In Situ Cryo-Electron Tomography of Translating Ribosomes in Chlamydomonas Chloroplasts: Polysome Architecture, Cotranslational Folding Intermediates, and Trigger Factor Binding

This study investigates in situ cryo-ET visualization of chloroplast ribosome polysome architecture, cotranslational folding intermediate conformations, and trigger factor chaperone binding geometry in Chlamydomonas reinhardtii within the context of structural cell biology and cryo-electron tomography, an area of growing scientific importance given its implications for chloroplast gene expression regulation, in situ structural biology methodology, and cotranslational chaperone mechanism dissection. Using cryo-FIB-SEM lamella preparation, cryo-ET at 300 kV (JEOL CRYO ARM 300) with tilt series ±65 degrees at 2-degree increments, SIRT reconstruction, subtomogram averaging (STA) of ribosomal subunits, and distance analysis of polysome geometry, we examine chloroplast ribosomes organizing into helical polysomes with 8-14 ribosome/polysome adapted for co-translational membrane protein insertion into thylakoid, with trigger factor binding at ribosome exit tunnel stabilizing nascent transmembrane domain folding intermediates in 2,840 subtomogram averages from 184 tilt series (48 lamellae, 6 cells) yielding 4.2 A overall resolution by STA with polysome architecture analyzed from 412 identified polysomes in segmented tomograms drawn from Chlamydomonas reinhardtii CC-124 cells grown in TAP medium under 12h:12h light:dark cycle, cryo-vitrified by plunge freezing, and cryo-FIB-SEM lamellae prepared at MPI Dortmund. Results indicate that chloroplast polysomes adopt stacked-disk architecture (mean 10.8 ribosomes/polysome, inter-ribosome distance 28.4 nm) with trigger factor bound at 68% of actively translating ribosomes; 42% of ribosomes show density consistent with cotranslational folding intermediates at exit tunnel (p < 0.001), with 68% trigger factor occupancy; 42% with folding intermediates; 10.8 ribosomes/polysome mean as the primary quantitative benchmark. Concordance between primary and confirmatory measurement approaches exceeded 93%, validating the analytical framework. These findings contribute empirically to structural cell biology and cryo-electron tomography and carry actionable implications for the design of programs and policies targeting chloroplast gene expression regulation, in situ structural biology methodology, and cotranslational chaperone mechanism dissection.