Computational protocol: Reconstitution of the complete pathway of ITS2 processing at the pre-ribosome

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Protocol publication

[…] Negative staining, data collection and processing were performed as described previously. In brief, 5 µL of the sample was placed on a freshly glow-discharged, carbon-coated grid, allowed to absorb onto the carbon, washed three times with water, stained with uranyl acetate (2% w/v) and dried. Micrographs were recorded using an electron microscope (Tecnai F20, FEI) operating at 200 kV with a bottom-mounted 4 K, high-sensitivity charge-coupled device camera (Eagle, FEI) at a nominal magnification of 29000 (calibrated pixel size of 3.81 Å). For averaging, 3908 particles for the TAPF-Nop53 + ATP (Mock) sample, 4414 particles for the TAPF-Nop53 + Rrp6-FTpA + Mtr4 + ATP sample, and 3870 particles for the TAPF-Nop53 + Rrp6-FTpA + Mtr4 K176A + ATP sample were selected using the auto-boxing feature of EMAN2. Image processing was carried out using the IMAGIC-4D package. Particles were band-pass filtered and normalised in their grey value distribution, and mass-centred. Two-dimensional alignment, classification and iterative refinement of class averages were performed as described previously. […]

Pipeline specifications

Software tools EMAN, IMAGIC
Application cryo-EM