Structural Insights into RubisCO Dynamics and Pyrenoid Architecture in Chlamydomonas reinhardtii

RubisCO is the key enzyme that catalyzes the conversion of atmospheric CO2 into organic carbon in photosynthetic organisms. To enhance RubisCO efficiency, eukaryotic algal species pack these enzymes within the pyrenoid, a specialized micro-compartment inside the chloroplast that enriches CO2 for carbon fixation. In the pyrenoid, RubisCO molecules are interconnected by linking peptides and form condensates through liquid-liquid phase separation (LLPS). Using a publicly available CryoET dataset of Chlamydomonas reinhardtii , we determine RubisCO structures at 6.5 Å resolution, allowing the trace of the additional densities corresponding to the RubisCO-linking peptides. Through quantitative analysis of RubisCO spatial distribution, we revealed predominant arrangement patterns within the pyrenoid. Additionally, we characterized the structure of pyrenoid minitubules and examined how RubisCOs interact with the membrane network inside the pyrenoid, which facilitates the delivery of substrates for carbon fixation. This work provides valuable structural insights into the LLPS process and enhances our quantitative understanding of carbon fixation within the pyrenoid.