Quantitative analysis of data reveals axoneme and spoke periodicity.

<p>(A) Quantitative analysis of the axonemal repeat periodicity (Ax repeat, stained n = 77, frozen n = 45, FFT n = 5), PFR repeat (PFR-repeat, stained n = 299, frozen n = 34, FFT = 6), periodicity of axoneme-PFR interface densities (Ax-PFR repeat, stained n = 102, frozen n = 82) and distance between spoke heads (S 1–2, stained n = 116, frozen = 48), (S 2–3, stained n = 112, frozen = 12),. Error bars show standard deviation. (B) TEM image of a frozen flagellar skeleton. Boxed regions of the axoneme (Black corner markers) and PFR (White corner markers) were used to produce the Fourier transform power spectrums shown in C and D respectively. The 9.5nm and 8.58nm (C and D respectively) positions are indicated.</p

Structure of the PFR.

<p>(<b>A</b>) CS slice showing transverse views of the flagellum skeleton (slice thickness 1nm) showing the proximal (P), intermediate (I) and distal (D) PFR zones. Densities extending across the PFR can be seen (white arrows). Yellow box indicates position of data in E–F. (B) Perspective view of tomogram data, with P, I and D zones indicated. (C) Coronal slices of Ax-PFR interface and PFR zones (as indicated) show raw data (top), data with laths and struts highlighted by solid and dashed lines, respectively (middle) and diagrams (bottom). Circles indicate lattice intersection points. (D) Top: Sagittal slice (3nm) through each of the zones showing parallel filaments in the intermediate zone (red arrow) and orthogonal filaments extending from the proximal through distal zones (blue arrow). Bottom: composite diagram showing PFR zones in projection with a LS periodicity of 48 nm and a structural repeat unit (shaded rectangles). (E) Surface rendering of region shown in A, cut through the Ax-PFR interface, P, I and D zones, as indicated. Insets show face-on views of each cut surface. (F) Enlargement of structure shown in E. Left: Same orientation as in E, shows twisting of structure (blue arrow) at the P-I zone interface and struts (red text). Middle: Same region shown in left rotated 40° to show wall-like intermediate lath. Right: same orientation as middle shows twisting of structure at I-D interface (blue arrow). D zone struts are indicated. (G) 3D graphical diagram of PFR orientated to reflect transverse (tilted slightly to reflect sample orientation in E) and coronal views and a rotated view showing the 3D construction. Zones are colored as shown in C. Twisting elements are indicated (blue arrows and “twist” label) Compasses in panels A, C and D show sample orientation, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025700#pone-0025700-g001" target="_blank">Fig. 1</a>. See also supporting <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025700#pone.0025700.s009" target="_blank">Videos V3</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025700#pone.0025700.s010" target="_blank">4</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025700#pone.0025700.s011" target="_blank">5</a>. Scale bars are 50nm.</p

Three-Dimensional Structure of the Trypanosome Flagellum Suggests that the Paraflagellar Rod Functions as a Biomechanical Spring

<div><p>Flagellum motility is critical for normal human development and for transmission of pathogenic protozoa that cause tremendous human suffering worldwide. Biophysical principles underlying motility of eukaryotic flagella are conserved from protists to vertebrates. However, individual cells exhibit diverse waveforms that depend on cell-specific elaborations on basic flagellum architecture. <i>Trypanosoma brucei</i> is a uniflagellated protozoan parasite that causes African sleeping sickness. The <i>T. brucei</i> flagellum is comprised of a 9+2 axoneme and an extra-axonemal paraflagellar rod (PFR), but the three-dimensional (3D) arrangement of the underlying structural units is poorly defined. Here, we use dual-axis electron tomography to determine the 3D architecture of the <i>T. brucei</i> flagellum. We define the <i>T. brucei</i> axonemal repeating unit. We observe direct connections between the PFR and axonemal dyneins, suggesting a mechanism by which mechanochemical signals may be transmitted from the PFR to axonemal dyneins. We find that the PFR itself is comprised of overlapping laths organized into distinct zones that are connected through twisting elements at the zonal interfaces. The overall structure has an underlying 57nm repeating unit. Biomechanical properties inferred from PFR structure lead us to propose that the PFR functions as a biomechanical spring that may store and transmit energy derived from axonemal beating. These findings provide insight into the structural foundations that underlie the distinctive flagellar waveform that is a hallmark of <i>T. brucei</i> cell motility.</p></div

Stained and frozen-hydrated flagellum skeletons provide consistent structural information.

<p>(A). Scanning electron microscopy image of a <i>T. brucei</i> procyclic cell. Inset shows cross-section TEM image. (B) Resin-embedded samples exhibit a range of orientations, cross-section (CS) and longitudinal-section (LS) are shown. (C) Frozen-hydrated samples in LS orientation show the axoneme (AX) and paraflagellar rod (PFR) side by side. (D) CS view from tomogram of stained samples (slice thickness≈50nm). Outer doublet microtubules (OD), radial spokes (S), outer arm dyneins (OAD), inner arm dyneins (IAD), central pair microtubules (CP) and nexin links (N) are indicated. Ax-PFR Connections are visible. The proximal (P), intermediate (I) and distal (D) PFR zones are indicated on both CS (D) and LS (E–F) views. LS views from tomograms of stained (E) and frozen (F) samples show repeating structures along the flagellum (≈3nm thick). Compasses (E,F) show sample orientation with respect to axoneme (A), PFR (P), base (B), tip (T) and left (L) and right (R) sides (viewed from flagellum base to tip). Scale bars 1 µm (A), 100nm (B–C, E–F) and 50nm (D).</p

Comparisons between corresponding cryoEM structures (green) and crystal structures (red) by superimposition.

<p>(a–d) The cryoEM-derived structures of the four subunits, namely subunits A (a), B (b), C (c), and D (d), from an asymmetric unit are similar to their crystal structure counterparts. Side chains are only shown for those residues that were resolved in the cryoEM structure of full-length core, but not in the crystal structure.</p

Direct observation of the HBc C-terminal ARD segments protruding outside the core.

<p>(a) Radially-colored, shaded surface representation of the HBV reconstruction filtered to 10Å resolution as viewed along a five-fold axis, showing some densities of the C-terminal ARD tails on the exterior of core (arrow). The two-fold, three-fold, local three-fold and five-fold axes are indicated by the labels 2, 3, L3, and 5, respectively. The density map is contoured at 1σ above the mean. (b) An enlarged view around a two-fold axis, showing a density ~20Å in length protruding outside the core through the local three-fold axis (arrow). The two-fold, three-fold, and local three-fold axes are indicated by the labels 2, 3, and L3, respectively. The ordered outer layer is colored in gray using atomic model, while the rest of density map is colored in green. The map is contoured at 1σ above the mean. (c) Density map (grey) superimposed with ribbon models of A (red) and B (blue) molecules, showing the positive-charged ARD density (green) interaction with the negative-charged D2 and E43 amino acids that line the local three-fold channel (L3). The ordered outer layer (transparent gray) is separated from the rest (green) of the structure by using atomic model. The density map is contoured at 1σ above the mean. (d, e) A slab (9.3 Å in thickness) across the local three-fold axis as viewed along a five-fold axis, showing densities (arrow) underneath one of the 12 vertices extending toward the adjacent local 3-fold axes and a possible pathway (red dotted line) for the ARD tail of molecule A protruding outside the core. While the ordered outer layer (gray) is contoured at 2σ above the mean in both maps, the rest (green) of the cryoEM map is contoured at 2σ in (d) and 1σ in (e), respectively. The five-fold and local three-fold axes are indicated by the labels 5 and L3, respectively.</p

Maps of HBV core reconstruction filtered to 10Å resolution.

<p>All density maps are contoured at 2σ above the mean. (a) Inside view of the radially colored 3D reconstruction map, showing the full-length HBV core has a double-layer structure: an outer layer composed of the N-terminal core assembly domain (residues 1-149) and an inner layer composed of the basic C-terminal ARD (residues 150-185) and its bound RNAs. (b) A gray scale view of a central map slice from (a), showing links (arrows) connecting the outer layer and the inner layer. The links are located around 2-fold axes. (c) Density map superimposed with ribbon model of subunit C (yellow), showing the ordered C-terminal residues (residues 143-146) resolved in our cryoEM structure are superimposable with or close to the linker density around the 2-fold axis. The two-fold axis is indicated by the number 2. The ordered outer layer (transparent gray) is separated from the rest (green) of the structure by using atomic model. (d) A close-up view of density map, showing densities underneath one of the 12 vertices do not connect with either of the outer layer (gray) and the inner layer (green). Instead, these densities (arrow) extend toward the adjacent local 3-fold axes along the space between the outer and inner layers. The five-fold and local three-fold axes are indicated by the labels 5 and L3, respectively.</p

CryoEM and 3D reconstruction of hepatitis B virus (HBV) core assembled from full-length HBV core proteins at 3.5Å resolution.

<p>(a, b) Representative focal-pair cryoEM images of core particles embedded in a thin layer of vitreous ice, recorded on Kodak So 163 film in an FEI Titan Krios cryo electron microscope operated at 300kV at liquid-nitrogen temperature. (c) Fourier transform of the cryoEM image in (a), showing contrast transfer function rings visible up to 1/3Å^-1. (d) Shaded surface representation of the core reconstruction at 3.5Å resolution as viewed along a five-fold axis. One asymmetric unit is segmented and color-coded. The A, B, C, and D subunits that form the asymmetric unit are colored in red, green, yellow, and blue, respectively. The two-fold, three-fold, and five-fold axes are indicated by the numbers 2, 3, and 5, respectively. (e) Resolution assessment of HBV 3D reconstruction based on the 0.143 criterion of reference-based Fourier shell correlation coefficient, showing that the effective resolution is ~3.5Å. (f) Atomic model (stick) of an α-helix is superimposed in its density map (mesh). (g) An atomic model of an asymmetric unit of the T=4 HBV core derived from the cryoEM structure viewed along a five-fold axis. The A, B, C, and D subunits that form the asymmetric unit are color-coded in red, green, yellow, and blue, respectively.</p

The paraflagellar rod repeat unit is synchronous with the axoneme repeat unit.

<p>Panels A to H show digital slices of tomograms. (A) Cross-section (CS) slice of a stained flagellum skeleton (also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025700#pone-0025700-g001" target="_blank">Fig. 1E</a>) shows positions of slices in panels B–G. (B, C) Slice through radial spokes (1–3), IAD and OAD. Raw data is shown in panel B, the same image with annotation is shown in C. Panel B inset shows a CS view of the same tomogram, with outer doublet microtubules (blue) position of slice (orange line). (D) Slice through outer doublet microtubules. Inset shows a CS view of the same tomogram, for slice position reference. A (At) and B (Bt) tubules of the outer doublet are shown, as are OAD motor domains and the IAD/NDRC complex. (E, F) Digital slices of stained (E) and frozen (F) samples bisect the central pair microtubules (CP), outer doublet microtubules (OD) or outer arm dyneins (OAD). Axoneme repeat is indicated (yellow lines). (G) Slice of frozen sample showing the OAD and IAD/NDRC with respect to the triplet spoke repeat along the axonemes. Slice of stained (H) and frozen (I) samples show radial spokes connected to central pair microtubules (CP) via filaments (red arrows). (J) Diagram summarizing the axoneme repeat. Abbreviations are as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025700#pone-0025700-g001" target="_blank">Fig. 1</a>. Compasses in panels A and F show sample orientation as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025700#pone-0025700-g001" target="_blank">Fig. 1</a>. Digital slice thickness is 50 nm (A) or 3 nm (B–H). Scale bars are 50nm.</p

Axoneme-PFR connectors.

<p>(A) CS slice shows positions of outer doublets (OD) 4, 5, 6 and 7 adjacent to the PFR. (B) Surface rendering of data (A) with relative orientation of transverse, sagittal and coronal planes. (C) Transverse, sagittal and coronal tomogram slices at the PFR interface with OD4 -7. Two identical transverse slices, unannotated (left) and annotated (right), are shown. Stained and frozen data are indicated. Ax-PFR connectors (pink arrows) connect the PFR (yellow) to OAD (white), and OD (dashed light blue). Positions of stained sagittal (dashed dark blue) and coronal (dashed red) views are shown. Length of the 96-nm axoneme repeat is shown (dashed orange). (D) Surface rendering of segmented tomograms show transverse (top) and sagittal (bottom) views of OD (light blue), OAD (dark purple), IAD (dark blue), connectors (pink) and PFR (yellow). (E,F) Diagrams summarize arrangement of Ax-PFR connectors. (E) Transverse and sagittal detail of OD-PFR interface, with connectors represented in pink, is shown. Connectors (.1-.3) present at a given OD-PFR interface are shown with corresponding OD number (4, 5, 6 or 7). (F) Diagram in transverse view shows the position of each Ax-PFR connector (Con.). Abbreviations are as defined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025700#pone-0025700-g001" target="_blank">Fig. 1</a>. Digital slice thickness is 50 nm (A) or 3 nm (C). Scale bars are 50nm.</p