Samples for cryo-EM were prepared by applying 4 μl of purified TRPA1 to 1.2/1.3 holey carbon grids (Quantifoil) and blotting for 8-12 sec. in a Vitrobot Mark IV (Thermo Fisher) prior to plunge freezing in liquid ethane. For multi-shot imaging, samples were prepared on 2/2 holey carbon grids (Quantifoil) and blotted for 6-8 s. Cryo-EM samples were imaged on Polara and Titan Krios microscopes (Thermo Fisher, see Extended Data Table 1 for details) equipped with the K2 Summit camera (Gatan). Movies were drift-corrected using MotionCor234 (link) and CTF parameters estimated with gctf35 (link). Particle images were selected from micrographs using gautomatch (MRC-LMB, https://www.mrc-lmb.cam.ac.uk/kzhang/ ) and extracted in Relion36 (link). 2- and 3-D classification of particle images was performed in cryoSPARC37 (link) and 3D maps refined in cryoSPARC and cisTEM38 (link). Conversion of data from cryoSPARC to Relion and generation of orientation distribution plots were performed using pyem39 (link). Directional Fourier shell correlations of cryo-EM maps were performed as previously described40 (link).
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Cryo-EM Imaging of TRPA1 Protein
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Other organizations : Howard Hughes Medical Institute, University of California, San Francisco
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- Preparation of cryo-EM samples by applying 4 μl of purified TRPA1 to 1.2/1.3 holey carbon grids (Quantifoil) and blotting for 8-12 sec. in a Vitrobot Mark IV (Thermo Fisher) prior to plunge freezing in liquid ethane
- Preparation of cryo-EM samples on 2/2 holey carbon grids (Quantifoil) and blotting for 6-8 s. for multi-shot imaging
- Cryo-EM imaging on Polara and Titan Krios microscopes (Thermo Fisher) equipped with the K2 Summit camera (Gatan)
- Drift-correction of movies using MotionCor2
- CTF parameter estimation using gctf
- Particle image selection from micrographs using gautomatch
- Particle image extraction using Relion
- 2- and 3-D classification of particle images using cryoSPARC
- 3D map refinement using cryoSPARC and cisTEM
- Conversion of data from cryoSPARC to Relion and generation of orientation distribution plots using pyem
- Directional Fourier shell correlations of cryo-EM maps
- Not explicitly mentioned
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