Purified 25.10C-pfGP-TD trimer was diluted to a working concentration of 0.8 mg/mL in a buffer containing 50 mM Tris pH 7.5 and 150 mM NaCl. Then, 3 μL protein was mixed with 1 μL LMNG detergent (0.02 mM), and 3 μL of this mixture was adsorbed on a Quantifoil 2/1 grid (QUANTIFOIL) that was previously plasma-cleaned for 30 s in a NanoClean Model 1070 (Fischione Instruments) with a ratio of 25% oxygen to 75% argon. Excess protein solution was blotted for 10 s before immediate plunge freezing into liquid ethane using an FEI Vitrobot (Thermo Fisher Scientific). Frozen grids were imaged using a Titan Krios (Thermo Fisher Scientific) equipped with a Gatan K2 detector and operated in super-resolution mode. Movies were collected using SerialEM (Mastronarde, 2005 (link)) at a magnification of 463,003× at the detector that corresponds to a calibrated pixel size of 0.548 Å/pixel. Micrographs were collected in a single session with a defocus range between 1.0 and 2.5 micron underfocus.
2 1 grid
Manufactured by Quantifoil
The Quantifoil 2/1 grid is a laboratory equipment used for specimen support in electron microscopy. It features a uniform array of holes with a diameter of 2 micrometers and a hole spacing of 1 micrometer, forming a regular grid pattern.
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2 protocols using 2 1 grid
1
Cryo-EM Structural Analysis of 25.10C-pfGP-TD Trimer
2
Cryo-EM Structural Analysis of 25.10C-pfGP-TD Trimer
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Purified 25.10C-pfGP-TD trimer was diluted to a working concentration of 0.8 mg/mL in a buffer containing 50 mM Tris pH 7.5 and 150 mM NaCl. Then, 3 μL protein was mixed with 1 μL LMNG detergent (0.02 mM), and 3 μL of this mixture was adsorbed on a Quantifoil 2/1 grid (QUANTIFOIL) that was previously plasma-cleaned for 30 s in a NanoClean Model 1070 (Fischione Instruments) with a ratio of 25% oxygen to 75% argon. Excess protein solution was blotted for 10 s before immediate plunge freezing into liquid ethane using an FEI Vitrobot (Thermo Fisher Scientific). Frozen grids were imaged using a Titan Krios (Thermo Fisher Scientific) equipped with a Gatan K2 detector and operated in super-resolution mode. Movies were collected using SerialEM (Mastronarde, 2005 (link)) at a magnification of 463,003× at the detector that corresponds to a calibrated pixel size of 0.548 Å/pixel. Micrographs were collected in a single session with a defocus range between 1.0 and 2.5 micron underfocus.
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