Abstract
A three-dimensional model of the dimeric reaction center-light harvesting I-PufX (RC-LH1-PufX) complex from Rhodobacter sphaeroides, calculated from electron microscope single particle analysis of negatively stained complexes, shows that the two halves of the dimer molecule incline toward each other on the periplasmic side, creating a remarkable V-shaped structure. The distribution of negative stain is consistent with loose packing of the LH1 ring near the 14th LH1 alpha/beta pair, which could facilitate the migration of quinone and quinol molecules across the LH1 boundary. The three-dimensional model encloses a space near the reaction center Q(B) site and the 14th LH1 alpha/beta pair, which is approximately 20 angstroms in diameter, sufficient to sequester a quinone pool. Helical arrays of dimers were used to construct a three-dimensional membrane model, which matches the packing lattice deduced from electron microscope analysis of the tubular dimer-only membranes found in mutants of Rba. sphaeroides lacking the LH2 complex. The intrinsic curvature of the dimer explains the shape and approximately 70-nm diameter of these membrane tubules, and at least partially accounts for the spherical membrane invaginations found in wild-type Rba. sphaeroides. A model of dimer aggregation and membrane curvature in these spherical membrane invaginations is presented.
Highlights
Photosynthetic bacteria provide an ideal system for investigating the conversion of light energy into chemical energy in nature
The bacterial photosynthetic membrane is composed of closely packed arrays of LH2 and reaction center (RC)-LH1 complexes; atomic force microscopy (AFM) of membranes from several photosynthetic bacteria has revealed the organization of these arrays [7, 8]
High resolution cryo-electron microscopy projection maps of the LH1-only complex reconstituted from B820 subunits from Rhodospirillum (Rsp.) rubrum, and of monomeric RC-LH1 core complexes both from wild and mutant type Rsp. rubrum, showed a closed LH1 ring consisting of 16 ␣/ subunits [11, 15, 16]
Summary
Cell Culture and Purification of the RC-LH1-PufX Dimer— An LH2Ϫ mutant strain of Rba. sphaeroides (DD13/DGa2 [pRKEH]) [34] was cultured in M22 medium anaerobically at 34 °C for 5 days. A band rich in tubular membranes was collected at the interface between 30 and 40% sucrose It was used for EM observation directly without further purification. Electron Microscopy—Isolated native tubular membranes, reconstituted two-dimensional crystals, and dimeric RC-LH1PufX samples were used for electron microscopy data collection. Image Processing—Images of isolated native tubular membranes and two-dimensional crystals were processed using the MRC suite of programs [36] and DigitalMicrograph (Gatan) as described [15, 26]. An initial three-dimensional reference model of the dimeric RC-LH1-PufX complex was produced by the use of three interpretable classes, viz. On the Z-X plane, the RC orientation was fitted against the side view of the three-dimensional reconstruction model of the dimer by rotating the RC around its x axis. Projection maps were calculated for various angles of tilt of the RC out of the membrane plane and compared qualitatively with the experimentally determined projection map of Ref. 22 using the CCP4 program suite [44]
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