The architecture of the DRC in WT axonemes

Previous work showed that the DRC is located in the distal half of the 96-nm repeat (Mastronarde et al., 1992; Gardner et al., 1994). By comparing the two most severe drc mutants, pf2 and pf3, which together lack at least seven DRC components (Table I), we show in this study that the WT DRC is more complex and extended than previously reported (Fig. 2 and see Fig. 4), with a total length of ~50 nm (Fig. 2, A, D, G, and J; Fig. 3 C; and Video 1).

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Figure 3.

Connections within domains of the DRC and between the DRC and other axonemal structures. (A–G) A tomographic slice (A) and surface-rendering visualizations (B–G) of averaged 96-nm repeats of WT (C) and pWT (A, B, and D–G) are shown. A total of 10 connections are shown from different orientations. The connections are colored in magenta (isosurface and numbers 1–10). The DRC is colored yellow in WT (C) and gold in pWT (A, B, and D–G). (A–C) The DRC is the only structure in addition to the dynein arms that forms a continuous connection to the B-tubule (B_t) of the neighboring MTD (connections 1a and 1b), demonstrating that the DRC linker is the nexin link. (A and B) Similar bottom views of the DRC, but shown as tomographic slice (A) and surface-rendering representation (B). The position of the dynein stalks (arrowheads) and the IC–LC complex of the I1 dynein are indicated. (C) The cross-sectional view of WT shows that the DRC connects neighboring MTDs, whereas the uncharacterized protrusion (light green) distal of the DRC does not reach the neighboring B-tubule. (D–G) Surface renderings of the DRC and its connections to various axonemal structures from different viewpoints: distal (D), proximal (E), bottom (F), and top (G). Note that to allow better views of the DRC, different axonemal structures surrounding the DRC have been made transparent (D and E) or removed (F and G). Summary of connections: 1a and 1b, proximal and distal lobe (pL and dL) of the nexin link to neighboring B-tubule; 2, base plate to B11 structure; 3, OID link to ODA; 4, linker to tail of IA4; 5, L1 protrusion to the IA5 dynein head; 6 and 7, internal connections within the DRC linking the proximal and distal lobes (6) and the distal lobe and the L1 protrusion (7); 8, base plate to RS base; 9, DRC linker to an uncharacterized density that connects to the distal end of the IC–LC complex of I1 dynein; 10, DRC linker to an uncharacterized structure distal of the DRC. The light green protrusion shows an uncharacterized structure that was previously proposed as candidate for the nexin link (Bui et al., 2008). A_t, A-tubule; BP, base plate protrusion; L1 and L2, linker protrusions 1 and 2. Bar, 20 nm.

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Figure 4.

Comparison of the DRC structure between WT and drc mutants. (A–T) The WT DRC is displayed in yellow (A, F, and P; and pWT in K), and the mutant DRCs are shown in different colors and ordered from left to right by increasing size of structural changes: sup-pf-4 (B, G, L, and Q), sup-pf-3 (C, H, M, and R), pf2 (D, I, N, and S), and pf3 (E, J, O, and T). The averaged 96-nm repeats are visualized by isosurface rendering and are shown from four different viewing directions: longitudinal close-up view from the front (slightly rotated toward the bottom view; A–E), cross-sectional overview from distal end (F–J), and longitudinal close-up from the top (K–O) and bottom (P–T). Different parts of the DRC and associated structures are missing in the mutants. In sup-pf-4, the structural changes are small, with only the L2 protrusion (G and L) and most of the distal lobe (dL) missing (B and L). In all of the other mutants, both the proximal and distal lobes (pL and dL) and the L2 protrusion are missing; i.e., these strains have no DRC connection to the B-tubule (B_t) of the neighboring doublet (M–O). In addition, the DRC linker, including the OID linker (F and K, red arrowheads) to the ODAs, is reduced in length to varying degrees (compare H–J with M–O). In pf3, the entire DRC base plate (E, J, and T), including its protrusion (BP; J and T), connections 2 and 8 (see Fig. S2 J), and the hole (red arrow/dashed red circle) through the B11 structure (T), is missing. The base plate protrusion is also lacking in pf2 (I). The defects in the assembly of IDAs also increase from left to right; i.e., IA4 (A–E, left arrows) is reduced in sup-pf-3 (C) and pf2 (D) and missing in pf3 (E), whereas IA5 (right arrows) is reduced in pf2 (D) and missing in pf3 (E). A-tubule (A_t) includes protofilament A2; B-tubule includes filaments B10 and -11. The light green protrusion in A is an uncharacterized structure previously proposed as a candidate for the nexin link (Bui et al., 2008). L1, linker protrusion 1.

Transformation of the pf2 mutant with a GFP-tagged version of the WT PF2 gene rescues the motility defects (Bower, R., personal communication). Averages of WT and the rescued strain, which we denote pseudo-WT (pWT), are almost identical along the 96-nm repeat (Fig. 2, compare A–C, G, and H with D–F and I). However, some structural details such as the dynein stalks and tails are more easily visualized in the pWT averages (Figs. 2 D and 3 B and Fig. S3). Other minor variances between the WT and pWT structures such as differences in the thickness of some connections can be accounted for by differences in resolution. Therefore, we used averaged tomograms from both strains to identify the major domains within the structure of the WT DRC (Figs. 2 and ​and33 and Fig. S2).

The DRC contains two distinct domains, a base plate that is attached to the A-tubule and a linker that connects to the B-tubule of the neighboring doublet

The DRC can be subdivided into two distinct parts (Fig. 2 and Video 1). The first part is the base plate, which is attached to the underside of the A-tubule that faces the axoneme center (Fig. 2 J). The second part is the linker, which extends away from the A-tubule toward the B-tubule of the neighboring MTD (Fig. 2, G and J; Fig. 3, A–E; and Fig. S2, D–F). The base plate is located distal to RS 2 and extends from the junction of the A- and B-tubules to protofilament 4 of the A-tubule (Fig. 2 J). The base plate appears to be directly connected to the B11 density at the A-tubule–B-tubule junction (Fig. 2, G and J; and Fig. S2, I and J), and it also has one short appendage, the base plate protrusion (Fig. 2, J and K). Just proximal to the base plate connection, a 3–4-nm hole penetrates the MT wall at the B11 density (Fig. 4, P–S; and Fig. S2, G–J). From the volume measured in the tomographic averages, we estimated the molecular mass of the base plate to be ~300–350 kD (see Materials and methods for details).

The DRC linker begins at protofilament A4 and projects into the gap between neighboring doublets (Figs. 2 J and 3 C). The central region contains at least three different protrusions (L1, L2, and the distal OID linker; Fig. 2, H–J), and its total mass is estimated at ~1–1.2 MD. The L1 protrusion connects to inner arm dynein IA5 (Fig. 2 J and Fig. S3), and the OID linker connects the DRC to the ODAs (Nicastro et al., 2006). As the DRC linker approaches the neighboring doublet, it branches into two distinct lobes (Fig. 2 K). The proximal lobe is at least twice the size of the distal lobe, but both lobes connect to protofilament B8 of the neighboring B-tubule (Fig. 3, A–E; Fig. S2, D–F; and Fig. S3). Therefore, the DRC forms a continuous connection from the A-tubule to the B-tubule of the neighboring MTD (Fig. 3, C–E; Fig. S2, D–F; and Figs. S3 and S5 N). As the DRC is the only structure besides the dynein arms that connects adjacent outer doublets (Fig. 3, A–C; and Fig. S3 and S5, A–D), we conclude that the DRC is in fact the nexin link (Fig. S5, M–O).

Connections between the DRC and other structures in the 96-nm repeat

The DRC makes 10 connections to structures in the 96-nm repeat (Fig. 3, Fig. S2, and Video 2). Connections 1–3 are mentioned in the previous section (1a and 1b, proximal and distal lobes; 2, connection to the B11 structure; 3, OID linker) and are described in greater detail in Fig. 3 and Fig. S2. Connection 4 is the attachment of the dynein tail of IA4 to the base plate (Fig. 3 F and Fig. S2, C and E), and connection 5 is the connection between the L1 protrusion and IA5 dynein head domain (Figs. 2 J and 3, D and F; and Fig. S2, C and E).

Connections 6–10 are thin and most easily observed in the averages with the best resolution (Fig. 3, D–G; and Fig. S2, B–F). The proximal and distal lobes of the B-tubule linker appear to be interconnected by a thin bridge (6; Fig. 3, F and G; and Fig. S2, C–F), and the distal lobe is also connected to the L1 protrusion (7; Figs. 2 K and 3, D, F, and G; and Fig. S2, D–F). In addition, the base plate appears to connect to the base of RS 2 (8; Fig. S2 J). In pWT and sup-pf-4, we observed a fine connection (9) between the DRC linker and another structure just proximal of the DRC that connects to the IC–LC complex of the I1 inner dynein (Fig. 3, E and F; Fig. S2, A, B, E, and F; and Fig. S3). Finally, the DRC connects on its distal side to an uncharacterized structure (10; Fig. 3, A, B, D, F, and G; and Fig. S2, E and F).

In sup-pf-4, the distal lobe and its connection to the neighboring B-tubule are reduced

Cryo-ET of sup-pf-4 axonemes demonstrates the sensitivity of our approach for revealing structural details within the DRC. The sup-pf-4 strain was isolated as a suppressor of RS paralysis, and it is most similar to WT with respect to its motility phenotype (Table II). It only lacks two polypeptides (Table I; Huang et al., 1982), but a previous EM study of sup-pf-4 did not identify any clear differences in the DRC structure (Gardner et al., 1994). However, cryo-ET of sup-pf-4 axonemes has revealed that most of the distal lobe of the linker (Fig. 4, B and L; and Fig. S5, E and F) as well as the entire L2 projection (Fig. 4, G and L) are missing. This significantly reduces the size of distal lobe connection (1b) to the adjacent B-tubule (Fig. 4, B and L; Fig. 5 B; and Fig. S4, C–F). No differences have been observed in any other DRC domains. The estimated mass of missing density in sup-pf-4 is ~300 kD.

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Figure 5.

DRC subunit localization and model of the DRC interactions. (A–J) Averaged repeats from WT (A, F, and H–J) and drc mutants (B–E) are visualized by isosurface rendering. (A–E) Bottom views of the DRC structure in WT (A), sup-pf-4 (B), sup-pf-3 (C), pf2 (D), and pf3 (E; coloring as in Fig. 4). The dashed white line represents the shape of the WT DRC for better comparison (C–E and G). (F–J) Proposed locations of the DRC subunits, as suggested by our WT mutant comparisons. We correlated previously published data on missing subunits (Huang et al., 1982; Piperno et al. 1994) with structural data on the presence or absence of specific DRC features in the different drc mutants (see Discussion section “Location of DRC subunits within the NDRC”). The suggested locations of DRC subunits are summarized and colored (see color legend in H) within the WT DRC structure (F and H–J), which is shown from the bottom (F), in cross section (H), and as and overview (I) and close-up (J) in a longitudinal front-bottom view. The WT mutant comparison also revealed DRC structures that could not be correlated to the published biochemical DRC components, suggesting that these densities are unknown DRC subunits (colored red in F–J and highlighted in G). Note that subunit DRC7 was not included in the model because of uncertainty about its presence or absence in different mutants. (K) Simplified model that summarizes the structural connections between the WT DRC with other axonemal structures, indicating potential regulatory interactions. The IDA includes IA2–6 and I1 dynein with IC–LC complex and 1-α- and 1-β-dyneins. A_t, A-tubule; BP, base plate protrusion; B_t, B-tubule; dL, distal lobe; L1, linker protrusion 1; pL, proximal lobe. Bar, 10 nm.

sup-pf-3 and pf2 show significant changes in all parts of the DRC linker

Previous studies have shown that the sup-pf-3 mutation is associated with more significant polypeptide deficiencies and structural defects than sup-pf-4 (Table I; Huang et al., 1982; Gardner et al., 1994). Consistent with these observations, the tomographic average of sup-pf-3 showed large deficiencies in the DRC linker region. Its mass was reduced by ~1 MD, and missing structures included the L2 projection and both the proximal and distal lobes of the linker (Fig. 4, C, H, and M; and Fig. 5 C). Thus, the entire connection to the neighboring B-tubule was lost, and the total length of the linker was reduced to ~36 nm (Fig. 4, H and M; Fig. 5 C; Fig. S4, G–J; and Fig. S5, G and H). Relative to WT, both the OID linker and the L1 projection were reduced in density in the tomographic averages (Fig. 4, C, H, and M), as was the density of IA4 (see the smaller size of the IA4 dynein head in Fig. 4 C).

The tomographic average of pf2 axonemes (Fig. 4, D, I, N, and S; Fig. 5 D; and Fig. S4, K–N) displayed larger deficiencies in the DRC structure than those of either sup-pf-4 or sup-pf-3 (compare Fig. 5 [B–D] with Fig. S5 [E–J]), which is consistent with previous studies (Table I; Mastronarde et al., 1992; Gardner et al., 1994). In pf2, the DRC linker was almost completely reduced, and the missing structures included the proximal and distal lobe, all three linker projections (L1, L2, and the OID linker), and a major part of the central DRC linker (Fig. 4, D, I, and N). Only a small density of ~70 kD was observed at the transition between linker and base plate (Fig. 4, D and I). The pf2 averages also showed that the base plate protrusion was missing (Fig. 4, D, I, and S). We estimate the mass of the DRC remaining in pf2 to be ~240 kD, or only ~15% of the WT DRC structure. In addition, the two inner arm dyneins most closely associated with the DRC were mostly (IA4) and slightly (IA5) reduced (Fig. 4 D). These observations are consistent with previous studies of inner arm defects in pf2 (Gardner et al., 1994; Piperno et al., 1994; Bui et al., 2008).

The DRC is the nexin link

The nexin link, originally seen by Gibbons (1963) in cross sections and later called the circumferential link (Fig. S5 M) is thought to be an elastic element involved in the conversion of sliding to bending (Satir, 1968, 1985), but the component proteins are unknown (Lindemann et al., 2005). Our images show that the DRC is the only structure, besides the IDAs and ODAs, that connects from the A-tubule of one doublet to the B-tubule of the neighboring doublet (Fig. 3, A–C; and Figs. S3 and S5) and therefore must be the nexin link. We suggest the term the nexin-DRC (NDRC).

The suggestion that the nexin link is in close proximity to or part of the DRC is not entirely new (Mastronarde et al., 1992; Gardner et al., 1994; Woolley, 1997; Nicastro et al., 2006). However, others have suggested that a density distal of the DRC is the nexin link (Bui et al., 2008). We also identify this distal density in our images (colored light green in Fig. 2, G–I; Fig. 3 C; and Fig. S2, D–F), but it is only ~17 nm in length, i.e., ~10 nm shy of connecting to the neighboring B-tubule (Fig. 3 C; Fig. S2, D–F; and Fig. S3). At our resolution, we see no evidence of connections between this distal density and the DRC or between this density and the neighboring B-tubule, which suggests that it is not part of the nexin link.

Location of DRC subunits within the NDRC

The drc mutants were previously characterized by SDS-PAGE, and to date, seven DRC subunits have been described as missing spots or bands in different mutants (Huang et al., 1982; Piperno et al., 1994; Piperno, 1995). By correlating published data with the distribution of missing densities in our images, we can identify the likely locations of the DRC subunits and other polypeptides whose assembly depends on their presence (Fig. 5, F–J; and Video 3). We also observed structures that cannot be easily correlated with any of the known subunits (Fig. 5 G and Table III). We estimate the total mass of the WT DRC to be ~1.4–1.5 MD. This is nearly three times larger than the sum of all seven DRC subunits (~550 kD; Piperno et al., 1994). Although there is no information about the stoichiometry of DRC1–7, it seems likely that the DRC contains other, unidentified proteins.

Table III.

Summary of structural components present or absent in WT and drc mutants

Structural component	Strain
	WT/pWT	sup-pf-4	sup-pf-3	pf2	pf3
DRC linker (1,000–1,200)
Nexin link connection to neighboring B-tubule
Proximal lobe (~350)	+	+	−	−	−
Distal lobe (~120)	+	+/−	−	−	−
Linker projections
L1 (50–80)	+	+	+/−	−	+/−
L2	+	−	−	−	−
OID linker	+	+	+/−	−	+/−
Central linker region
Central linker	+	+	+/−	−	+/−
Docking subunit (~60)	+	+	+	+	+
DRC base plate (300–350)
Base plate protrusion (~80)	+	+	+	−	−
B11-A3 section	+	+	+	+	−
Connection to B11	+	+	+	+	−
B-tubule hole	+	+	+	+	−
IDAs
IA4	+	+	+/−	+/−	−
IA5	+	+	+	+/−	(−)

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+ indicates that the component is present; +/− indicates that the component reduced; (−) indicates that the component is almost completely reduced; − indicates that the component is lacking. Molecular mass is indicated in parentheses (in kilodaltons).

Averages of sup-pf-4 lack most of the distal lobe and portion of the B-tubule connection (1b) and the L2 projection of the DRC linker (Fig. 4, B, G, and L; and Fig. 5 B). These structures are missing in all drc mutants (Fig. 4, L–O; and Fig. 5, B–E). sup-pf-4 axonemes lack two subunits, DRC5 (40 kD) and -6 (29 kD); these are also missing in all other drc mutants (Table I; Huang et al., 1982; Piperno et al., 1994). Thus, DRC5 and -6 must be located within the distal lobe of the B-tubule connector and/or the L2 projection (Fig. 5, F and H–J, yellow). However, the estimated loss of mass in sup-pf-4 of ~200–300 kD is greater than the combined mass of DRC5 and -6 (70 kD). Thus, either one or both subunits must be present in multiple copies, or there are additional unidentified DRC subunits whose assembly depends on the presence of DRC5 or -6 (Table III). Perhaps because the proximal lobe of the linker and B-tubule connector is unchanged in sup-pf-4 (Fig. 4, B and L), only minor changes in swimming behavior have been observed (Brokaw and Luck, 1985; Gardner et al., 1994).

The structural changes in the other three drc mutants are larger. pf2 axonemes lack at least five DRC subunits (DRC3–7) but retain DRC1 and -2 (Huang et al., 1982; Piperno et al., 1994). These observations suggest that DRC3, -4, and -7 are located in the missing linker, whereas DRC1 and -2 are located in the base plate structure that remains in pf2 and that reaches from the B-tubule junction to protofilament A4 (Fig. 5, F and H–J, blue). Consistent with this hypothesis, the base plate structure is missing in pf3 axonemes, which lack DRC1 and -2 (Table I). However, two features of the base plate are probably not associated with either DRC1 or -2. The first is the ~80-kD base plate projection, which is missing in pf2 (Fig. 4 I) even though DRC1 and -2 are present (Tables I and III). The second is an ~60-kD structure located near protofilament A4 that is present in both pf2 and pf3 (Fig. 5, D and E). We refer to the latter density as the docking subunit (Table III) because it appears to be the last contact of the DRC linker with the A-tubule. If one subtracts this subunit from the total estimated mass of the DRC structure in pf2, the remaining ~190 kD corresponds well with the estimated size of the DRC1–DRC2 complex (Tables I and III). The identity of the docking subunit, which is present in all of the mutants, is unknown.

Comparison of pf2 and sup-pf-3 indicates that DRC4 plays a pivotal role in the assembly of the DRC linker. pf2 mutations are null alleles in the DRC4 gene, whereas sup-pf-3 is an unusual DRC4 mutation that results in the assembly of truncated DRC4 subunits (Rupp and Porter, 2003; Bower, R., personal communication). Loss of DRC4 blocks the assembly of DRC3–7 in pf2 (Piperno et al., 1994). sup-pf-3 also lacks DRC3–6, but studies vary regarding the extent of these defects (Huang et al., 1982; Piperno et al., 1994; Piperno, 1995). The truncated forms of DRC4 in sup-pf-3 appear to be sufficient for the assembly of a larger part of the DRC linker as compared with pf2 (Fig. 4, H vs. I). These results place DRC4 near the base of the linker, close to the unknown ~60-kD docking subunit (Fig. 5, F and H–J). Consistent with this hypothesis, transformation of pf2 with WT or epitope-tagged versions of DRC4 rescued the pf2 motility defects and restored the missing DRC structures (Fig. 2, D–F and I; Rupp and Porter, 2003)

The pf3 averages show the largest defects; the entire base plate and both linker lobes are missing, and only a small portion of the linker close to the A-tubule remains (Figs. 4 J and 5 E). We estimate the size of the pf3 linker to be ~130–200 kD, compared with 1.2 MD for the WT linker, which means a loss of >80% of the WT mass. However, the pf3 linker appears too small to contain the remaining DRC subunits (DRC3, -4, and -7 and the docking subunit). One possibility is that the loss of the base plate results in increased flexibility of the remaining DRC structures. Disorganization, structural flexibility, or polypeptides that are present only in a subset of the repeats could reduce the apparent size of the structures in the final average. In addition, the absence of the pf3 gene product appears to destabilize the assembly of several other axonemal polypeptides that may contribute to the assembly of the DRC linker (Table I).

To summarize, we predict that DRC1 and -2 are located within the base plate, DRC5 and -6 (and perhaps additional polypeptides) are located in the distal lobe of the B-tubule connector, and DRC3, -4, and -7 are located within the central region of the linker (Fig. 5, F and H–J). However, this means that the proximal lobe (estimated at ~350 kD) and a small portion of the distal branch (~50 kD) cannot be correlated with any known DRC subunits, even though they are missing in sup-pf-3, pf2, and pf3 (Fig. 5 G and Table III). We predict that at least 500 kD, i.e., approximately one third of the total mass of the DRC, is comprised of DRC components that were not previously detected as missing in earlier 1D or 2D PAGE analyses. Efforts to identify these other DRC subunits are underway.

The nexin link as a regulator of dynein activity

The conclusion that the DRC subunits are major components of the nexin link has important implications for nexin function. One surprising observation of our tomograms is that none of the drc mutants display defects in the arrangement of the nine outer MTDs, even though the nexin links no longer connect neighboring doublets (Fig. 4, M–O). These results indicate that other structures such as the dynein arms and/or RSs are sufficient to maintain axoneme integrity in the absence of the nexin links. More importantly, our findings agree well with the hypothesis that the primary functions of the nexin link are signal transduction and local restriction of interdoublet sliding (Lindemann et al., 2005). In the field, the DRC and nexin link were regarded as two distinct structures with different functions, dynein regulation versus limiting doublet sliding, we now realize that both functions are performed by one and the same complex, the NDRC. This conclusion is not only important for future work, but it might require reevaluation of previous studies, as e.g., protease treatment could have not only weakened the nexin linkage but also interfered with dynein regulation.

It is still unclear what happens to the NDRC links during interdoublet sliding. Do the nexin links stretch and extend (Warner, 1976; Olson and Linck, 1977), e.g., by protein unfolding (Lindemann et al., 2005), or do they release and reattach after sliding along the neighboring B-tubule (Bozkurt and Woolley, 1993; Minoura et al., 1999)? One model is that the nexin link has elastic properties that contribute to the passive (i.e., non ATP dependent) recovery of the axoneme after bending. However, Bozkurt and Woolley (1993) found no evidence of stretched nexin links in bull sperm and suggested instead a displacement mechanism. We did not find indications of stretching of the nexin link in bent regions of our axoneme samples. However, this is not conclusive, as our samples were not actively beating and any curvature present was presumably passively imposed during blotting and plunge freezing. Future studies using actively beating axonemes may resolve this important question.

Preparation of cryo samples

Cryo samples were prepared using Quantifoil grids (Quantifoil Micro Tools GmbH) with holey carbon support film (copper; 200 mesh; R2/2) and a homemade guillotine device (plunge freezer). After glow discharging grids for 30 s at −40 mA, 10-nm colloidal gold (Sigma-Aldrich) was applied and dried onto the grid for later use as fiducial markers in the tilt series alignment process. 3 µl of axoneme sample and 1 µl of a 10-fold–concentrated 10-nm colloidal gold solution were applied to the grid, briefly mixed, and blotted from the front side for 1.5–2.5 s with filter paper. Immediately after this, the grid was plunge frozen into liquid ethane that was cooled by liquid nitrogen to achieve vitrification of the axoneme sample. All samples were stored in liquid nitrogen until examination by cryo-EM.

EM

All electron micrographs were digitally recorded using a sensitive 2,000 × 2,000 charge-coupled device camera (Gatan, Inc.) on a transmission electron microscope (Tecnai F30; FEI, Inc.) with field-emission gun, high tilt stage, and postcolumn energy filter (Gatan, Inc.). Cryo samples were loaded into the microscope using a cryo-transfer holder (Gatan, Inc.) and kept below devitrification temperature at all times. Tilt series were recorded of intact, noncompressed axonemes located within holes in the carbon film. For a single-axis tilt series, typically 60–100 images were recorded at a magnification of 13,500 while tilting the sample from about −65 to 65° with 1.5–2.5° tilting increments. The defocus values ranged from −6 to −8 µm, and the specimen exposure was limited to a total electron dose of ~100 e/Ų to minimize radiation damage of the sample. The microscope was operated at 300 keV, and imaging was performed under low dose mode conditions using the energy filter in the zero-loss mode with a slit width of 20 eV to enhance contrast. Acquisition of overview maps and tilt series was accomplished using the microscope control program SerialEM (Mastronarde, 2005).

Image processing

The tilt series images were aligned, reconstructed into tomograms, and analyzed using the IMOD software package (Kremer et al., 1996). Repetitive structures, i.e., the 96-nm repeat unit of the axoneme, were aligned and averaged with missing wedge compensation using the software program PEET (particle estimation for ET; Nicastro et al., 2006). Details about number of tomograms and particles included in the 3D averaged structures as well as the resulting image resolutions using the 0.5 Fourier shell correlation criterion (Böttcher et al., 1997) are summarized in Table II. Contrast transfer function and modulation transfer function corrections were calculated using the IMOD software (Xiong et al., 2009). The UCSF Chimera package (Pettersen et al., 2004) was used for 3D visualization by isosurface rendering, analysis, mass estimations, and the calculation of difference maps (Fig. S4). Mass estimations of the DRC complex and subvolumes were calculated using the average density of 1.43 g/cm³ for proteins (Quillin and Matthews, 2000) and after normalizing the isosurface-rendering threshold to the mass of MTs.

We wish to thank Raqual Bower for providing the pf2 rescue strain and discussions of unpublished data and Claudia G. Vasquez and Matthew R. Rebesco for their help with tomogram reconstruction and image processing. We are grateful to Dr. Chen Xu for his excellent management of the EM facility at Brandeis University and to Dr. David DeRosier for proofreading the manuscript.

This work was supported by grants from the National Institutes of Health (GM083122 to D. Nicastro and GM55667 to M.E. Porter) and the National Science Foundation (DMR-MRSEC-0820492) and by a Pew Scholars Award (to D. Nicastro).