The presence of the structure αK40 loops 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.

Cryo-EM data collection refinement

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

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) Inter-PF rotation angles for each PF across all three strains (WT, K40R, MEC17-KO). (B-G) Comparison of inter-PF rotation angle change between A3 and A4, showing minor changes. (H-M) Comparison of inter-PF 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.