Overall structure of the basal body triplet
Purified basal bodies in near-native state were visualized by cryo-ET. In most of the tomograms, two cylindrically shaped basal bodies were visible since they are tethered laterally by the distal striated fibres (Hoops et al, 1984
). The basal bodies have a reproducible size, with an average diameter of 260 nm and an overall length of 600 nm (). The MT triplets start from the proximal end (the bottom in ) and span about 400 nm longitudinally towards the distal end where the C-tubules of the triplets terminate. The A- and B-tubules continue as a doublet for about 150 nm before they reach the transitional plate, a hallmark of the transition zone where the axoneme will assemble (Cavalier-Smith, 1974
; O'Toole et al, 2003
; Geimer and Melkonian, 2004
). We did not observe the cartwheel structures in our tomograms, presumably because they were lost during purification. Electron dense structures, known as ‘A-tubule feet', are consistently visible in our tomograms along the wall of the triplets projecting towards the lumen (Cavalier-Smith, 1974
; Geimer and Melkonian, 2004
), showing characteristic 8- and 16-nm periodicity (). The region decorated with A-tubule feet starts at about 100 nm from the triplet minus (proximal) end, spans about 250 nm longitudinally and terminates at about 45 nm before the triplets become doublets. This section forms the central core of the basal body and was used for tomogram subvolume averaging in order to obtain a higher-resolution structure of the basal body.
Figure 1 Cryo-ET reconstruction of the basal body triplet. (A) A cross-section of tomographic reconstructed volume containing a basal body. The section is through the centre of the basal body barrel. The proximal end is at the bottom and the distal end is at the (more ...)
We obtained an averaged MT triplet at 33 Å resolution (; Supplementary Figure S1
). The resolution of the averaged structure is sufficiently high to allow us to discern each PF in the longitudinal projection of the triplet (). The A-tubule is a complete MT composed of 13 PFs, numbering starts clockwise from the basal body luminal side as A1 to A13 following the Tilney-Linck convention (Linck and Stephens, 2007
). PF A10 is the site where the B-tubule joins with the A-tubule. PFs A10 to A13 are referred to as partition PFs shared between A- and B-tubule. The B-tubule is composed of 10 PFs that are numbered from the outside surface of the triplet clockwise as B1 to B10. The C-tubule also has 10 PFs. PF C1 starts as a branch from PF B4 and they are numbered clockwise as C1 to C10. PFs B5 to B8 form the partition shared by the B- and C-tubules.
The A-tubule has an elliptical shape with a substantial variation of curvature along the MT wall (). The longest axis running across the internal diameter of the A-tubule is between PFs A3 and A10 with a diameter of ~247 Å. The shortest axis is ~201 Å between PFs A6 and A13. This change of diameters is equivalent to a 10% distortion of an intact 13-PF MT and is similar to the A-tubule in the axoneme doublet where ~8% distortion has been observed (Sui and Downing, 2006
). Interestingly, the variation of curvature is non-isotropic along the wall of the A-tubule. The highest curvature is at PFs A9 and A10, followed by the next-highest curvature at PFs A2 and A3, where large lateral gaps are observed between PFs. The smallest curvature is at the partition site from A11 to A13. In contrast to the A-tubule, the B- and C-tubules have a rather smooth and uniform curvature with diameters about 260 Å. The diameter and the curvature of the B- and C-tubules indicate that both would form a circular MT with 15 PFs if they formed complete rings, similar to the B-tubule in the axoneme doublet (Sui and Downing, 2006
Building a pseudo-atomic model of the triplet
In our structure, the 4-nm periodicity between individual tubulin monomers can be easily resolved along most of the PFs of the basal body (Supplementary Figure S2
). The z
-rise (longitudinal rise) of tubulins between adjacent PFs, varies from 10 to 12 Å, consistent with previous theoretical and experimental data from MTs with different PF numbers (Chrétien and Wade, 1991
; Sui and Downing, 2010
). This allowed us to fit the atomic structure of α/β tubulin into the EM density map and build a pseudo-atomic model of the tubulin core of the triplet (). Previous studies have shown that in α/β tubulin, the M-loop and H1-S2/H2-S3 loops provide the main lateral interactions between adjacent PFs and we have kept these contacts in our model (Nogales et al, 1999
; Li et al, 2002
; Sui and Downing, 2010
). The overall fit is excellent at the current resolution. In the A-tubule, due to the variation in local curvature, the closest lateral interactions are within partition PFs A11 to A13. Conversely, the interfaces between PFs A2/A3 and PFs A9/A10 have less contact due to large local curvature.
The fitting of tubulin PFs into the B- and C-tubule density resulted in uniform tubulin lateral interactions. We have modelled PF B1 as a tubulin PF that makes unusual lateral contact with the outside surface of PF A10. Since the distal half of PF C1 exhibits an 8-nm interval with a gap (), it is most likely occupied by non-tubulin protein in this region. Therefore, we did not fit tubulin monomers into the PF C1 position anywhere along its length. This longitudinal change in PF C1 will be described in detail below.
Figure 4 Longitudinal structural variations observed in the triplets. (A) Subvolumes are divided into eight groups and averaged according to their longitudinal position in the basal body. The relative positions of the group are highlighted with the yellow boxes. (more ...)
Non-tubulin components associated with the triplet
Proteomic studies have shown that, besides tubulin, the basal body contains nearly 50 non-tubulin components (Keller et al, 2005
). In order to find where they bind, how the tubules are associated to form a triplet and how the triplets are connected in the basal body, we used the pseudo-atomic model of the tubulins within the triplet as a mask to subtract its density from the 3D density map. The resulting difference map shows novel density features that must correspond to the locations of most of the non-tubulin accessory proteins ().
Figure 2 Non-tubulin densities associated with the basal body triplet. (A) A calculated difference map based on the pseudo-atomic model of the triplet. The density contributed by tubulin is coloured in blue and the non-tubulin proteins associated with the triplet (more ...)
Just as in the axoneme doublet structure (Nicastro et al, 2006
; Sui and Downing, 2006
), we also observed densities decorating on the internal wall of A- and B-tubules in the triplet, but in a more asymmetric manner. In the A- and B-tubule, there are filamentous densities running across PFs A1 to A4 and B4 to B6, respectively, with an 8-nm periodicity (). The densities follow the rise of the tubulin helical repeat inside the lumen, forming lateral cross-links between neighbouring tubulin monomers at the lumen side. Likely, these are Tektin family proteins that stabilize adjacent PFs and fine-tune local curvature of the tubule (Amos, 2008
). We also observed a cone-shaped density attached on the luminal side of PF A5 with 8-nm periodicity (*
in ), likely to stabilize the A-tubule or as the part of structure connecting to the neighbouring triplet. Interestingly, similar density has been consistently observed in the axoneme doublet (Nicastro et al, 2006
; Sui and Downing, 2006
; Movassagh et al, 2010
). Across from the cone-shaped density, at PF A6 a stub-like density projects out every 8 nm on the outside wall of the A-tubule (). The stub connects to the C-tubule from the neighbouring triplet and will be described in detail in the next section. Meanwhile, density was observed on the outside wall of the A-tubule running across PFs A8 to A10 with an 8-nm longitudinal spacing (). Since both PFs A10 and B1 are at the junction of A- and B-tubules, this density presumably will stabilize the linkage between the A- and B-tubules at the outside wall of triplet.
Remarkably, a large Y-shaped density was observed lying horizontally as a ridge along the luminal side of the A- and B-tubules ( and ). It spans about 380 Å, nearly the entire inner circumference of the A- and B-tubules. It projects radially about 170 Å towards the centre of the basal body barrel. The estimated mass of this structure is 1.1 MDa. We have divided this structure into four parts and assigned them as a central stem with three arms, termed armA, armB and armC (). The central stem binds directly to both A- and B-tubule. On the right side in , it fits into the groove between PFs A1 and A2, which is likely the position of the seam in the A-tubule (Song and Mandelkow, 1995
). After rising longitudinally ~40 Å, the left side of the stem binds to PF B10 in the B-tubule. Here, the stem extends laterally, running across the entire outer surface of PF B10 and partial surface of PF B9. Together, this central stem fills in the gap between the A- and B-tubule and cross-links the two tubules at their closest distance (~82 Å). It makes substantial interactions with both tubules at their luminal joint, with an estimated total contact area between the stem and the A- and B-tubule of ~5200 Å2
. Multiple copies of the stem stack longitudinally with an axial repeat of 8 nm, forming a left-handed spiral-shaped filament viewed from the outside of the basal body (). This filament might account for the 11th PF of the B-tubule as previously observed (Tilney et al, 1973
Figure 3 Link between the A- and B-tubules in the basal body triplet. (A) Two views showing a large Y-shaped structure lying horizontal between the A- and B-tubules at the luminal side of the basal body. A stack of these structures forms a longitudinal filament (more ...)
ArmA rotates about 108° counterclockwise relative to the stem and extends 190 Å towards the direction of A-tubule (). At its end, armA connects longitudinally to the neighbouring armAs, resulting in a filament along the basal body barrel with an 8-nm periodicity. ArmB is 210 Å long and it is about 115° clockwise relative to the stem (). At the end of armB, the neighbouring two arms join, forming in a bifurcated structure with 16-nm periodicity (). This contributes to the 16-nm axial repeat pattern projecting towards the lumen of the basal body barrel as observed in the tomograms (). ArmC is branched 90° from the armB, with a hook-shaped structure. Like armA, a stack of armC also connects longitudinally to form a filament with 8-nm periodicity (). In conclusion, this large Y-shaped structure forms a scaffold on the luminal side of the basal body cross-linking and probably stabilizing both A- and B-tubule.
In addition to the large density that connects the A- and B-tubules as described above, we also observed relative weak filamentous densities that connect PFs B10 and A13 on the inside of the tubule structure ( and ). The connection, which spans about 90 Å, forms a ladder with 4-nm periodicity. Each rung of the ladder is tilted, with a rise about 4 nm from one end at PF B10 to the other end at PF A13, further stabilizing the linkage between A- and B-tubule.
Longitudinal structural variations along the basal body
To reveal the structural variations of the basal body, we have divided the triplet subvolumes into eight groups according to their longitudinal positions. Only subvolumes within each group were averaged (). To our surprise, groups from the proximal half of basal body (groups 1–4) have distinct structural variations compared with the groups from the distal half (groups 5–8). The variations mainly reside on the C-tubule. From group 4 to group 5, there is an emergence of density in the lumen of the C-tubule. In addition, the periodicity at PF C1 changes from 4 to 8 nm. To analyse these changes in greater detail, we re-divided the subvolumes into two groups, the proximal half (sum of 972 subvolumes from groups 1–4) and the distal half (sum of 972 subvolumes from groups 5–8) and recalculated their averaged structure, respectively. The resulting two EM density maps, as shown in , show significant differences. In the proximal half, the C1 PF, like any other MT PFs in the C-tubule, exhibits a 4-nm axial repeat. In contrast, the distal half has longitudinally disconnected density with 8-nm periodicity. In addition, the density with 8-nm spacing at C1 extends further laterally across the outside surface of PF C2, C3 and C4, forming a continuous hook-shaped arm with an estimated mass of 270 kDa. This arm forms a stable linker between the B- and C-tubules at the outside surface of the basal body.
The distal half of the basal body additionally contains a stack of crescent-shaped filamentous structures (). These filaments span ~136 Å in the lumen of the C-tubule. One end of the filament anchors to PF C3, while the other end attaches to the junction between PFs C6 and C7. The estimated mass of this elongated filament is about 46 kDa. Interestingly, the filament is tilted and descends about 20 Å running from PF C3 to PFs C6/C7. Assuming the triplet C-tubule has the MT B-surface lattice and can be seen as a bundle of tubulin helices (red arrows in ) (Chrétien and Wade, 1991
; Song and Mandelkow, 1995
), the tilting of the filament is in the opposite direction to the tilting of the tubulin helices, indicating that the luminal filament most likely attaches to one tubulin helix at the C3 end and attaches to the adjacent tubulin helix running underneath at the C6/C7 end. These attachments suggest that the filament has intrinsic polarity with two ends making unique contacts with the C-tubule. The luminal densities in the MT have been reported previously and presumably stabilize the local MT structure (Garvalov et al, 2006
; Cyrklaff et al, 2007
). The filamentous structure that we describe in the lumen of the C-tubule could function as a reinforcement beam to enhance C-tubule rigidity in the distal half of the basal body.
The emergence of the luminal filaments at the distal half of the basal body coincides with a structural change at PF C1. These transitions take place near the middle of the basal body, about 225 nm from the triplet minus (proximal) end (). The two structures also overlap spatially as the hook-shaped arm runs across PFs C1 to C4 and the luminal filament spans PF C3 and PF C6/C7. It is conceivable that they might interact directly to participate in coordinated assembly of the basal body C-tubule.
Building a basal body model
Most basal bodies and centrioles are composed of nine triplets interconnected circumferentially to make a barrel-shaped structure. In order to find out how the triplets are linked, we reconstructed the entire basal body based on the averaged triplet structure. Since the basal bodies were visualized in their near-native state and subvolumes containing triplet segments were computationally extracted from the tomogram for alignment and averaging, their spatial coordinates in the context of the entire basal body volume are known. By using the known subvolume coordinates, we replaced the subvolumes in each basal body with the averaged triplet structures (as shown in ) and reconstructed entire longitudinal segments of the basal body without imposing nine-fold symmetry. The results as shown in represent the basal body proximal and distal ends, respectively. To our surprise, a comparison of the two ends reveals that the inter-triplet linkages are significantly different (black arrows in the right panel of , respectively). At the proximal end, a pointed stub-like density projects from PF A6 and connects to the neighbouring triplet at the end of the extended tail of the C-tubule. This inter-triplet linker is about 130 Å long from the base of the stub to the tip of the extended tail. In contrast, at the distal end, the A6 stub connects directly to the outside wall of the neighbouring C-tubule at PFs C7 and C8 with an approximate length of 170 Å.
Figure 5 Building the basal body model. (A, B) Cross-section views of the reconstructed basal body at the proximal end (A) and at the distal end (B). Depending on the longitudinal position in the basal body, the proximal or the distal half averaged triplet structure (more ...)
To further analyse the change of this inter-triplet linkage, we measured the angle between two adjacent triplets. As shown in , a nonagon is created by connecting nine triplet centres in the basal body (red lines), in this case the centres of B-tubule. Meanwhile, a vector (yellow arrow) can be drawn across PFs A6–B6–C5/C6, indicating orientation for each triplet. The angle between the triplet vector and the nearest side of nonagon defines the relative angle of the triplet. Eight basal bodies were used for the angle measurement and they are summarized in . At the proximal end of the basal body, the average angle between the triplet vector and the nearest side of nonagon is 10.9°. However, this angle decreases gradually and reaches 1.0° at the distal end of the basal body. The gradual change of the angle indicates that each triplet twists about its centre at 0.04°/nm in a left-handed fashion from the proximal end to the distal end. The diameter of the basal body barrel and the relative location of the triplets remain constant in our models. This observation is consistent with previous studies on mammalian centrioles, where a twist angle of up to 25° has been observed and the diameter of the basal body barrel remained constant in the absence of EDTA (Anderson, 1972
; Paintrand et al, 1992
). This longitudinal left-handed twist of the triplet along the basal body results in a structural change at the inter-triplet linker. Since this twist proceeds gradually, it suggests that the inter-triplet linker is flexible. Alternatively, there might be multiple attachment sites for the inter-triplet linker running across the wall of the C-tubule. The mechanism for this transition awaits molecular details achieved only from higher-resolution structures.