Chlamydomonas chlororibosome architecture mapped by cryo-ET and purified ribosome cryo-EM
A new report in Nature Plants combines in situ cryo-electron tomography with high-resolution single-particle cryo-EM to reveal how chloroplast ribosomes in Chlamydomonas reinhardtii are reshaped by a small-subunit extension encoded in chloroplast DNA. The study targets both native membrane-associated ribosome states and functional tRNA-containing conformations, linking ultrastructure to translation-relevant dynamics.
For the in situ component, tilt-series data were retrieved from EMPIAR-1183026 and acquired on a Titan Krios G4 (300 kV) with an energy filter (10 eV slit) and a Falcon 4i detector. Dose-symmetric tilt schemes covered ±60° with 2–3° steps, while target focus varied in defined increments to maximize reconstruction quality. Raw dose-fractionated EER movies were motion-corrected and CTF-estimated, then reconstructed using AreTomo, followed by Icecream denoising on odd/even frame reconstructions.
Particle detection relied on 3D template matching (pyTOM-match-pick) after binning and low-pass filtering a ribosome reference from SPA. Candidate positions were imported into RELION for multiple rounds of 3D classification without alignment, removing false positives such as membrane segments. Successive rounds of CTF refinement, Bayesian polishing, and re-extraction at higher sampling improved map quality to ~6 Å, with focused refinements yielding sub-6 Å structures for major ribosomal regions.
To probe functional and spatial heterogeneity, the team further classified particles at different binning levels to isolate membrane-bound ribosomes and distinct tRNA states. Membrane-bound particles were separated via masked, alignment-free 3D classification, producing a final ~9.29 Å reconstruction. For translation-state analysis, masked classification with high T separated six tRNA-related conformational states, setting the stage for state-specific architectural comparisons.
Complementing tomography, the study purified chloroplast ribosomes from 2 liters of cultured Chlamydomonas and prepared grids for cryo-EM at both 200 kV (Glacios) and 300 kV (Titan Krios). Data were collected in EPU, processed independently in cryoSPARC, and merged at the final stages. Resolution for the SSU extension class reached ~2.6–2.7 Å after refinement strategies that included reference-based motion correction and local/global CTF refinement.
Model building used ModelAngelo de novo sequencing fed into AlphaFold2-based model retrieval, then rigid-body fitting into cryo-EM maps and iterative refinement in COOT with PHENIX real_space_refine. rRNA models were adapted from bacterial templates, while ions, ligands, and RNA modifications were curated by homology and manual inspection.
Finally, a dedicated polysome workflow represented ribosome contacts as graph components using Euclidean KDTree neighbor searches and SO(3) orientation constraints, accepting polysomes by both distance windows (200–320 nm) and angular similarity thresholds. Membrane orientation was quantified by extracting membrane normals from fitted membrane density and tracking deviations on tangent-plane projections.
Subject of Research:
Chloroplast ribosome structural reshaping by a small-subunit extension in Chlamydomonas
Article Title:
Chloroplast-encoded small subunit extensions reshape the Chlamydomonas chlororibosome.
Article References:
Waltz, F., Lehner, P.A., Van der Stappen, P. et al. Chloroplast-encoded small subunit extensions reshape the Chlamydomonas chlororibosome. Nat. Plants (2026). https://doi.org/10.1038/s41477-026-02361-1
Keywords:
cryo-ET, chloroplast ribosome, SSU extension, tRNA states, polysomes, membrane-bound ribosomes, cryo-EM, Titan Krios
