1 was collected on a Glacios (ThermoFisher). Data for all other structures was collected on a Titan Krios (ThermoFisher) equipped with a Quantum Gatan Imaging Filter energy filter (Gatan Inc.) operating in zero-loss mode with a 20-eV slit width. Both microscopes were equipped with a K-2 Summit Direct Detect camera (Gatan Inc.). On the Glacios, movies were collected in counted mode with a pixel size of 1.16 Å/pixel, with 50 frames and a total dose of 65 electrons/ Å2. On the Titan Krios, movies were collected in superresolution mode with a pixel size of 0.525 Å/pixel, with 50 frames and a total dose of 90 electrons/ Å2. Data collection was automated using Leginon (61 (link)).
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