Computational protocol: Structural Characterization of the Mechanosensitive Channel Candidate MCA2 from Arabidopsis thaliana

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Protocol publication

[…] Purified MCA2-6H was embedded in a thin layer of vitreous ice on a thin carbon film covered on a holey carbon grid. Commercially available holey carbon grids (R1.2/1.3 grids, Quantifoil MicroTools GmbH, Jena, Germany) were covered with an amorphous carbon film (about 5-nm thickness), dried in air, and kept in a desiccator in advance. The carbon film-covered Quantifoils were hydrated by glow-discharge at low air pressure just before the preparation of specimens. A total of 2.5 µl of MCA2-6H (0.05 mg/ml) was then applied, the excess solution was blotted with filter paper at 20°C with 100% humidity, and the grid was quickly placed into liquid ethane cooled by liquid nitrogen using Vitrobot Mark IV (FEI Company Eindhoven, the Netherlands). The grid was transferred into a cryo-EM, JEM-3100FFC (JEOL Co., Ltd, Tokyo, Japan) equipped with a field emission gun operated at an acceleration of 300 kV voltage, a Zernike phase plate, and an in-column (omega-type) energy filter. The temperature of specimens was maintained at ∼55 K as reported . The Zernike phase plate was made of a vacuum-evaporated amorphous carbon film ∼27 nm thick which was optimized for 300 kV electrons . The hole in the center of the film was 0.7 µm in diameter. The phase plate was kept at a temperature above 150°C to prevent beam-induced contamination . Cryo-EM observations were performed with the Zernike phase plate inserted at the back focal plane of an objective lens to obtain in-focus Zernike phase contrast (ZPC) images. Data was collected at a nominal magnification of 60,000 in a zero-loss energy filtering mode with a 20 eV slit width at an electron dose of 20 e-/Å2. Images were recorded using the 2k×2k CCD camera, Megascan-795 (Gatan, Pleasanton, CA) with a pixel size of 0.3 nm.Image processing and 3D reconstruction were performed using the EMAN version 1.9 , IMAGIC and SPIDER software packages. A total of 8,265 particles from CCD images were selected manually and boxed using the BOXER program from the EMAN package. Particles were then centered, low-pass filtered at 6 Å, and high-pass filtered for two Fourier pixels for initial image processing. A reference-free two-dimensional (2D) classification was performed with 8,265 particles into 200 classes by refine2d.py in the EMAN software package to visualize size variations in particles picked by semi-automated particle picking using the BOXER. A 3D model was generated using the startcsym command by applying C4 symmetry, which was suggested by the chemical cross-linking analysis. The initial model for projection matching refinement was made from this model by applying a low-pass filter to 70 Å and threshold masking using SPIDER. All micrographs were then filtered using Matlab script to suppress low-frequency signals as described elsewhere . Particles were re-boxed from filtered micrographs with a box-size of 80×80 pixels. A reference-free 2D classification was performed for these particles using IMAGIC. Multivariate statistical analysis and multi-reference alignment were performed for 50, 200, 200, and 50 classes. Projection matching refinement was performed with the refine command in the EMAN package, applying C4 symmetry. Particles were classified into 133 classes corresponding to a 6-degree angular step during the iterative refinement procedure. The final 3D map was reconstructed after eight iterations. The resolution of the final map was estimated by the standard 0.5 criterion of the Fourier shell correlation between two maps made separately from two halves of the data set. The map was low-pass filtered to 20 Å and rendered into surface views with CHIMERA . […]

Pipeline specifications

Software tools EMAN, IMAGIC, SPIDER
Application cryo-EM
Organisms Arabidopsis thaliana