Subvolumes were extracted from 2× binned data in 100 × 100 × 100 px uniformly distributed along the length of each tube, spaced by 17 px (fig. S2A). Initial Euler angles were assigned on the basis of the centroid position of each volume relative to the tube axis [“backbone” as defined in Dynamo (57)]. Initial averaging was performed via six-dimensional search (16 iterations), applying no symmetry. The resulting average map of the tube segment was then used to determine the positions of CA hexamers throughout the length of each tube. Subvolumes of 64 × 64 × 64 px centered on these positions were then reextracted from the 2× binned tomograms and assigned initial Euler angles in reference to the tube axis. An initial hexamer search template was generated by averaging the subvolumes using only azimuthal refinement, applying sixfold symmetry. Subtomogram averaging was then performed separately for each tube, applying twofold symmetry, a low-pass filter of 30 Å, and default masks in Dynamo. Upon convergence, lattice maps were generated as described (58) and visually examined. Particles that were clearly misaligned and/or had very low cross-correlation values were discarded. After another round of averaging and examination, 9684 subvolumes were extracted from unbinned tomograms (128 × 128 × 128 px) and split into even/odd subsets. The two subsets were treated independently from this point forward. For each subset, an initial template was generated by averaging all particles according to the Euler angles and positions determined from the previous refinement. Four iterations of refinement were performed, applying a soft-edged spherical mask of 35-px radius and progressively narrower angular and positional search ranges. The final map was calculated with a low-pass filter of 20 Å.

Note: The content above has been extracted from a research article, so it may not display correctly.



Q&A
Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.



We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.