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Protocol publication

[…] O2). Frozen-hydrated grids were prepared using a Vitrobot Mark IV (FEI, Hillsboro, OR, USA). 3 μl of sample (5 mg/ml GluA2-GSG1L with or without 0.3 mM antagonist ZK200775) was applied to the plasma-treated grids using 3.0 s blotting time, 3 blot force, 30.0 s wait time and 100% humidity at 22 ˚C. For the open state, 4 mg/ml GluA2-STZ was incubated for 15 minutes with 100 μM CTZ, then 3 μl of this was quickly added and pipetted up-and-down in a mixture of 0.5 μl 700 mM glutamate (final concentration 100 mM). 3 μl of this mixture was added to the grid, blotted as described above., The GluA2-GSG1L data (apo/ZK) were collected on a Tecnai F30 Polara (FEI) microscope operating at 300 kV, using Leginon with a Gatan K2 Summit electron detection (DED) camera (Gatan, Pleasanton, CA, USA) in counting mode with a pixel size of 0.98 Å. Data were collected across 40 frames (0.2 s per frame), with a dose rate of 8 e− pixel−1 s−1 (total dose of ~67 e− Å−2), within defocus range −1.5 μm to −3.5 μm, Cs 2.26 mm. The GluA2-STZ data were collected on a Titan Krios microscope (FEI) operating at 300 kV, using Leginon with a post-GIF quantum energy filter (20 eV slit) and Gatan K2 Summit direct camera DED camera in counting mode operating at a pixel size of 1.08 Å/pixel. A dose rate of 8 e− physical pixel−1 s−1 (total dose of ~55 e− Å2) was used across 40 frames (0.2 ms per frame), within the defocus range −1.0 μm to −3.0 μm., All frame alignment and dose-weighting was carried out in MotionCor2. CTF correction, with gCTF, was performed on non-dose-weighted micrographs, with all further processing on dose-weighted micrographs using RELION 2.0. From 2,938 micrographs for GluA2-GSG1L bound to ZK, 541,261 particles were picked. The data was binned to 3.92 Å/pixel and subject to 2D classification, which resulted in cleaning the particle pool to 513,406 particles. These particles were then subject to 3D classification without symmetry into ten classes and the GluA2-2xGSG1LZK model low-pass filtered to 40 Å as a reference. Three classes, with two showing different ECD conformations (i.e., state 1 and state 2) and two auxiliary subunits bound were chosen for additional 3D sub-classification. This resulted in a group of 234,426 particles, which were unbinned to a pixel size of 1.96, and subject to classification with the GluA2 […]

Pipeline specifications

Software tools Leginon, MotionCor2, Gctf, RELION
Diseases Disease, Nervous System Diseases
Chemicals alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid, Glutamic Acid