Abstract
Monomeric and dimeric PufX-containing core complexes have been purified from membranes of wild-type Rhodobacter sphaeroides. Reconstitution of both samples by detergent removal in the presence of lipids leads to the formation of two-dimensional crystals constituted of dimeric core complexes. Two-dimensional crystals were further analyzed by cryoelectron microscopy and atomic force microscopy. A projection map at 26-A resolution reveals that core complexes assemble in an "S"-shaped dimeric complex. Each core complex is composed of one reaction center, 12 light-harvesting 1 alpha/beta-heterodimers, and one PufX protein. The light-harvesting 1 assemblies are open with a gap of density of approximately 30-A width and surround oriented reaction centers. A maximum density is found at the dimer junction. Based on the projection map, a model is proposed, in which the two PufX proteins are located at the dimer junction, consistent with the finding of dimerization of monomeric core complexes upon reconstitution. This localization of PufX in the core complex implies that PufX is the structural key for the dimer complex formation rather than a channel-forming protein for the exchange of ubiquinone/ubiquinol between the reaction center and the cytochrome bc1 complex.
Highlights
In purple photosynthetic bacteria, highly organized transmembrane pigment-protein complexes perform absorption of light and its conversion into chemical energy
LH1-reaction center (RC)-PufX core complexes were extracted from membranes of semiaerobically grown wild type R. sphaeroides using an octyl -D-glucopyranoside (OG)/sodium cholate mixture [22, 23]
The monomeric and dimeric LH1-RC-PufX bands purified in the OG/cholate sucrose gradient were dialyzed against buffers containing different detergents
Summary
Materials—All phospholipids and detergents were of highest purity and were purchased from Avanti Polar Lipids and from Fluka or Anatrace, respectively. Before two-dimensional crystallization, the monomeric or dimeric core complexes purified on an OG/cholate sucrose gradient, were dialyzed against a buffer containing 50 mM glycyl-glycine, pH 7.8, 0.1% 1-S-dodecyl--D-thiomaltoside (DOTM). Two-dimensional crystals were either negatively stained with 1% uranyl acetate, or quick frozen in liquid ethane for cryo-EM analysis. The latter were transferred into the electron microscope using a Gatan cryo transfer system. The AFM was operated in contact mode applying forces of Ͻ0.2 nN at scan frequencies around 3 Hz. Image Processing—Cryo-EM images of two-dimensional crystals recorded on the CCD camera were treated using the MRC image processing package [30, 31]. Density contouring is at 0.25 root mean square with densities above the mean contoured by solid lines
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