Image of PhoE Porin in projection at 3.5Å resolution

Abstract

The outer membrane of Gram-negative bacteria, such as E. coli, contains a high density of a family of pore-forming proteins, which are often called porins. One member of the family, PhoE porin, is expressed under conditions of phosphate starvation, and has been shown to favor the transport of phosphate-containing compounds and negatively charged molecules. The three-dimensional (3-D) structure of PhoE porin obtained from two-dimensional crystals, negatively stained with uranyl acetate, has been determined to a resolution of about 18Å. The projected structure of glucose-embedded PhoE porin has also been obtained to a resolution of about 6.5Å. Based on these results and previous spectroscopic studies, a 3-D model of PhoE porin has been proposed, consisting of a trimer of extended funnel-like structures. Each funnel-like structure comprises a large water filled vestibule leading into a narrower extension. The vestibule wall is formed by β-sheet which has a large fraction of the β-strands oriented perpendicular to the membrane plane. In this report, we present an extension of the resolution to 3.5Å in the projection map of PhoE porin. At this resolution we can confirm that the β-strands of the vestibule are predominantly normal to the membrane plane.We have recorded high resolution images of trehalose-embedded PhoE porin using both the conventional flood illumination technique and the spot scan illumination method. Images with high quality as judged from optical diffraction were digitized and processed to correct for crystal distortions, lens aberrations and illumination tilt. Phases obtained from several high quality images were used to determine the phases of the reflections used in reconstructing the projected map. The amplitudes of the reflections needed to generate the reconstruction were obtained from the 3-D diffraction data set of PhoE porin, which has been determined to a resolution of about 2.8Å. A typical electron diffraction pattern of PhoE porin at zero tilt angle is shown in Figure 1.