The presence of the structure αK40 loop in Tetrahymena thermophila DMT.

(A) Surface rendering of the DMT viewed from the tip of the cilia, with MIPs colored in the 48-nm repeat cryo-EM electron density map of Tetrahymena. (B) Relative location of the αK40 loop (dashed black line) and the lateral contacts of tubulins. Color: α-tubulin, green; β-tubulin: blue. (C-F) Cryo-EM map and models of the fully structured αK40 loops in PF A3 (C-D) and the fully structured (E) and partially structured (F) αK40 loops in PF B10. The red arrows point to the location of the αK40 loops. (G) Bar graph showing the composition of visible full (missing no more than two residues from residues 37 to 48) and partial loops (missing 3-5 residues) in both the A- and B-tubules. (H) 48-nm repeat surface rendering of selected PFs with MIPs that interact with visible full and partial αK40 loops colored in red, indicating that αK40 loops are structured in regions with many MIPs.

Comparison of αK40 loop conformation.

(A) Superimposed view of all the orientations of all the visible full and partial αK40 loops, showing their orientation. (B) Interaction of K40 loop and DM10 domains from RIB72A (3 domains, blue), RIB72B (3 domains, purple) and CFAP67 (1 domain, cyan). Black box represents the view in (C). (C) Zoom in view of DM10 domains and K40 loop interaction. Asterisk (*) denotes the conserved aromatic residue potentially interact with the K40 loop. (D) Alternative view of DM10 domains interacting with K40 loop. (E) Multiple sequence alignment of DM10 domains from RIB72A, RIB72B and CFAP67. (F) Cryo-EM map (left) and model (right) of the inner junction region of Tetrahymena to show the interaction of the full αK40 loop with CFAP52. (G) Cryo-EM map (left) and model (right) of the inner junction region of Chlamydomonas to show the interaction of the full αK40 loop with CFAP52. (H) Superimposed view of the Chlamydomonas αK40 loop (gray) onto the Tetrahymena αK40 loop of B10.

Comparison of DMT structures from WT, MEC17-KO and K40R mutants

(A) Comparison of the cryo-EM density maps of the DMT from WT, K40R, and MEC17-KO strains of Tetrahymena to show that the MIPs are intact in all three species. (B-G). Models of the full αK40 loops in PF A1 (B-D) and PF A4 (E-G) from WT, K40R, and MEC17-KO strains. (H-I) Models of the αK40 loops from A1 (H) and A4 (I) superimposed from WT, K40R, and MEC17-KO species.

Deacetylation affects the inter-PF angles in the DMT.

(A) Rotation angles for each PF across all three strains (WT, K40R, MEC17-KO). (B-D) Comparison of rotation angle change between A1 and A2, showing minor changes. (E-G) Comparison of rotation angle change between A3 and A4, showing minimal changes. Red arrows point to the changes in αK40 loop shape between each species. (H-J) Comparison of rotation angle change between B9 and B10, showing significant changes.

Molecular dynamic simulations of the acetylated and non-acetylated αK40 loops.

(A) All-atom simulations of αK40 loop clusters in different conformations of acetylated (pink) and base/non-acetylated (blue) indicate that acetylated conformations adopt higher frames and are less flexible. (B) RMSD and probability of each cluster simulated in A. (C) Molecular dynamics coarse grain model of the inner junction region of Tetrahymena; each amino acid is 1 bead. (D) Graph showing the energy difference (in kcal/mol) between base (non-acetylated) and acetylated αK40 to show that each acetylated αK40 has slightly lower energy than the non-acetylated αK40.

Mass spectrometry of WT, K40R and MEC17-KO mutants.

(A) Bar graph showing the abundance of MIPs based upon quantitative values (normalized total spectra) from mass spectrometry. Asterisk (*) indicates a significant difference with p < 0.05. (B) Proteins upregulated in RIB72A/B, K40R, and MEC17-KO mutants compared with the WT. (C) Proteins downregulated in RIB72A/B, K40R, and MEC17-KO mutants compared to the WT. (D) Proteins only found in the mass spectrometry of WT when compared with K40R and MEC17-KO mutants. (E) Downregulated proteins in both K40R and MEC17-KO mutants compared to WT. (F) F. Proteins in both K40R and MEC17-KO mutants but are absent in WT. (G) Upregulated proteins in both K40R and MEC17-KO mutants compared to WT.

Models of acetylation contribution in the DMT

In the case of more MIPs, such as in the A-tubule, the MIP-αK40 interaction dominates the contribution to the lateral interaction; therefore, deacetylation does not affect the structures significantly. With fewer MIPs, such as in the B-tubule, acetylation contribution to the lateral interaction becomes significant and therefore can contribute to the stabilization of the tubulin lattice.

Cryo-EM data collection refinement

Refinement statistics of WT, K40R and MEC17 48nm models.

Purification and structure determination of DMT.

(A) Purification scheme of the axoneme in this study. (B) A typical cryo-EM image of the Tetrahymena DMT. (C) Gold-standard Fourier shell correlation of the 48-nm repeat cryo-EM maps of the WT, K40R, and MEC17-KO Tetrahymena strains.

Diverse structures of the αK40 loops.

Cryo-EM maps and models of partial and full αK40 loops in A2 (A-C), A10 (D-F), B1 (G-I), from WT, K40R, and MEC17-KO Tetrahymena strains.

Interdimer distance within the same PF measured from cryo-EM density maps of WT, K40R, and MEC17-KO Tetrahymena species.

Mass spectrometry analysis of WT, K40R and MEC17-KO

(A) Radial spoke protein abundance in mass spectrometry triplicates of WT, K40R, and MEC17-KO Tetrahymena species. Asterisk (*) indicates a significant difference with p < 0.05. (B) Volcano plot showing up- and downregulated proteins in K40R mutants compared against WT, y-axis showing the −Log10 p values, and x-axis showing the Log2-fold change where negative and positive values suggest potential downregulation and upregulation, respectively. (C) Volcano plot showing up- and downregulated proteins in MEC17-KO mutants compared to WT.

Count of full, partial, and missing αK40 loops based upon the K40R cryo-EM density maps, along with possible MIP interactions for αK40 loops in each PF.

Inter-PF angles between subsequent PF obtained from cryo-EM maps of WT, K40R, and MEC17-KO Tetrahymena cilia.

ANOVA test p values and significance of the interdimer distances of tubulin subunits within each PF in WT, K40R, and MEC17-KO Tetrahymena cilia.

Proteins downregulated at least twofold in the K40R mutant compared to the WT.

Proteins upregulated at least twofold in the K40R mutant compared to WT

Proteins downregulated at least twofold in the MEC17-KO mutant compared to the WT

Proteins upregulated least twofold in the MEC17-KO mutant compared to the WT