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Pipeline publication

[…] ive rounds of mode 3 (global search) alignment included data in the resolution range from 30 Å to 300 Å. Next, the 4×-binned image stack was aligned against the common reference using mode 1 (local refinement), including data up to a high-resolution limit of 12 Å (20 Å in case of 30SΔS1•RNAP)., The refined parameters were then used for classification and refinement of the 2x-binned stack into 8 classes in 40 rounds, using resolutions from 12- to 300 Å, to separate the RNAP-containing classes. This step was done with three-dimensional (3D) masks that included RNAP with or without S1. In both cases, the resulting 30S•RNAP classes contained prominent S1 density. The 3D masks were created using IMOD and EMAN2 () by generating a density map, low-pass filtered to 20 Å, from our initial atomic models of RNAP with or without S1 (RNAP mask was used for 30SΔS1•RNAP data). The mask was applied to reference volumes in FREALIGN, with the volume outside of the mask weighted by a factor of 1.0 (). A five-pixel cosine edge was used on the mask and the masking filter function. This classification for the 30S•RNAP data set revealed 5 classes containing 30S and RNAP and 2 classes of free 30S subunit. For the classes bound with RNAP, particles with >50% occupancy were extracted from the 2×-binned stack. The new stack was sub-classified and refined into 4 classes in 60 rounds with the 3D mask, including RNAP and S1 (outside of the mask was down-weighted by a factor of 0.1) to resolve the RNAP, using data between 12- and 300 Å resolution. This classification resulted in 6.7- and 7.9 Å resolution maps (FSC = 0.14 […]

Pipeline specifications

Software tools IMOD, FSC, Frealign