The folding and assembly of RuBisCO, the most abundant enzyme in nature, needs a series of chaperones, including the RuBisCO accumulation factor Raf1, which is highly conserved in cyanobacteria and plants. Here, we report the crystal structures of Raf1 from cyanobacteria Anabaena sp. PCC 7120 and its complex with RuBisCO large subunit RbcL. Structural analyses and biochemical assays reveal that each Raf1 dimer captures an RbcL dimer, with the C-terminal tail inserting into the catalytic pocket, and further mediates the assembly of RbcL dimers to form the octameric core of RuBisCO. Furthermore, the cryo-electron microscopy structures of the RbcL–Raf1–RbcS assembly intermediates enable us to see a dynamic assembly process from RbcL₈Raf1₈ to the holoenzyme RbcL₈RbcS₈. In vitro assays also indicate that Raf1 can attenuate and reverse CcmM-mediated cyanobacterial RuBisCO condensation. Combined with previous findings, we propose a putative model for the assembly of cyanobacterial RuBisCO coordinated by the chaperone Raf1.

RuBisCO是自然界中最丰富的酶，其折叠和组装需要一系列的伴侣，包括RuBisCO积累因子Raf1，该因子在蓝细菌和植物中高度保守。在这里，我们报告从蓝细菌鱼腥藻Raf1的晶体结构。PCC 7120及其与RuBisCO大亚基RbcL的复合体。结构分析和生化分析表明，每个Raf1二聚体均捕获一个RbcL二聚体，其C端尾部插入催化口袋，并进一步介导RbcL二聚体的组装以形成RuBisCO的八聚体核心。此外，RbcL–Raf1–RbcS组装中间体的低温电子显微镜结构使我们能够看到从RbcL ₈ Raf1 ₈到全酶RbcL的动态组装过程。₈ RbcS ₈。体外测定还表明，Raf1可以减弱和逆转CcmM介导的蓝细菌RuBisCO缩合。结合以前的发现，我们提出了一个由伴侣Raf1协调的蓝细菌RuBisCO组装的推定模型。