Projection structure by single-particle electron microscopy of secondary transport proteins GltT, CitS, and GltS.

The structure of three secondary transporter proteins, GltT of Bacillus stearothermophilus, CitS of Klebsiella pneumoniae, and GltS of Escherichia coli, was studied. The proteins were purified to homogeneity in detergent solution by Ni(2+)-NTA affinity chromatography, and the complexes were determined by BN-PAGE to be trimeric, dimeric, and dimeric for GltT, CitS, and GltS, respectively. The subunit stoichiometry correlated with the binding affinity of the Ni(2+)-NTA resin for the protein complexes. Projection maps of negatively stained transporter particles were obtained by single-particle electron microscopy. Processing of the GltT particles revealed a projection map possessing 3-fold rotational symmetry, in good agreement with the trimer observed in the crystal structure of a homologous protein, Glt(Ph) of Pyrococcus horikoshii. The CitS protein showed up in two main views: as a kidney-shaped particle and a biscuit-shaped particle, both with a long axis of 160 A. The latter has a width of 84 A, the former of 92 A. Symmetry considerations identify the biscuit shape as a top view and the kidney shape as a side view from within the membrane. Combining the two images shows that the CitS dimer is a protein with a strong curvature at one side of the membrane and, at the opposite side, an indentation in the middle at the subunit interface. The GltS protein was shaped like CitS with dimensions of 145 A x 84 A. The shapes and dimensions of the CitS and GltS particles are consistent with a similar structure of these two unrelated proteins.

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