Novel approaches for the lipid sponge phase crystallization of the Rhodobacter spnaeroides photosynthetic reaction center

With the recent developments in the field of free-electron-laser-based serial femtosecond crystallography, the necessity to obtain a large number of highquality crystals has emerged. In this work crystallization techniques were selected, tested and optimized for the lipid mesophase crystallization of the Rhodobacter sphaeroides membrane pigment-protein complex, known as the photosynthetic reaction center (RC). Novel approaches for lipid sponge phase crystallization in comparatively large volumes using Hamilton gas-tight glass syringes and plastic pipetting tips are described. An analysis of RC crystal structures obtained by lipid mesophase crystallization revealed non-native ligands that displaced the native electron-transfer cofactors (carotenoid spheroidene and a ubiquinone molecule) from their binding pockets. These ligands were identified and were found to be lipids that are major mesophase components. The selection of distinct co-crystallization conditions with the missing cofactors facilitated the restoration of spheroidene in its binding site.