Cryo-EM data processing
Unless stated otherwise all processing was performed with RELION (v2.1b.1)43. Movies were down sampled in Fourier space by a factor of 2 and summed after correction of beam-induced motion by MotionCor244. CTF estimation was performed by CTFFIND4.145 and micrographs with ice contamination were discarded by visual inspection of the power spectra. Initially ~1000 particles were manually picked and subjected to reference-free 2D classification to serve as templates for auto picking. A total of ~220,000 particles were extracted from summed micrographs and subjected to multiple rounds of 2D classification. A representative subset of class averages was selected for initial model generation in EMAN2.246 using the common line method. The initial model was low pass filtered to 20 Å and particles were subjected to 3D classification, giving rise to two classes of C22 and C21 symmetry. Initial refinement with C22 symmetry led to a 4.2 Å map. These initial refinements were used to create a solvent mask, which was low pass filtered to 15 Å for subsequent refinements. This final subset of 58,000 particles was selected for masked movie refinement and particle polishing with C22 symmetry, where the MTF of the detector was used to determine a b-factor of −180 Å2. High resolution features were enhanced by sharpening with this b-factor for the purposes of map visualisation. The global resolution was estimated by the Gold Standard 0.143 criterion when comparing the Fourier shell correlation between two independent half maps47. The local variation of resolution was further analysed using blocres using a search box size of 20 voxels and FSC criterion of 0.548.