Carboxysomes are anabolic bacterial microcompartments that play an essential role in CO2 fixation in cyanobacteria. This self-assembling proteinaceous organelle uses a polyhedral shell constructed by hundreds of shell protein paralogs to encapsulate the key CO2-fixing enzymes Rubisco and carbonic anhydrase. Deciphering the precise arrangement and structural organization of Rubisco enzymes within carboxysomes is crucial for understanding carboxysome formation and overall functionality. Here, we employed cryo-electron tomography and subtomogram averaging to delineate the three-dimensional packaging of Rubiscos within β-carboxysomes in the freshwater cyanobacterium Synechococcus elongatus PCC7942 grown under low light. Our results revealed that Rubiscos are arranged in multiple concentric layers parallel to the shell within the β-carboxysome lumen. We also detected Rubisco binding with the scaffolding protein CcmM in β-carboxysomes, which is instrumental for Rubisco encapsulation and β-carboxysome assembly. Using Quantification conCATamer (QconCAT)-based quantitative mass spectrometry, we determined the absolute stoichiometric composition of the entire β-carboxysome. This study provides insights into the assembly principles and structural variation of β-carboxysomes, which will aid in the rational design and repurposing of carboxysome nanostructures for diverse bioengineering applications.

中文翻译：

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Synechococcus elongatus PCC7942 中天然 β-羧基体的 Rubisco 包装和化学计量组成


羧基体是合成代谢细菌微区室，在蓝细菌的 CO2 固定中起着重要作用。这种自组装的蛋白质细胞器使用由数百个壳蛋白旁系物构建的多面体壳来封装关键的 CO2 固定酶 Rubisco 和碳酸酐酶。破译 Rubisco 酶在羧基体中的精确排列和结构组织对于理解羧基体的形成和整体功能至关重要。在这里，我们采用冷冻电子断层扫描和亚断层照片平均来描绘在弱光下生长的淡水蓝藻 Synechococcus elongatus PCC7942 中 Rubiscos 在β-羧基体内的三维包装。我们的结果表明，Rubiscos 排列在 β-羧基体腔内的多个平行于壳的同心层中。我们还检测到 Rubisco 与 β-羧基体中的支架蛋白 CcmM 结合，这有助于 Rubisco 包封和 β-羧基体组装。使用基于定量 conCATamer （QconCAT） 的定量质谱法，我们确定了整个 β-羧基体的绝对化学计量组成。本研究提供了对 β-羧基体的组装原理和结构变化的见解，这将有助于羧基体纳米结构的合理设计和重新利用，用于各种生物工程应用。