The resolution of subtomogram averages calculated from cryo-electron tomograms (cryo-ET) of crowded cellular environments is often limited owing to signal loss in, and misalignment of, the subtomograms. By contrast, single-particle cryo-electron microscopy (SP-cryo-EM) routinely reaches near-atomic resolution of isolated complexes. We report a method called ‘tomography-guided 3D reconstruction of subcellular structures’ (TYGRESS) that is a hybrid of cryo-ET and SP-cryo-EM, and is able to achieve close-to-nanometer resolution of complexes inside crowded cellular environments. TYGRESS combines the advantages of SP-cryo-EM (images with good signal-to-noise ratio and contrast, as well as minimal radiation damage) and subtomogram averaging (three-dimensional alignment of macromolecules in a complex sample). Using TYGRESS, we determined the structure of the intact ciliary axoneme with up to resolution of 12 Å. These results reveal many structural details that were not visible by cryo-ET alone. TYGRESS is generally applicable to cellular complexes that are amenable to subtomogram averaging.
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The TYGRESS reconstructions have been deposited in the Electron Microscopy Data Bank under accession code EMD-9023. All other data that support the findings of this study are available in the manuscript or its Supplementary Information. Raw image data (that is, HD images and corresponding tilt series) used to generate the TYGRESS average and figures in this study are available from the corresponding author upon request.
TYGRESS source code and documentation are available on Code Ocean (https://doi.org/10.24433/CO.2034333.v1). The TYGRESS program is also available at https://www.utsouthwestern.edu/labs/nicastro/tygress/. A user manual is available as a Supplementary Protocol (https://doi.org/10.21203/rs.2.16083/v1).
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We thank C. Xu for training and for maintaining the electron microscopy facility at Brandeis University; J. Heumann and D. Mastronarde for technical advice concerning cryo-ET and subtomogram averaging; A. Rohou and S. C. Harrison for helpful discussions; T. Ni, J. Pinskey and G. Riddihough for critically reading the manuscript; and R. Zhang for providing the EM structure of the tubulin dimer for docking. This work was supported by funding from the National Institutes of Health (grant R01 GM111506 to D.N.) and the Cancer Prevention and Research Institute of Texas (grant RR140082 to D.N.). N.G. is an investigator of the Howard Hughes Medical Institute.
The authors declare no competing interests.
Peer review information Allison Doerr was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.
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Integrated supplementary information
(a and b) A typical HD image of a single axoneme (a) and one of its particles (b, a single 96 nm repeat, cut out and zoomed-in from the red box in a) show no clear features to enable particle picking because of the overlap of many structures in the projection image. (c-f) In the corresponding tomogram slice, many prominent particle features, such as radial spokes and microtubule walls (‘RS’ and ‘MT’ in f) are well-defined to help pick repeating particles (orange dot in f) in 3D (red box area in e). In (c and d) the locations for all picked particles are shown as colored dots. Each color represents one of the 9 DMTs. (g and h) After the conversion of 3D coordinates into 2D, all particles can be picked on the HD image (g); the particle shown in (f) is centered at the upper orange dot (h). Scale bars: 100 nm (a, c, e, and g); 50 nm (b, f, and h).
Supplementary Fig. 2 The determination of the defocus value succeeded for TYGRESS HD images but failed for regular cryo-ET LD images.
(a-c) An HD image of Tetrahymena thermophila axonemes recorded at 0° tilt with a defocus setting of −2.5 µm (a), its Fourier transform (FT) (b), and its averaged power spectrum (c, right), fitting to the theoretical Thon rings (c, left). (d-e) A corresponding LD image (0° tilt, defocus setting −8 µm) (d) and its Fourier transform (e). The electron dose of each image is indicated in the bottom left corner of the images. Strong layer lines diffracted from the repeating structures of the axoneme and dark Thon rings (indicated by dashed lines) are visible in the HD image (b) but not in the LD image of the same sample (d and e). This causes the defocus detection to fail for the LD image. Scale bar: 200 nm.
(a-c) Diagrams of intact axoneme (a) and a selected DMT with associated complexes (b) viewed in cross-section (viewed from proximal). The nexin-dynein regulatory complex (N-DRC) links neighboring DMTs. (c) A longitudinal diagram of a 96-nm-long axonemal unit that repeats along the DMT; each repeat unit contains four outer dynein arms (ODAs), six single-headed inner dynein arms (IDAs: a, b, c, d, e and g), and one double-headed IDA (I1 or dynein f) anchored to the A-tubule (At). Other labels: B-tubule (Bt), central pair complex (CPC), and radial spokes (RSs 1–3); microtubule polarity from proximal to distal.
(a) Cross-sectional slices of isosurface renderings of the 96-nm axonemal repeat at three different locations showing the locations of the ODA ruler-like structure (OA-R, dark red), 96-nm axonemal ruler (AR, red), and the IDA ruler-like structure (IA-R, magenta), as well as their interactions between radial spokes (RS1-3, light blue) and inner dynein arms (IDA, rose). (b-g) EM slices (b-d) and 3D isosurface renderings (e-g) of the TYGRESS reconstructed 96-nm axonemal repeat (b, c, e and f) and a 16-nm DMT repeat (d and g) show e.g. the inner junction (IJ) that consists of FAP20 (gray arrowheads and coloring) and PACRG (black arrowheads and coloring) that repeat with 8 nm periodicity, whereas their connections with protofilament A13 have a 16 nm periodicity (as indicated in c), as well as an additional density extending from the N-DRC base plate (purple arrowheads and coloring). The white line in (b) indicates the location of the EM slices shown in (c and d). The microtubule protofilaments numbers of the A- and B-tubules are labelled with black and white numbers in (b and c), respectively. The hole in the IJ is indicated by white arrowheads. The MAPs and MIPs in (e and f) are colored according to the coloring used in Figs. 3 and 4. Scale bars: 10 nm.
Supplementary Fig. 5 Structural characteristics of MIPs 1-9 in intact axonemes resolved using TYGRESS.
Cross-sectional (left column) and longitudinal (middle column) EM slices, and longitudinal views of 3D isosurface renderings (right column) of the 96-nm axonemal repeat show MIPs 1-9. The MIPs are colored and numbered according to their locations in the cross-section (see Fig. 4d, e). MIPs present at similar locations in the cross-sectional view but in various locations in longitudinal views are further distinguished by letters (a-e). MIP periodicities are indicated by numbers in brackets on the left. White lines in the cross-sections indicate the locations of the EM slices shown in the middle column. The protofilament numbers of the A- and B-tubules are indicated by black and white labels, respectively. Scale bars: 10 nm.
Cross-sectional (left column) and longitudinal (middle column) EM slices of the 96-nm axonemal repeat show the eleven resolved filamentous MIPs. The protofilament numbers of the A- and B-tubules are indicated by black and white labels, respectively. The dark blue arrows highlight the corresponding MIPs. White lines in the cross-sections show the locations of the corresponding longitudinal EM slices. Scale bars: 10 nm.
Supplementary Figs. 1–6, Supplementary Tables 1–2 and Supplementary Protocol.
The TYGRESS reconstruction of the 96-nm axonemal repeat from Tetrahymena thermophila cilia. The TYGRESS reconstruction of the 96-nm axonemal repeat from Tetrahymena thermophila cilia shows unprecedented details of the microtubule doublet and associated complexes, including the MIPs. The video starts with longitudinal electron-microscopy slices followed by cross-sectional electron-microscopy slices through the 3D reconstructed axonemal repeat from intact ciliary axonemes. The video ends in an isosurface rendering representation that visualizes the doublet microtubule and MIPs in 3D. AR, axonemal ruler; MIP10, filamentous microtubule inner protein 10 .
Three-dimensional visualization by isosurface rendering of the 96-nm axonemal repeat from Tetrahymena thermophila cilia; 3D visualization by isosurface rendering of the 96-nm axonemal repeat from Tetrahymena thermophila cilia that were averaged using the TYGRESS method. At the beginning of the video, the proximal end of the axonemal repeat is on the left side. Radial spokes (RSs; blue); inner junction (IJ, gray); axonemal ruler (AR, red); ODA ruler-like structure (OA-R, dark red); IDA ruler-like structure (IA-R, magenta); nexin–dynein regulatory complex (N-DRC, purple, yellow, dark green and blue).
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Song, K., Shang, Z., Fu, X. et al. In situ structure determination at nanometer resolution using TYGRESS. Nat Methods 17, 201–208 (2020). https://doi.org/10.1038/s41592-019-0651-0
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Subnanometer-resolution structure determination in situ by hybrid subtomogram averaging - single particle cryo-EM
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