Projection structure of NhaA, a secondary transporter from Escherichia coli, at 4.0 angstrom resolution

Electron cryomicroscopy of frozen-hydrated two-dimensional crystals of NhaA , a Na+/H+ antiporter from Escherichia coli predicted to have 12 transmembr ane alpha-helices, has facilitated the calculation of a projection map of N haA at 4.0 Angstrom resolution. NhaA was homologously expressed in E.coli w ith a His(6) tag, solubilized in dodecyl maltoside and purified in a single step using Ni2+ affinity chromatography. Two-dimensional crystals were obt ained after reconstitution of purified protein with E.coli lipids. The proj ection map reveals that this secondary transporter has a highly asymmetric structure in projection. NhaA exhibits overall dimensions of similar to 38 x 48 Angstrom with a ring-shaped density feature probably corresponding to a bundle of tilted helices, adjacent to an elongated region of density cont aining several peaks indicative of transmembrane helices. Two crystal forms with p22(1)2(1) symmetry show tightly packed dimers of NhaA which differ i n the interactions between adjacent dimers. This work provides the first di rect glimpse into the structure of a secondary transporter.