Structure of a functional obligate complex III2IV2 respiratory supercomplex from Mycobacterium smegmatis.

In the mycobacterial electron-transport chain, respiratory complex III passes electrons from menaquinol to complex IV, which in turn reduces oxygen, the terminal acceptor. Electron transfer is coupled to transmembrane proton translocation, thus establishing the electrochemical proton gradient that drives ATP synthesis. We isolated, biochemically characterized, and determined the structure of the obligate III2IV2 supercomplex from Mycobacterium smegmatis, a model for Mycobacterium tuberculosis. The supercomplex has quinol:O2 oxidoreductase activity without exogenous cytochrome c and includes a superoxide dismutase subunit that may detoxify reactive oxygen species produced during respiration. We found menaquinone bound in both the Qo and Qi sites of complex III. The complex III-intrinsic diheme cytochrome cc subunit, which functionally replaces both cytochrome c1 and soluble cytochrome c in canonical electron-transport chains, displays two conformations: one in which it provides a direct electronic link to complex IV and another in which it serves as an electrical switch interrupting the connection.

Dead-end complex, lipid interactions and catalytic mechanism of microsomal glutathione transferase 1, an electron crystallography and mutagenesis investigation.

Microsomal glutathione transferase 1 (MGST1) is a detoxification enzyme belonging to the Membrane Associated Proteins in Eicosanoid and Glutathione Metabolism (MAPEG) superfamily. Here we have used electron crystallography of two-dimensional crystals in order to determine an atomic model of rat MGST1 in a lipid environment. The model comprises 123 of the 155 amino acid residues, two structured phospholipid molecules, two aliphatic chains and one glutathione (GSH) molecule. The functional unit is a homotrimer centered on the crystallographic three-fold axes of the unit cell. The GSH substrate binds in an extended conformation at the interface between two subunits of the trimer supported by new in vitro mutagenesis data. Mutation of Arginine 130 to alanine resulted in complete loss of activity consistent with a role for Arginine 130 in stabilizing the strongly nucleophilic GSH thiolate required for catalysis. Based on the new model and an electron diffraction data set from crystals soaked with trinitrobenzene, that forms a dead-end Meisenheimer complex with GSH, a difference map was calculated. The map reveals side chain movements opening a cavity that defines the second substrate site.

Two-Dimensional Crystallization Procedure, from Protein Expression to Sample Preparation.

Membrane proteins play important roles for living cells. Structural studies of membrane proteins provide deeper understanding of their mechanisms and further aid in drug design. As compared to other methods, electron microscopy is uniquely suitable for analysis of a broad range of specimens, from small proteins to large complexes. Of various electron microscopic methods, electron crystallography is particularly well-suited to study membrane proteins which are reconstituted into two-dimensional crystals in lipid environments. In this review, we discuss the steps and parameters for obtaining large and well-ordered two-dimensional crystals. A general description of the principle in each step is provided since this information can also be applied to other biochemical and biophysical methods. The examples are taken from our own studies and published results with related proteins. Our purpose is to give readers a more general idea of electron crystallography and to share our experiences in obtaining suitable crystals for data collection.

Structure of potassium channels.

Potassium channels ubiquitously exist in nearly all kingdoms of life and perform diverse but important functions. Since the first atomic structure of a prokaryotic potassium channel (KcsA, a channel from Streptomyces lividans) was determined, tremendous progress has been made in understanding the mechanism of potassium channels and channels conducting other ions. In this review, we discuss the structure of various kinds of potassium channels, including the potassium channel with the pore-forming domain only (KcsA), voltage-gated, inwardly rectifying, tandem pore domain, and ligand-gated ones. The general properties shared by all potassium channels are introduced first, followed by specific features in each class. Our purpose is to help readers to grasp the basic concepts, to be familiar with the property of the different domains, and to understand the structure and function of the potassium channels better.

A Refined Single-Particle Reconstruction Procedure to Process Two-Dimensional Crystal Images from Transmission Electron Microscopy.

Single-particle reconstruction (SPR) and electron crystallography (EC), two major applications in electron microscopy, can be used to determine the structure of membrane proteins. The three-dimensional (3D) map is obtained from separated particles in conventional SPR, but from periodic unit cells in EC. Here, we report a refined SPR procedure for processing 2D crystal images. The method is applied to 2D crystals of melibiose permease, a secondary transporter in Escherichia coli. The current procedure is improved from our previously published one in several aspects. The "gold standard Fourier shell correlation" resolution of our final reconstruction reaches 13 Å, which is significantly better than the previously obtained 17 Å resolution. The choices of different refinement parameters for reconstruction are discussed. Our refined SPR procedure could be applied to determine the structure of other membrane proteins in small or locally distorted 2D crystals, which are not ideal for EC.

Free RCK arrangement in Kch, a putative escherichia coli potassium channel, as suggested by electron crystallography.

The ligand-gated potassium channels are stimulated by various kinds of messengers. Previous studies showed that ligand-gated potassium channels containing RCK domains (the regulator of the conductance of potassium ion) form a dimer of tetramer structure through the RCK octameric gating ring in the presence of detergent. Here, we have analyzed the structure of Kch, a channel of this type from Escherichia coli, in a lipid environment using electron crystallography. By combining information from the 3D map of the transmembrane part of the protein and docking of an atomic model of a potassium channel, we conclude that the RCK domains face the solution and that an RCK octameric gating ring arrangement does not form under our crystallization condition. Our findings may be applied to other potassium channels that have an RCK gating ring arrangement.

The projection structure of Kch, a putative potassium channel in Escherichia coli, by electron crystallography.

The kch gene, the only potassium channel gene in Escherichia coli, has the property to express both full-length Kch and its cytosolic domain (RCK) due to a methionine at position 240. The RCK domains are believed to form an octameric ring structure and regulate the gating of the potassium channels after having bound certain ligands. Several different gating ring structures have been reported for the soluble RCK domains, however, these were studied isolated from their transmembrane parts. We previously reported an octameric structure of Kch in solution by electron microscopy and single particle reconstruction, composed of two tetrameric full-length proteins through RCK interaction. To exclude the effect of the detergent, we have now performed an electron crystallographic study of the full-length Kch in membrane bound form. Well-ordered two-dimensional crystals were grown in a natural phospholipid environment. A projection map merged from the fifteen best images extended to 6Å resolution. The c12 two-sided plane group of the two-dimensional crystals showed that Kch crystallized as two symmetrically related overlapping layers. The arrangement suggests that the two layers of RCK domains are shifted with respect to each other and the RCK octameric gating ring of Kch does not form under the crystallization condition.