## 3 Results and Discussion  
**The active state of the receptor was well preserved throughout the simulation. ** As all members of the G protein-coupled receptor (GPCR) superfamily, human \(\beta_{2}\)AR shares the 7 TM structural motif, which consists of seven transmembrane-spanning alpha helices connected by loop regions at the intra- and extracellular sides of the membrane (See Figure 1a). Among other loop regions, the intracellular loop 3 (ICL3) plays a critical role in the recognition of G proteins [28]. In addition, there exists an allosteric coupling between ICL3 and the extracellular regions of the receptor, which incorporate the orthosteric binding site [37; 38; 39]. Conformational changes observed at the intracellular part affect the extracellular part, specifically the binding site, which holds key residues such as _Asp1_13 on transmembrane helix 3 (TM3), _Ser_203, _Ser_204, _Ser_207 on TM5, _Phe_289, _Asn_293 on TM6, and _Asn_312 on TM7. Figure 1: (**a**) Representation of human \(\beta_{2}\)AR in a complex with Gs complex in a membrane environment (created via VMD visualization tool [30]), (**b**) Intracellular views of 20 snapshots of ICL3 (**c**) side views of \(\beta_{2}\)AR-Gs complex for 4 different snapshots colored from red (initial) to white (intermediate), to blue (final) during simulation (**d**) RMSD profiles of ICL3 and transmembrane regions, (**e**) RMSF profiles (**f**) RMSD profile of extracellular parts of TM3 and TM5 helices, (**g**) RMSD profiles of intracellular parts of TM6, (**h**) RMSD of NPxX motif of TM7 with respect to the inactive state (PDB id:2 RH1) versus ionic lock distance (_Arg13C\({}_{\alpha}\)-Leu27C\({}_{\alpha}\)_), (**i**) distance profile for _Asp13C\({}_{\alpha}\)-Ser207C\({}_{\alpha}\)_ residue pair. The most important conformational changes observed in two independent runs were summarized in Figure 1. The active state of the receptor was well characterized by an approximately 11 A outward movement in the cytoplasmic end of TM6, and consequently, the adjoining ICL3 was pushed aside towards the lipid molecules (See Figure 1a-c). This unique conformation of the active state was only preserved in the presence of a G protein, which displaces TM6 and ICL3 outward for easy access to the receptor's binding cavity. Aligned snapshots of the receptor indicated high mobility in ICL3 in both runs, slightly enhanced in the second run. RMSD profiles of transmembrane helices 3, 5, 6, and 7 (TM3, TM5, TM6, and TM7) indicated that their initial states were well preserved throughout the simulation within the boundaries characterizing the active state of the receptor (Figure 1d-h). On the other hand, the distance between \(\alpha\)-Carbons of two key residues at the orthosteric binding site, _Asp_113, and _Ser_207, displayed a slight increase from a range of 10-12 A up to 13-16 A in both runs, especially more noticeable in Run #1 (See Figure 1i). Our simulations were conducted with no ligand attached at the orthosteric binding site. Thus, the increase in the distance between these two key residues clearly indicated the tendency of the cavity to expand a bit in the absence of any agonist attached, irrespective of the fact that the active state was well preserved throughout the receptor, especially at the G protein binding site. Previously, we attempted to simulate the active state in its free form, i.e., its G protein partner removed (PDB id: 3 SN6), and observed that the initially opened and swept away ICL3 region and its adjoining transmembrane helix 6 (H6) swiftly changed position towards the core of the receptor at the very early stages of the simulation (in the first 50 ns), closed itself towards the core of the receptor, and blocked the G protein binding cavity. Moreover, in a simulation study conducted by [PERSON] et al. [37], bond restraints were employed at the orthosteric binding site to preserve certain key distances between TM3 and TM5 within the experimentally reported range that represented the active state.