Purified Mla complex at ~1 mg/ml was applied to glow-discharged holey grids, blotted for 6 s, and plunged in liquid ethane using a Vitrobot (FEI). Images were acquired on a FEI Tecnai G2 F20 200 kV Cryo-TEM equipped with a Gatan K-2 Summit Direct Electron Detector camera with a pixel size of 1.26 Å/pixel. 500 micrographs were collected using Leginon (Suloway et al., 2005 (link)) spanning a defocus range of −1 to −2 µm.
Movie frames were aligned with MotionCorr2 (Zheng et al., 2017 (link)) and the defocus parameters were estimated with CTFFIND4 (49). 333 high-quality micrographs were selected by manual inspection, from which ~ 55,000 particles were picked with DOG in Appion (Lander et al., 2009 (link)). Particle stacks were generated in Appion using a box size of 200 pixels. Several successive rounds of 2D and 3D classification were performed in Relion 2 (Scheres, 2012 (link); Kimanius et al., 2016 (link)) using an initial model generated by Common Lines in EMAN2 (53) leading to a final stack of ~14,000 particles for 3D structure refinement in Relion.
Movie frames were aligned with MotionCorr2 (Zheng et al., 2017 (link)) and the defocus parameters were estimated with CTFFIND4 (49). 333 high-quality micrographs were selected by manual inspection, from which ~ 55,000 particles were picked with DOG in Appion (Lander et al., 2009 (link)). Particle stacks were generated in Appion using a box size of 200 pixels. Several successive rounds of 2D and 3D classification were performed in Relion 2 (Scheres, 2012 (link); Kimanius et al., 2016 (link)) using an initial model generated by Common Lines in EMAN2 (53) leading to a final stack of ~14,000 particles for 3D structure refinement in Relion.
Free full text: Click here
