Figure 1: The lipidic cubic phase of 9.7 MAG facilitates the crystallization of T4L-rhodopsin-arrestin complex.

(a) Temperature-composition phase diagram of 9.7 MAG/water system. The phase diagram was constructed based on small- and wide-angle X-ray scattering measurements made in the heating direction. Sample preparation and X-ray scattering measurements and analysis were as previously described^10,11. The phases observed include the lamellar crystalline (Lc, or solid phase; yellow), the fluid isotropic (FI or liquid phase; grey), and the following liquid crystalline phases: lamellar liquid crystal (L_α; brown), cubic-Ia3d (green) and cubic-Pn3m (purple). A separate aqueous phase observed in equilibrium with the solid or liquid crystalline phases is indicated by Aq. The phase diagram shows that the solid Lc phase stabilizes under equilibrium conditions below ~8 °C. The latter is some 10 °C below that observed with 9.9 MAG (monoolein)¹¹ and is similar to what was found with 7.9 MAG³⁶. This low solidification temperature enabled use in the current project of 9.7 MAG as a host lipid for LCP-SFX data collection in an evacuated sample chamber at 20 °C, where evaporative cooling created problems for measurements with 9.9 MAG but not with 7.9 MAG¹⁴. The maximum water carrying capacity of 9.7 MAG resides at ~50%(w/w) water which is considerably greater and smaller than that of 9.9 MAG¹¹, and 7.7 MAG³⁷, respectively. These observations indicate that the cubic mesophase of 9.7 MAG has larger aqueous channels compared to 9.9 MAG that are more like those of 7.7 MAG. This is consistent with 9.7 MAG supporting the growth of rhodopsin-arrestin-T4L crystals where the complex has sizable extra-membrane bulk best accommodated in a large aqueous channel. This parallels the observations made with 7.7 MAG as a host lipid for the β₂AR-Gs complex structure determination³⁰. (b) Cartoon representation of the T4L-rhodopsin-arrestin complex protein (rhodopsin is in blue, arrestin in green, and T4L is omitted) reconstituted in the LCP of 9.7 MAG, with the rhodopsin embedded in the bilayer of the LCP and the arrestin accommodated in the aqueous channel. Components of the precipitant solution are proposed to locally stabilize a lamellar domain into which the protein preferentially partitions from the LCP which acts as a reservoir. It is in the lamellar domain that nucleation and crystal growth take place (figure adapted from Li et al.³⁸).