Figure 3. Structure of the DNAH7 MTBD.

(A) Partial sequence alignment of human cytoplasmic dynein-1 (DYHC1) and human Axonemal Dynein 7 (DNAH7). DNAH7 has a 13-residue insert between H2 and H3 compared to cytoplasmic dynein called the flap. Full MTBD sequence alignment in Figure 3—figure supplement 1 (B) A model of DNAH7 (violet) was refined into the SRS⁺-DNAH7_2758-2896 cryo-EM density (grey, lowpass filtered to 5 Å). (C) A comparison between the microtubule-bound DNAH7 model (violet) and the low-affinity Flagellar dynein C NMR structure (Kato et al., 2014, 2RR7, ensemble chain 8, docked to the same density, white). As with cytoplasmic dynein, the only major conformational changes between the two is the upward movement of CC1/H1 (purple and grey for DNAH7 and 2RR7 respectively) (D) The SRS⁺-DNAH7_2758-2896 cryo-EM density (MTBD in purple, α-tubulin in green, β-tubulin in blue) thresholded at a low level, revealing the extension of the flap to contact the adjacent protofilament (E) Rotation of the DNAH7 MTBD relative to cytoplasmic dynein-1. The tubulin was aligned in the cytoplasmic and axonemal dynein models. This reveals a rotation around the base of H6/CC2 towards the adjacent protofilament in DNAH7 (violet) compared to cytoplasmic dynein-1 (white). (F) Close-up view of the flap of the DNAH7 MTBD model with its corresponding density. (G) Corresponding view to F, without the electron density. (H) The DNAH7 map lowpass filtered to 10 Å reveals a new connecting density that cannot be explained by the flap (DNAH7 model docked, violet). (I) The extra density observed in H is not seen in the cytoplasmic dynein-1 density filtered to the same resolution (J) The C-terminal tail of β-tubulin could potentially explain the density in H, given its length and possible binding site in a positively charged pocket on the top surface of DNAH7 (surface coloured by coloumbic potential) (K) Cytoplasmic dynein-1 (surface coloured by coloumbic potential) does not have the same positive patch on DNAH7, suggesting that the C-terminal tail would not dock in the same way.

Figure 3—figure supplement 1. Sequence alignments of dynein microtubule-binding domains.

(A) Full sequence alignment for the MTBD of human cytoplasmic dynein-1 (DYHC1) and human DNAH7. (B) A sequence alignment of the region between H2 and H3 of the MTBDs of the human dynein heavy chains. DNAH1, 3, 5, 7 and 8 all have a 18/19 residue long flap, while all other dyneins have a much shorter loop. (C) Equivalent to B for Chlamydomonas reinhardtii axonemal dynein heavy chain sequences.

Figure 3—figure supplement 2. Assessment of the DNAH7 MTBD structure.

(A) FSC curve for the final, symmetrized (with refined operators) DNAH7 map (FSC_0.143 cut-off, gold-standard). (B) The refined DNAH7 model docked into density, at a low threshold to visualise the flap (C) A comparison of our newly refined high-affinity cytoplasmic dynein-1 and DNAH7 MTBD models. (D) MTBD density from one protofilament of the asymmetric DNAH7 final map. (E) Corresponding view to D, following symmetrization of the DNAH7 map using the regular, circular operators used for the previous structures. The decorating density is much weaker, suggesting that the averaging was incoherent (F) Corresponding view to D, following symmetrization of the DNAH7 map using local symmetry operators refined in Relion. This finds the optimal position for each mask, and as such accounts for any deviations in curvature. The density is now much strong, and contains higher quality MTBD features than the asymmetric map. (G) Binarised representation of classes B and C, aligned at a seam-adjacent protofilament. (H) Results from 3D classification of DNAH7 when larger Tau value and local refinements were used (see Materials and methods), generating classes ‘A’ and ‘B’ (indicated) (I) Class A with protofilament coordinates. An ellipse was fit to the coordinates (black line), from which the ellipticity is shown. (J) As for H, but with Class B (K) As for H, but with the refined SRS-DYNC1H1_3260-3427 map.