Extended Data Fig. 2: Cryo-EM image processing and map resolution and quality for CCP5 focused refinement.
From: Tubulin code eraser CCP5 binds branch glutamates by substrate deformation
a. Raw micrograph of CCP5 decorated microtubules. b. Initial image processing was performed in RELION. 4643 movies were motion corrected and summed; high-quality micrographs were selected based on CTF estimation and visual inspection for manual picking of microtubule segments. A microtubule RELION-based pipeline (MiRP) was used to determine protofilament number, microtubule polarity, identify seam position and generate a C1 reconstruction. CCP5, gold, binds every 80 Å along the microtubule lattice, grey. Protofilament refinement with a custom mask that covered one protofilament yielded a map of higher quality for tubulin, but not CCP5. c. Focused classification of CCP5 generated three classes with good definition. Refinement of these three classes resulted in maps with resolutions of 3.1 Å (class #1), 3.4 Å (class #2) and 3.6 Å (class #3). See Methods for additional details. d. Fourier shell correlation (FSC) curves for the single protofilament reconstruction and three CCP5 focused reconstructions indicate their nominal resolution. FSC estimates were calculated at the 0.143 criterion. e. Microtubule reconstruction using the single protofilament refinement protocol color-coded by local resolution. f. CCP5 focused reconstructions color-coded by local resolution for class #1, #2 and #3. g, h. Cryo-EM maps showing fit of the CCP5 X-ray apo model (g) and the local map quality (h) for classes #1, #2 and #3.
