The supplementary figures and tables provide detailed insights into the stability and structure of the β2AR-Gs complex, particularly in the presence of nucleotide analogs, pH, and nanobodies. Key findings include: 1. **Stability of the β2AR-Gs Complex**: - **Nucleotides**: GDP and GTPγS cause dissociation of the complex, while pyrophosphate and foscarnet, which mimic nucleotide phosphate groups, do not. - **Purification**: The complex is stable in 20, 100, and 500 mM NaCl but dissociates at 2.5 M NaCl. - **Nanobodies**: Nb35 and Nb37 bind to separate epitopes on the Gs heterotrimer, forming stable complexes that are insensitive to GTPγS treatment. 2. **Crystal Structure**: - **Mesophase Crystals**: Crystals of the T4L-β2AR-Gs-Nb35 complex are observed in sponge-like mesophase. - **Electron Density**: Detailed views of electron density for residues in the β2AR-Gs interface, including interactions between TM3, TM7, and the Gαs domain. - **Rigid Body Motion**: The GαsAH domain shows rigid body movement without significant intradomain conformational changes upon GTPγS binding. 3. **Purification and Stability**: - **Purification Procedures**: A flow chart outlines the steps for preparing the β2AR-Gs complex with Nb35, including SDS-PAGE, gel filtration, and anion exchange chromatography. - **Purity and Homogeneity**: The purity and homogeneity of the β2AR-Gs complex are confirmed through various purification techniques. - **Stabilizing Agents**: MNG-3 stabilizes the β2AR-Gs complex, enhancing its stability compared to DDM. 4. **Intermolecular Interactions**: - **Intermolecular Interactions**: Supplementary tables detail potential intermolecular interactions within the β2AR-Gs interface. These findings collectively contribute to a deeper understanding of the structural and functional stability of the β2AR-Gs complex, which is crucial for its role in signal transduction pathways.The supplementary figures and tables provide detailed insights into the stability and structure of the β2AR-Gs complex, particularly in the presence of nucleotide analogs, pH, and nanobodies. Key findings include: 1. **Stability of the β2AR-Gs Complex**: - **Nucleotides**: GDP and GTPγS cause dissociation of the complex, while pyrophosphate and foscarnet, which mimic nucleotide phosphate groups, do not. - **Purification**: The complex is stable in 20, 100, and 500 mM NaCl but dissociates at 2.5 M NaCl. - **Nanobodies**: Nb35 and Nb37 bind to separate epitopes on the Gs heterotrimer, forming stable complexes that are insensitive to GTPγS treatment. 2. **Crystal Structure**: - **Mesophase Crystals**: Crystals of the T4L-β2AR-Gs-Nb35 complex are observed in sponge-like mesophase. - **Electron Density**: Detailed views of electron density for residues in the β2AR-Gs interface, including interactions between TM3, TM7, and the Gαs domain. - **Rigid Body Motion**: The GαsAH domain shows rigid body movement without significant intradomain conformational changes upon GTPγS binding. 3. **Purification and Stability**: - **Purification Procedures**: A flow chart outlines the steps for preparing the β2AR-Gs complex with Nb35, including SDS-PAGE, gel filtration, and anion exchange chromatography. - **Purity and Homogeneity**: The purity and homogeneity of the β2AR-Gs complex are confirmed through various purification techniques. - **Stabilizing Agents**: MNG-3 stabilizes the β2AR-Gs complex, enhancing its stability compared to DDM. 4. **Intermolecular Interactions**: - **Intermolecular Interactions**: Supplementary tables detail potential intermolecular interactions within the β2AR-Gs interface. These findings collectively contribute to a deeper understanding of the structural and functional stability of the β2AR-Gs complex, which is crucial for its role in signal transduction pathways.