Figure - PMC

Skip to main content

An official website of the United States government

Here's how you know

Here's how you know

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

View full-text article in PMC

. 2024 Apr 12;31(7):1134–1144. doi: 10.1038/s41594-024-01281-y

Search in PMC
Search in PubMed
View in NLM Catalog
Add to search

© The Author(s) 2024

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

PMC Copyright notice

Extended Data Fig. 1 — a, (left) Representative electron micrograph at 13,000x magnification of the enriched SPB sample on lacey grids. Similar SPB structures were observed in more than 500 independent tomograms. (right) Slice through a tomogram with red circles indicating centre of the dipole geometry used to pick γTuRCs, the arrows define the z-axis. Blue circles indicate positions along which microtubules were picked as a filament, the z-axis is pointed away from the SPB. Representative of more than 500 tomograms. b, Custom data processing pipeline, also outlined in the Methods section. Tilt-series (TS) were preprocessed in Warp and aligned using dautoalign4warp (Dynamo). Bin8 tomograms were generated in Warp and particle picking was performed in Dynamo, with γTuRC picked as dipoles and MTs picked as filaments. Sub-tomogram volumes were extracted in Warp and refined using RELION. Both γTuRC and MT particles were co-refined in M to give a final consensus refinement map. To identify and correct particle misalignments, a manual procedure (the Fixalign procedure) was developed: γTuRC particles were classified without alignment to identify classes where the seam of the γTuRC 3D class (full black line) was misaligned with respect to the seam of the initial γTuRC reference (dotted line). Misalignment was corrected by manually realigning 3D class averages in ChimeraX and applying the corresponding transformations to the respective particle stacks of the misaligned 3D class averages (arrow from full black line to dotted line). The Fixalign procedure improved density of γTuSCs-1 and γTuSCs-7 substantially (see also panel d). Next, A custom-built Napari sub-boxer plugin was used to sub-box and mask γTuRC particles into individual γTuSCs with two rows of α/β-tubulin to expand the dataset and improve the resolution. Sub-boxed γTuSCs were subjected to 3D refinement in RELION 3.1. WBP: weighted back projection. c, Fourier-shell correlation (FSC) curves for the reconstructions used in this study. d, Bottom view of the γTuRC before and after manually correcting misalignment of the γTuRC helical register (Fixalign). Prior to manual realignment the density for γTuSC-1 and γTuSC-7 was diffuse, but improved substantially after realignment and further 3D refinements.