Where in the cell is my protein?

The application of cryo-correlative light and cryo-electron microscopy (cryo-CLEM) gives us a way to locate structures of interest in the electron microscope. In brief, the structures of interest are fluorescently tagged, and images from the cryo-fluorescent microscope (cryo-FM) maps are superimposed on those from the cryo-electron microscope (cryo-EM). By enhancing cryo-FM to include single-molecule localization microscopy (SMLM), we can achieve much better localization.

The 3D structure of villin as an unusual F-Actin crosslinker.

Villin is an F-actin nucleating, crosslinking, severing, and capping protein within the gelsolin superfamily. We have used electron tomography of 2D arrays of villin-crosslinked F-actin to generate 3D images revealing villin's crosslinking structure. In these polar arrays, neighboring filaments are spaced 125.

A comparison of subjective and mathematical estimations of fetal heart rate variability.

Objectives: To develop a computerized algorithm to quantify fetal heart rate (FHR) variability and compare it to perinatologists' interpretation of FHR variability.

Methods: FHR variability was calculated using data from 30 women who had a fetal scalp electrode placed for a clinical indication, and compared to the assessment of FHR variability from four perinatologists who interpreted paper tracings of the same data. Inter-rater reliability was calculated and receiver-operator curve analysis was done.

Concatenated metallothionein as a clonable gold label for electron microscopy.

Localization of proteins in cells or complexes using electron microscopy has mainly relied upon the use of heavy metal clusters, which can be difficult to direct to sites of interest. For this reason, we would like to develop a clonable tag analogous to the clonable fluorescent tags common to light microscopy. Instead of fluorescing, such a tag would initiate formation of a heavy metal cluster.

The three-dimensional structure of the flagellar rotor from a clockwise-locked mutant of Salmonella enterica serovar Typhimurium.

Three-dimensional reconstructions from electron cryomicrographs of the rotor of the flagellar motor reveal that the symmetry of individual M rings varies from 24-fold to 26-fold while that of the C rings, containing the two motor/switch proteins FliM and FliN, varies from 32-fold to 36-fold, with no apparent correlation between the symmetries of the two rings. Results from other studies provided evidence that, in addition to the transmembrane protein FliF, at least some part of the third motor/switch protein, FliG, contributes to a thickening on the face of the M ring, but there was no evidence as to whether or not any portion of FliG also contributes to the C ring. Of the four morphological features in the cross section of the C ring, the feature closest to the M ring is not present with the rotational symmetry of the rest of the C ring, but instead it has the symmetry of the M ring.

Self-assembly of receptor/signaling complexes in bacterial chemotaxis.

Escherichia coli chemotaxis is mediated by membrane receptor/histidine kinase signaling complexes. Fusing the cytoplasmic domain of the aspartate receptor, Tar, to a leucine zipper dimerization domain produces a hybrid, lzTar(C), that forms soluble complexes with CheA and CheW. The three-dimensional reconstruction of these complexes was different from that anticipated based solely on structures of the isolated components.

Ewald sphere correction for single-particle electron microscopy.

Most algorithms for three-dimensional (3D) reconstruction from electron micrographs assume that images correspond to projections of the 3D structure. This approximation limits the attainable resolution of the reconstruction when the dimensions of the structure exceed the depth of field of the microscope. We have developed two methods to calculate a reconstruction that corrects for the depth of field.

How to make a curved Drosophila bristle using straight actin bundles.

This, our Inaugural Article as Academy Members, is ironically our swan song from the field of the actin cytoskeleton. By reviewing what we have learned and what we think is going on during development, we hope to lure you, the reader, into applying your skills to the bristle cell. The processes of the assembly and disassembly of actin bundles is laid out in time and space in an organism that lends itself to genetic manipulation.

Gold nanocluster formation using metallothionein: mass spectrometry and electron microscopy.

Clonable contrasting agents for light microscopy, such as green fluorescent protein, have revolutionized biology, but few such agents have been developed for transmission electron microscopy (TEM). As an attempt to develop a novel clonable contrasting agent for TEM, we have evaluated metallothionein, a small metal-binding protein, reacted with aurothiomalate, an anti-arthritic gold compound. Electro spray ionization and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry measurements show a distribution of gold atoms bound to individual metallothionein molecules.

A partial atomic structure for the flagellar hook of Salmonella typhimurium.

The axial proteins of the bacterial flagellum function as a drive shaft, universal joint, and propeller driven by the flagellar rotary motor; they also form the putative protein export channel. The N- and C-terminal sequences of the eight axial proteins were predicted to form interlocking alpha-domains generating an axial tube. We report on an approximately 1-nm resolution map of the hook from Salmonella typhimurium, which reveals such a tube made from interdigitated, 1-nm rod-like densities similar to those seen in maps of the filament.

Three-dimensional structure and organization of a receptor/signaling complex.

Transmembrane signaling in bacterial chemotaxis has become an important model system for experimental and theoretical studies. These studies have provided a wealth of detailed molecular structures, including the structures of CheA, CheW, and the cytoplasmic domain of the serine receptor Tsr. How these three proteins interact to form the receptor/signaling complex remains unknown.

Structure of the bacterial flagellar hook and implication for the molecular universal joint mechanism.

The bacterial flagellum is a motile organelle, and the flagellar hook is a short, highly curved tubular structure that connects the flagellar motor to the long filament acting as a helical propeller. The hook is made of about 120 copies of a single protein, FlgE, and its function as a nano-sized universal joint is essential for dynamic and efficient bacterial motility and taxis. It transmits the motor torque to the helical propeller over a wide range of its orientation for swimming and tumbling.

Order, disorder, and perturbations in actin-aldolase rafts.

Actin-aldolase rafts provide insights into the use of rafts as models for three-dimensional actin bundles. Although aldolase has three twofold axes, filaments in actin-aldolase rafts were not strictly related by a twofold axis. Interfilament angles were on average +15 degrees off the expected 180 degrees, and most rafts appeared handed; that is, rows of cross-bridges were tilted in a clockwise direction off the perpendicular.

Myosin isoforms show unique conformations in the actin-bound state.

Crystallographic data for several myosin isoforms have provided evidence for at least two conformations in the absence of actin: a prehydrolysis state that is similar to the original nucleotide-free chicken skeletal subfragment-1 (S1) structure, and a transition-state structure that favors hydrolysis. These weak-binding states differ in the extent of closure of the cleft that divides the actin-binding region of the myosin and the position of the light chain binding domain or lever arm that is believed to be associated with force generation. Previously, we provided insights into the interaction of smooth-muscle S1 with actin by computer-based fitting of crystal structures into three-dimensional reconstructions obtained by electron cryomicroscopy.

Variable symmetry in Salmonella typhimurium flagellar motors.

Electron cryomicroscopy of rotor complexes of the Salmonella typhimurium flagellar motor, overproduced in a nonmotile Escherichia coli host, has revealed a variation in subunit symmetry of the cytoplasmic ring (C ring) module. C rings with subunit symmetries ranging from 31 to 38 were found. They formed a Gaussian distribution around a mean between 34 and 35, a similar number to that determined for native C rings.

Microtubule structure at 8 A resolution.

We have obtained a 3D reconstruction of intact microtubules, using cryoelectron microscopy and image processing, at a resolution of about 8 A, sufficient to resolve much of the secondary structure. The level of detail in the map allows docking of the tubulin structure previously determined by electron crystallography, with very strong constraints, providing several important insights not previously available through docking tubulin into lower-resolution maps. This work provides an improved picture of the interactions between adjacent protofilaments, which are responsible for microtubule stability, and also suggests that some structural features are different in microtubules from those in the zinc sheets with which the tubulin structure was determined.

Subunit organization in a soluble complex of tar, CheW, and CheA by electron microscopy.

The Salmonella and Escherichia coli aspartate receptor, Tar, is representative of a large class of membrane receptors that generate chemotaxis responses by regulating the activity of an associated histidine protein kinase, CheA. Tar is composed of an NH(2)-terminal periplasmic ligand-binding domain linked through a transmembrane sequence to a COOH-terminal coiled-coil signaling domain in the cytoplasm. The isolated cytoplasmic domain of Tar fused to a leucine zipper sequence forms a soluble complex with CheA and the Src homology 3-like kinase activator, CheW.