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Pipeline publication

[…] MDB: 2990; ref. ) was filtered to 60 Å and used as initial reference for 3D classification. At every step of classification, the data were grouped into two volumes (Supplementary Fig. ). After three rounds of successive 3D classification with K = 2, a stable class with 16,190 particles yielded a well-defined volume (Supplementary Fig. ). The second data set (from K2 Summit) consisting of 71,519 particles was sorted according to the same protocol and gave rise to additional 10,663 particles. The two data sets were combined after rescaling the second data set to a pixel size of 1.38 Å. The volume was further refined with a molecular shape mask (5 pixel Gaussian fall-off) and post-processed in Relion with C2 symmetry imposed. The resolution of the final volume was determined to be 7.9 Å based on the Fourier shell correlation (FSC) = 0.143 criterion (Supplementary Fig. ). During post-processing, the volume was corrected for the modulation transfer function of the detector and the effect of mask was checked by phase randomization. The final map was sharpened with a B-factor value of −130 Å2. The local resolution analysis of the map was carried out using the program ResMap., The final, combined set of 26,853 particles was used for 3D classification without any alignment to two classes based on the molecular shape mask covering only the Lst8 and Avo1 part of the map. The classification converged with one volume populated with 94.6% of the particles and a featureless second volume from the residual data. Three-dimensional auto refinement using the 94.6% particles of first volume did not yield an improved map. Second, focused 3D refinement alone was performed with the mask described above. This also did not improve the density in this part of the map. This may be because the region of interest is very small compared to the total volume of the map and has a […]

Pipeline specifications

Software tools RELION, FSC, ResMap