The 3.7 A projection map of the glycerol facilitator GlpF: a variant of the aquaporin tetramer

Abstract

GlpF, the glycerol facilitator protein of Escherichia coli, is an archetypal member of the aquaporin superfamily. To assess its structure, recombinant histidine-tagged protein was overexpressed, solubilized in octylglucoside and purified to homogeneity. Negative stain electron microscopy of solubilized GlpF protein revealed a tetrameric structure of approximately 80 A side length. Scanning transmission electron microscopy yielded a mass of 170 kDa, corroborating the tetrameric nature of GlpF. Reconstitution of GlpF in the presence of lipids produced highly ordered two-dimensional crystals, which diffracted electrons to 3.6 A resolution. Cryoelectron microscopy provided a 3.7 A projection map exhibiting a unit cell comprised of two tetramers. In projection, GlpF is similar to AQP1, the erythrocyte water channel. However, the major density minimum within each monomer is distinctly larger in GlpF than in AQP1.

Fig. 1. TEM and STEM of solubilized GlpF. (A) Overview of negatively stained solubilized GlpF in TEM. Randomly oriented particles, often presenting a square shape, are predominant, but complexes of approximately twice the size are also seen (arrowheads). Scale bar corresponds to 450 Å. (B) Selected square-shaped particles. Image side length corresponds to 230 Å. Large inset: 4-fold symmetrized average after reference-free single particle analysis. Scale bar corresponds to 20 Å. (C) Dark-field STEM image of freeze-dried solubilized GlpF recorded at 3.3 e/Ų. Scale bar corresponds to 100 Å. (D) Result of STEM mass measurements. The mass histogram comprising 2073 measurements was fitted with two significant Gauss peaks at 170 kDa (total error of ± 12 kDa) and 327 ± 20 kDa, respectively, and a minor peak at 511 ± 31 kDa.

Fig. 2. Two-dimensional (2D) crystal of GlpF reconstituted in the presence of lipid. (A) The rectangular double-layered crystals were usually mono-crystalline and well ordered and had sizes of several micrometres in diameter. Scale bar corresponds to 2 µm. (B) The electron diffraction pattern of a frozen–hydrated 2D crystal demonstrates the excellent crystallinity of such rectangular crystals. Two different lattices of the double-layered crystal are overlaid. Two hair crosses indicate the superimposed lattices. Diffraction orders marked by arrowheads correspond to a resolution of 3.6 Å. The scale bar represents 1/10 Å–¹.

Fig. 3. The P4-symmetrized 3.7 Å projection structure of GlpF. The map was calculated by merging eight electron micrographs after unbending crystal distortions and correcting the transfer function. The phase residuals indicated significant information up to 3.7 Å resolution (see Table I). A negative temperature factor of –20 Ų was applied. Scale bar corresponds to 50 Å.

Fig. 4. Comparison of GlpF and AQP1 at 4 Å resolution. (A) GlpF monomer. (B) AQP1 monomer. Overlaid crosses mark the position of density maxima found in AQP1. The depression in the AQP1 monomer, thought to represent the pore, is marked by an X. The most prominent difference revealed by the GlpF monomer is the central depression of ∼10 Å diameter, which is significantly larger than the one in AQP1 that exhibits a complex shape. Further differences are seen in the surrounding density maxima. They correspond to the projection of overlapping highly tilted helices. The minima around the 4-fold axes and between the monomers are rather similar. The image side length corresponds to 41 Å.