Structures of the Insecticidal Toxin Complex Subunit XptA2 Highlight Roles for Flexible Domains.

The Toxin Complex (Tc) superfamily consists of toxin translocases that contribute to the targeting, delivery, and cytotoxicity of certain pathogenic Gram-negative bacteria. Membrane receptor targeting is driven by the A-subunit (TcA), which comprises IgG-like receptor binding domains (RBDs) at the surface. To better understand XptA2, an insect specific TcA secreted by the symbiont X. nematophilus from the intestine of entomopathogenic nematodes, we determined structures by X-ray crystallography and cryo-EM. Contrary to a previous report, XptA2 is pentameric. RBD-B exhibits an indentation from crystal packing that indicates loose association with the shell and a hotspot for possible receptor binding or a trigger for conformational dynamics. A two-fragment XptA2 lacking an intact linker achieved the folded pre-pore state like wild type (wt), revealing no requirement of the linker for protein folding. The linker is disordered in all structures, and we propose it plays a role in dynamics downstream of the initial pre-pore state.

Detecting consistent common lines in cryo-EM by voting.

The single-particle reconstruction problem of electron cryo-microscopy (cryo-EM) is to find the three-dimensional structure of a macromolecule given its two-dimensional noisy projection images at unknown random directions. Ab initio estimates of the 3D structure are often obtained by the "Angular Reconstitution" method, in which a coordinate system is established from three projections, and the orientation of the particle giving rise to each image is deduced from common lines among the images. However, a reliable detection of common lines is difficult due to the low signal-to-noise ratio of the images. In this paper we describe a global self-correcting voting procedure in which all projection images participate to decide the identity of the consistent common lines. The algorithm determines which common line pairs were detected correctly and which are spurious. We show that the voting procedure succeeds at relatively low detection rates and that its performance improves as the number of projection images increases. We demonstrate the algorithm for both simulative and experimental images of the 50S ribosomal subunit.

Holey carbon micro-arrays for transmission electron microscopy: a microcontact printing approach.

We have used a microcontact printing approach to produce high quality and inexpensive holey carbon micro-arrays. Fabrication involves: (1) micromolding a poly(dimethylsiloxane) (PDMS) elastomer stamp from a microfabricated master that contains the desired array pattern; (2) using the PDMS stamp for microcontact printing a thin sacrificial plastic film that contains an array of holes; (3) floating the plastic film onto TEM grids; (4) evaporating carbon onto the plastic film and (5) removing the sacrificial plastic film. The final holey carbon micro-arrays are ready for use as support films in TEM applications with the fidelity of the original microfabricated pattern. This approach is cost effective as both the master and the stamps have long-term reusability. Arbitrary array patterns can be made with microfabricated masters made through a single-step photolithographic process.

Three-dimensional structure of the type 1 inositol 1,4,5-trisphosphate receptor at 24 A resolution.

We report here the first three-dimensional structure of the type 1 inositol 1,4,5-trisphosphate receptor (IP(3)R). From cryo-electron microscopic images of purified receptors embedded in vitreous ice, a three-dimensional structure was determined by use of standard single particle reconstruction techniques. The structure is strikingly different from that of the ryanodine receptor at similar resolution despite molecular similarities between these two calcium release channels. The 24 A resolution structure of the IP(3)R takes the shape of an uneven dumbbell, and is approximately 170 A tall. Its larger end is bulky, with four arms protruding laterally by approximately 50 A and, in comparison with the receptor topology, probably corresponds to the cytoplasmic domain of the receptor. The lateral dimension at the height of the protruding arms is approximately 155 A. The smaller end, whose lateral dimension is approximately 100 A, has structural features indicative of the membrane-spanning domain. A central opening in this domain, which is occluded on the cytoplasmic half, outlines a pathway for calcium flow in the open state of the channel.