Three-dimensional electron diffraction of PhoE porin to 2.8 A resolution.

A three-dimensional set of electron diffraction intensities of PhoE porin embedded in trehalose extending to 2.8 A resolution has been collected and analyzed. The strongest high-resolution intensities are distributed as a figure of revolution about the z*-axis and are located primarily in a resolution range of 4.5 A to 5.0 A. Within this region, centered near 4.8 A resolution the brightest intensities are clustered about inclination angles of 35 degrees and 0 degrees from the a*, b* plane. This distribution of intensities indicates that the beta-sheet in PhoE porin is arranged to form a cylinder-like structure that contains major populations of beta-sheet strands tilted an average of 35 degrees and 0 degrees with respect to the membrane plane normal. This cylindrical structure has been seen previously in the high-resolution projection map of PhoE as an elliptical ring of high density.

Two-dimensional crystallization and projection structure of KcsA potassium channel.

Potassium channels are integral membrane proteins that play a crucial role in regulating diverse cell functions in both electrically excitable and non-excitable cells. Molecular cloning has revealed a diverse family of genes that encode these proteins, and a variety of experimental strategies have defined functional domains. We have cloned, over-expressed and purified the KcsA potassium channel to homogeneity and reconstituted this channel protein with phospholipids to form two-dimensional crystals. The crystals belong to plane group p4 and have unit cell dimensions of a=b=48 A. A projection map at 6 A resolution has been obtained by electron crystallography. The map shows that the protein is a homotetramer, having a low-density region on the 4-fold axis that is the site of the ion conduction pathway. Each monomer contains density features that are consistent with the molecular model of a truncated form of KcsA recently determined by X-ray crystallography.

Structure and functional mechanism of porins.

Cellular organisms such as gram-negative bacteria are enclosed by a dual lipid bilayer system. The outer membranes of the dual bilayer envelopes predominantly contain large numbers of water-filled transmembrane protein channels known as porins. The recent availability of the molecular structures of several bacterial porins has provided the opportunity for comparing the results of a wide range of functional studies with the atomic level structural details of these membrane channels. Taken together, the structure and function data present the most comprehensive set of boundary conditions available for the evaluation of theory and models predicting the characteristics of solute transport through membrane protein channels. In this paper, we review the high-resolution structure data from the bacterial porins, as well as recent theoretical studies, in the context of biophysical and biochemical observations and discuss the molecular mechanisms responsible for the transport of solutes through porin channels. Particular emphasis has been placed on the features and roles of common structural elements, channel sterics and electrostatics, and voltage-dependent gating. A model for water-coordinated transport, providing a qualitative view of the porin transport mechanism, is also described.

Structure of PhoE porin in projection at 3.5 A resolution.

The structure of PhoE porin in projection normal to the membrane plane has been determined to a resolution of about 3.5 A by electron crystallographic techniques. The purified protein was reconstituted with lipid to form two-dimensional crystals. High resolution images and electron diffraction patterns of these specimens embedded in trehalose were recorded to obtain respectively the structure factor phase information and the more accurate values of the amplitude. The projection map shows interesting features that are not seen in the earlier map at 6.5 A. Details of the trimeric ring-like structures in our earlier map are now resolved. Each ring-like structure consists of "beads" with interbead spacings of about 4-6 A. These beads are interpreted as the projections of beta-strands along the strands' axes. At the center of the trimeric structure, there is a low density region that we proposed previously to be the location of lipopolysaccharide. Within each ring-like structure, there are complicated features which may play an important role in the size, selectivity, and stability of the channel.

Structural architecture of an outer membrane channel as determined by electron crystallography.

Porins are a family of membrane channels commonly found in the outer membranes of Gram-negative bacteria where they serve as diffusional pathways for waste products, nutrients and antibiotics, and can also be receptors for bacteriophages. Porin channels have been shown in vitro to be voltage-gated. They can exhibit slight selectivities for certain solutes; for example PhoE porin has some selectivity for anionic and phosphate-containing compounds. Unlike many known membrane proteins which often contain long stretches of hydrophobic segments that are believed to traverse the membrane in a helical conformation, porins are found to have charged residues distributed almost uniformly along their primary sequences and have most of their secondary structure in a beta-sheet conformation. We have made crystalline patches of PhoE porin embedded in a lipid bilayer and have used these to determine the structure of PhoE porin by electron crystallography to a resolution of 6A. The basic structure consists of a trimer of elliptically shaped, cylindrical walls of beta sheet. Each cylinder has an inner lining, formed by parts of the polypeptide, that defines the channel size. The structure provides a clue as to how deletions of segments of polypeptide, which are found in certain mutants, can result in an actual increase in the channel size.

Crystallization and preliminary X-ray crystallographic analysis of water channel AQP1.

Aquaporin-1 (AQP1), a water channel from bovine red blood cells has been deglycosylated, purified to homogeneity and crystallized in a form suitable for X-ray crystallographic study. Crystals are grown using polyethylene glycol as precipitant and belong to the tetragonal space group I422, with unit-cell parameters a = b = 93.4, c = 180.4 A. The crystals diffract beyond 2.2 A resolution.