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 introduction of cryo-SMLM increased the yield of photons from fluorophores, which can benefit localization efforts. Dahlberg and Moerner (2021, Annual Review of Physical Chemistry, 72, 253-278) have a recent broad and elegant review of super-resolution cryo-CLEM. This paper focuses on cryo(F)PALM/STORM for the cryo-electron tomography community. I explore the current challenges to increase the accuracy of localization by SMLM and the mapping of those positions onto cryo-EM images and maps. There is much to consider: we need to know if the excitation of fluorophores damages the structures we seek to visualize. We need to determine if higher numerical aperture (NA) objectives, which add complexity to image analysis but increase resolution and the efficiency of photon collection, are better than lower NA objectives, which pose fewer problems. We need to figure out the best way to determine the axial position of fluorophores. We need to have better ways of aligning maps determined by FM with those determined by EM. We need to improve the instrumentation to be easier to use, more accurate, and ice-contamination free. The bottom line is that we have more work to do.

High-numerical-aperture cryogenic light microscopy for increased precision of superresolution reconstructions.

Superresolution microscopy has fundamentally altered our ability to resolve subcellular proteins, but improving on these techniques to study dense structures composed of single-molecule-sized elements has been a challenge. One possible approach to enhance superresolution precision is to use cryogenic fluorescent imaging, reported to reduce fluorescent protein bleaching rates, thereby increasing the precision of superresolution imaging. Here, we describe an approach to cryogenic photoactivated localization microscopy (cPALM) that permits the use of a room-temperature high-numerical-aperture objective lens to image frozen samples in their native state. We find that cPALM increases photon yields and show that this approach can be used to enhance the effective resolution of two photoactivatable/switchable fluorophore-labeled structures in the same frozen sample. This higher resolution, two-color extension of the cPALM technique will expand the accessibility of this approach to a range of laboratories interested in more precise reconstructions of complex subcellular targets.

3D reconstruction from electron micrographs a personal account of its development.

Prior to the development of 3D reconstruction, images were interpreted in terms of models made from simple units like ping-pong balls. Generally, people eye-balled the images and with other knowledge about its structure, such as the number of subunits, proposed models to account for the images. How was one to know if the models were correct and to what degree they faithfully represented the true structure? The analysis of electron micrographs of negatively stained viral structures led to the answers and 3D reconstruction.

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 125.9 +/- 7.1 A apart, offset axially by 17 A, with one villin crosslink per actin crossover. More than 6500 subvolumes containing a single villin crosslink and the neighboring actin filaments were aligned and classified to produce 3D subvolume averages. Placement of a complete villin homology model into the average density reveals that full-length villin binds to different sites on F-actin from those used by other actin-binding proteins and villin's close homolog gelsolin.

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. RESULTS: Correlation between the computer algorithm's assessment of variability and the perinatologists' assessment (0.27-0.68) was similar to the inter-rater reliability between perinatologists (0.33-0.72). CONCLUSIONS: A computer-based algorithm can assess FHR variability as well as expert clinicians.

Why phase errors affect the electron function more than amplitude errors.

If Fexp(ialpha) are the set of structure factors for a structure f, the amplitudes can be converted to those of an uncorrelated structure g (amplitude swapping) by multiplying each F by the positive number G/F. Correspondingly, the image f is convoluted with k, the Fourier transform of G/F; k has a large peak at the origin, so that f * k approximately f. For swapped phases, the image f is convoluted with l, the Fourier transform of exp(iDeltaalpha), where Deltaalpha, the phase difference between F and G, is a random variable; l does not have a large peak at the origin, so that f * l does not resemble f. The paper provides quantitative descriptions of these arguments.

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. To test the feasibility of such a tag, we exploited the metal-binding protein, metallothionein (MT). We created a chimeric protein by fusing one or two copies of the MT gene to the gene for maltose binding protein. These chimeric proteins bound many gold atoms, with a conservative value of 16 gold atoms per copy of metallothionein. Visualization of gold-labeled fusion proteins by scanning electron microscopy required one copy of metallothionein while transmission electron microscopy required two copies. Images of frozen-hydrated samples of simple complexes made with anti-MBP antibodies hint at the usefulness of this method.

Bacterial flagellum: visualizing the complete machine in situ.

Electron tomography of frozen-hydrated bacteria, combined with single particle averaging, has produced stunning images of the intact bacterial flagellum, revealing features of the rotor, stator and export apparatus.

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. We suggest that this inner feature arises from a domain of FliG. We present a hypothetical docking in which the C-terminal motor domain of FliG lies in the C ring, where it can interact intimately with FliM.

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. We found that analogous complexes self-assembled with a monomeric cytoplasmic domain fragment of the serine receptor without the leucine zipper dimerization domain. These complexes have essentially the same size, composition, and architecture as those formed from lzTar(C). Thus, the organization of these receptor/signaling complexes is determined by conserved interactions between the constituent chemotaxis proteins and may represent the active form in vivo. To understand this structure in its cellular context, we propose a model involving parallel membrane segments in receptor-mediated CheA activation in vivo.

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. Either method applied to synthetic data representing a large virus yields a higher resolution reconstruction than a method lacking this correction.

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. Unlike previous reports, these data show gold binding occurred as the addition of single atoms without retention of additional ligands. Moreover, under certain conditions, MALDI spectra show gold binding ratios of greater than 1:1 with the cysteine residues of metallothionein. Together, this may hint at a gold-binding mechanism similar to gold nanocluster formation. Finally, metallothionein-gold complexes visualized in the TEM show a range of sizes similar to those used as current TEM labels, and show the potential of the protein as a clonable TEM label in which the gold cluster is grown on the label, thereby circumventing the problems associated with attaching gold clusters.

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. The cell provides every process you could want: filament nucleation, growth of microvilli, joining of microvillar bundles into modules, assembly of modules into bundles, time-dependent use of at least two crossbridging proteins, filament turnover, treadmilling, disassembly, and filament translocation.

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. Atomic models for the two outer domains of the hook subunit were docked into the corresponding outermost features of the map. The N and C termini of the hook subunit fragment are positioned next to each other and face toward the axis of the hook. The placement of these termini would permit the residues missing in the fragment to form the rod-like features that form the core domain of the hook. We also fit the hook atomic model to an approximately 2-nm resolution map of the hook from Caulobacter crescentus. The hook protein sequence from C. crescentus is largely homologous to that of S. typhimurium except for a large insertion (20 kDa). According to difference maps and our fitting, this insertion is found on the outer surface of the hook, consistent with our modeling of the hook.

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. By using EM and single-particle image analysis, we present a three-dimensional reconstruction of the receptor/signaling complex. The complex contains CheA, CheW, and the cytoplasmic portion of the aspartate receptor Tar. We observe density consistent with a structure containing 24 aspartate-receptor monomers and additional density sufficient to house the expected four CheA monomers and six CheW monomers. Within this bipolar structure are four groups of three receptor dimers that are not threefold symmetric and are therefore unlike the symmetric trimers observed in the x-ray crystal structure of the cytoplasmic domain of the serine receptor. In the latter, the interdimer contacts occur in the signaling domains near the hairpin loop. In our structure, the signaling domains within trimers appear spaced apart by the presence of CheA and CheW. This structure argues against models where one CheA and one CheW bind to the outer face of each of the dimers in the trimer. This structure of the receptor/signaling complex provides an additional basis for understanding the architecture of the large arrays of chemotaxis receptors, CheA, and CheW found at the cell poles in motile bacteria.

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. Here we report a partial atomic model of the hook obtained by X-ray crystallography of FlgE31, a major proteolytic fragment of FlgE lacking unfolded terminal regions, and by electron cryomicroscopy and three-dimensional helical image reconstruction of the hook. The model reveals the intricate molecular interactions and a plausible switching mechanism for the hook to be flexible in bending but rigid against twisting for its universal joint function.

The role actin filaments play in providing the characteristic curved form of Drosophila bristles.

Drosophila bristles display a precise orientation and curvature. An asymmetric extension of the socket cell overlies the newly emerging bristle rudiment to provide direction for bristle elongation, a process thought to be orchestrated by the nerve dendrite lying between these cells. Scanning electron microscopic analysis of individual bristles showed that curvature is planar and far greater near the bristle base. Correlated with this, as development proceeds the pupa gradually recedes from the inner pupal case (an extracellular layer that encloses the pupa) leading to less bristle curvature along the shaft. We propose that the inner pupal case induces elongating bristles to bend when they contact this barrier. During elongation the actin cytoskeleton locks in this curvature by grafting together the overlapping modules that comprise the long filament bundles. Because the bristle is curved, the actin bundles on the superior side must be longer than those on the inferior side. This is accomplished during grafting by greater elongation of superior side modules. Poor actin cross-bridging in mutant bristles results in altered curvature. Thus, the pattern of bristle curvature is a product of both extrinsic factors-the socket cell and the inner pupal case--and intrinsic factors--actin cytoskeleton assembly.

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. We can account for both the deviation of the angle from 180 degrees and the handedness of the rafts by a steric constraint due to the lipid layer. We further found that the axial spacings of cross-bridges varied significantly from raft to raft. We suggest that this difference arises from variations in the twist of the filaments that nucleate raft formation; that is, filaments added to a raft adopt the symmetry of those in the raft. We conclude that the organization of filaments in rafts can be modulated by outside factors such as the lipid layer and that the variable twist of filaments in the nucleating core of the raft are imposed on all the filaments in the raft. These results provide a measure of the potential for polymorphism in actin assemblies.

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. Here, we analyze the conformations of actin-bound chicken skeletal muscle S1. We conclude that both myosin isoforms in the nucleotide-free, actin-bound state can achieve a more tightly closed cleft, a more downward position of the lever arm, and more stable surface loops than those seen in the available crystal structures, indicating the existence of unique actin-bound conformations.

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. C-ring diameter scaled with the number of subunits, indicating that the elliptical-shaped subunits maintained constant intersubunit spacing. Taken together with evidence that the M ring does not correspondingly increase in size, this finding indicates that rotor assembly does not require strict stoichiometric interactions between the M- and C-ring subunits. Implications for motor function are discussed.