Using Cryogenic Electron Tomography (cryoET) to Determine Rubisco Polymerization Constants in α-Carboxysomes

Area of Science:

• Biochemistry
• Structural Biology
• Microbiology

Background:

• Bacteria microcompartments (BMCs) are protein shells enclosing enzymes, with α-carboxysomes (α-CBs) sequestering Rubisco for carbon fixation.
• Rubisco's inefficiency is partly due to its low substrate selectivity and slow turnover, despite its crucial role in carbon fixation.
• The fibrillar polymerization of Rubisco within α-CBs is observed but its biophysical basis and quantitative parameters remain unclear.

Purpose of the Study:

• To develop a quantitative method for analyzing Rubisco polymerization within α-CBs using cryo-electron tomography data.
• To determine the binding constants and energies governing Rubisco fibril assembly and maintenance *in situ*.
• To provide a tool for evaluating biomolecular interactions of Rubisco and other proteins within cellular compartments.

Main Methods:

• Utilized cryo-electron tomography (cryoET) to obtain high-resolution structural data of α-CBs and Rubisco.
• Developed a computational approach to convert tomography-derived volumes and particle positions into polymerization binding curves.
• Applied the method to calculate Rubisco polymerization nucleus size (n) and equilibrium polymerization constant (Kpol).

Main Results:

• Successfully converted cryoET data into quantitative polymerization binding curves for Rubisco within α-CBs.
• Determined key polymerization constants (n and Kpol) for Rubisco, providing critical biophysical insights.
• Demonstrated that the modeling approach is consistent with *in situ* constraints like concentration-dependent binding and confinement.

Conclusions:

• A novel method enables quantitative biochemical analysis of protein polymerization *in situ* using cryoET.
• This approach provides critical insights into Rubisco organization and regulation within α-CBs.
• The developed method is applicable to studying *in vitro* and *in vivo* interactions of various proteins and polymers.