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

Abstract

Electron cryomicroscopy of frozen‐hydrated two‐dimensional crystals of NhaA, a Na⁺/H⁺ antiporter from Escherichia coli predicted to have 12 transmembrane α‐helices, has facilitated the calculation of a projection map of NhaA at 4.0 Å resolution. NhaA was homologously expressed in E.coli with a His₆ tag, solubilized in dodecyl maltoside and purified in a single step using Ni²⁺ affinity chromatography. Two‐dimensional crystals were obtained after reconstitution of purified protein with E.coli lipids. The projection map reveals that this secondary transporter has a highly asymmetric structure in projection. NhaA exhibits overall dimensions of ∼38×48 Å with a ring‐shaped density feature probably corresponding to a bundle of tilted helices, adjacent to an elongated region of density containing several peaks indicative of transmembrane helices. Two crystal forms with p22₁2₁ symmetry show tightly packed dimers of NhaA which differ in the interactions between adjacent dimers. This work provides the first direct glimpse into the structure of a secondary transporter.