high-resolution-single-particle-analysis-from-electron-cryo

High-resolution Single Particle Analysis from Electron Cryo-microscopy Images Using SPHIRE

SPHIRE (SPARX for High-Resolution Electron Microscopy) is a novel open-source, user-friendly software suite for the semi-automated processing of single ...

Max Planck Institute of Molecular Physiology

Hemocyanins are giant extracellular proteins that transport oxygen in the hemolymph of many molluscs. Molluscan hemocyanins are cylindrical decamers or didecamers of a 350-400 kDa subunit that contains seven or eight different covalently linked globular functional units (FUs), arranged in a linear manner. Each FU carries a single copper active site and reversibly binds one dioxygen molecule. As a consequence, the decamer can carry up to 70 or 80 O(2) molecules. Although complete sequence information is now available from several molluscan hemocyanins, many details of the quaternary structure are still unclear, including the topology of the 10 subunits within the decamer. Here we show 3D reconstructions from cryo-electron micrographs of the hemocyanin decamer of Nautilus pompilius (Cephalopoda) and Haliotis tuberculata (Gastropoda) at a resolution of 11A (FSC(1/2-bit) criterion). The wall structure of both hemocyanins is very similar and shows, as in previous reconstructions, three tiers with 20 functional units each that encircle the cylinder wall, and the 10 oblique minor and major wall grooves. However, the six types of wall FUs of the polypeptide subunit, termed a-b-c-d-e-f, are now for the first time individually discernable by their specific orientation, shape, and connections. Also, the internal collar complex of the decamers shows superior resolution which, in this case, reveals striking differences between the two hemocyanins. The five arcs (FU-g pairs) of the central collar (in both hemocyanins) and the five slabs (FU-h pairs) of the peripheral collar (only present in Haliotis hemocyanin), as well as their connections to the wall and to each other are now more clearly defined. The arc is attached to the wall through a feature termed the anchor, a previously undescribed structural element of the hemocyanin wall.

Comparative 11A structure of two molluscan hemocyanins from 3D cryo-electron microscopy.

Hemocyanins are giant extracellular oxygen carriers in the hemolymph of many molluscs. Nautilus pompilius (Cephalopoda) hemocyanin is a cylindrical decamer of a 350 kDa polypeptide subunit that in turn is a "pearl-chain" of seven different functional units (FU-a to FU-g). Each globular FU has a binuclear copper centre that reversibly binds one O(2) molecule, and the 70-FU decamer is a highly allosteric protein. Its primary structure and an 11 A cryo-electron microscopy (cryo-EM) structure have recently been determined, and the crystal structures of two related FU types are available in the databanks. However, in molluscan hemocyanin, the precise subunit pathway within the decamer, the inter-FU interfaces, and the allosteric unit are still obscure, but this knowledge is crucial to understand assembly and allosterism of these proteins. Here we present the cryo-EM structure of Nautilus hemocyanin at 9.1 A resolution (FSC(1/2-bit) criterion), and its molecular model obtained by rigid-body fitting of the individual FUs. In this model we identified the subunit dimer, the subunit pathway, and 15 types of inter-FU interface. Four interface types correspond to the association mode of the two protomers in the published Octopus FU-g crystal. Other interfaces explain previously described morphological structures such as the fenestrated wall (which shows D5 symmetry), the three horizontal wall tiers, the major and minor grooves, the anchor structure and the internal collar (which unexpectedly has C5 symmetry). Moreover, the potential calcium/magnesium and N-glycan binding sites have emerged. Many interfaces have amino acid constellations that might transfer allosteric interaction between FUs. From their topologies we propose that the prime allosteric unit is the oblique segment between major and minor groove, consisting of seven FUs from two different subunits. Thus, the 9 A structure of Nautilus hemocyanin provides fundamentally new insight into the architecture and function of molluscan hemocyanins.

Nautilus pompilius hemocyanin: 9 A cryo-EM structure and molecular model reveal the subunit pathway and the interfaces between the 70 functional units.

Hemocyanins are blue copper-containing respiratory proteins in the hemolymph of many arthropods and molluscs. Molluscan hemocyanins are decamers, didecamers, or multidecamers of a 340- to 400-kDa polypeptide subunit containing seven or eight globular functional units (FUs; FU-a to FU-h), each with an oxygen-binding site. The decamers are short 35-nm hollow cylinders, with their lumen narrowed by a collar complex. Our recently published 9-A cryo-electron microscopy/crystal structure hybrid model of a 3.4-MDa cephalopod hemocyanin decamer [Nautilus pompilius hemocyanin (NpH)] revealed the pathway of the seven-FU subunit (340 kDa), 15 types of inter-FU interface, and an asymmetric collar consisting of five "arcs" (FU-g pairs). We now present a comparable hybrid model of an 8-MDa gastropod hemocyanin didecamer assembled from two asymmetric decamers [isoform keyhole limpet hemocyanin (KLH) 1 of the established immunogen KLH]. Compared to NpH, the KLH1 subunit (400 kDa) is C-terminally elongated by FU-h, which is further extended by a unique tail domain. We have found that the wall-and-arc structure of the KLH1 decamer is very similar to that of NpH. We have traced the subunit pathway and how it continues from KLH1-g to KLH1-h to form an annulus of five "slabs" (FU-h pairs) at one cylinder edge. The 15 types of inter-FU interface detected in NpH are also present in KLH1. Moreover, we have identified one arc/slab interface, two slab/slab interfaces, five slab/wall interfaces, and four decamer/decamer interfaces. The 27 interfaces are described on the basis of two subunit conformers, yielding an asymmetric homodimer. Six protrusions from the cryo-electron microscopy structure per subunit are associated with putative attachment sites for N-linked glycans, indicating a total of 120 sugar trees in KLH1. Also, putative binding sites for divalent cations have been detected. In conclusion, the present 9-A data on KLH1 confirm and substantially broaden our recent analysis of the smaller cephalopod hemocyanin and essentially solve the gastropod hemocyanin structure.

Keyhole limpet hemocyanin: 9-A CryoEM structure and molecular model of the KLH1 didecamer reveal the interfaces and intricate topology of the 160 functional units.

The allosteric respiratory protein hemocyanin occurs in gastropods as tubular di-, tri- and multimers of a 35 x 18 nm, ring-like decamer with a collar complex at one opening. The decamer comprises five subunit dimers. The subunit, a 400 kDa polypeptide, is a concatenation of eight paralogous functional units. Their exact topology within the quaternary structure has recently been solved by 3D electron microscopy, providing a molecular model of an entire didecamer (two conjoined decamers). Here we study keyhole limpet hemocyanin (KLH2) tridecamers to unravel the exact association mode of the third decamer. Moreover, we introduce and describe a more complex type of hemocyanin tridecamer discovered in fresh/brackish-water cerithioid snails (Leptoxis, Melanoides, Terebralia).

Molluscan mega-hemocyanin: an ancient oxygen carrier tuned by a ~550 kDa polypeptide.

Membrane fusion within the eukaryotic endomembrane system depends on the initial recognition of Rab GTPase on transport vesicles by multisubunit tethering complexes and subsequent coupling to SNARE-mediated fusion. The conserved vacuolar/lysosomal homotypic fusion and vacuole protein sorting (HOPS) tethering complex combines both activities. Here we present the overall structure of the fusion-active HOPS complex. Our data reveal a flexible ≈30-nm elongated seahorse-like structure, which can adopt contracted and elongated shapes. Surprisingly, both ends of the HOPS complex contain a Rab-binding subunit: Vps41 and Vps39. The large head contains in addition to Vps41 the SNARE-interacting Vps33, whereas Vps39 is found in the bulky tip of its tail. Vps11 and Vps18 connect head and tail. Our data suggest that HOPS bridges Ypt7-positive membranes and chaperones SNAREs at fusion sites.

Molecular architecture of the multisubunit homotypic fusion and vacuole protein sorting (HOPS) tethering complex.

Photorhabdus luminescens is an insect pathogenic bacterium that is symbiotic with entomopathogenic nematodes. On invasion of insect larvae, P. luminescens is released from the nematodes and kills the insect through the action of a variety of virulence factors including large tripartite ABC-type toxin complexes (Tcs). Tcs are typically composed of TcA, TcB and TcC proteins and are biologically active only when complete. Functioning as ADP-ribosyltransferases, TcC proteins were identified as the actual functional components that induce actin-clustering, defects in phagocytosis and cell death. However, little is known about the translocation of TcC into the cell by the TcA and TcB components. Here we show that TcA in P. luminescens (TcdA1) forms a transmembrane pore and report its structure in the prepore and pore state determined by cryoelectron microscopy. We find that the TcdA1 prepore assembles as a pentamer forming an α-helical, vuvuzela-shaped channel less than 1.5 nanometres in diameter surrounded by a large outer shell. Membrane insertion is triggered not only at low pH as expected, but also at high pH, explaining Tc action directly through the midgut of insects. Comparisons with structures of the TcdA1 pore inserted into a membrane and in complex with TcdB2 and TccC3 reveal large conformational changes during membrane insertion, suggesting a novel syringe-like mechanism of protein translocation. Our results demonstrate how ABC-type toxin complexes bridge a membrane to insert their lethal components into the cytoplasm of the host cell. We believe that the proposed mechanism is characteristic of the whole ABC-type toxin family. This explanation of toxin translocation is a step towards understanding the host-pathogen interaction and the complex life cycle of P. luminescens and other pathogens, including human pathogenic bacteria, and serves as a strong foundation for the development of biopesticides.

A syringe-like injection mechanism in Photorhabdus luminescens toxins.

A convenient PCR cloning strategy allows one to prepare hundreds of picomoles of circular single-stranded DNA molecules, which are suitable as scaffolds for the assembly of DNA origami structures. The method is based on a combination of site-directed mutagenesis and site- and ligation-independent cloning protocols, with simultaneous insertion of a nicking endonuclease restriction site on a double-stranded plasmid of desired length and sequence.

A facile method for preparation of tailored scaffolds for DNA-origami.

Septins are guanine nucleotide-binding proteins that polymerize into filamentous and higher-order structures. Cdc42 and its effector Gic1 are involved in septin recruitment, ring formation and dissociation. The regulatory mechanisms behind these processes are not well understood. Here, we have used electron microscopy and cryo electron tomography to elucidate the structural basis of the Gic1-septin and Gic1-Cdc42-septin interaction. We show that Gic1 acts as a scaffolding protein for septin filaments forming long and flexible filament cables. Cdc42 in its GTP-form binds to Gic1, which ultimately leads to the dissociation of Gic1 from the filament cables. Surprisingly, Cdc42-GDP is not inactive, but in the absence of Gic1 directly interacts with septin filaments resulting in their disassembly. We suggest that this unanticipated dual function of Cdc42 is crucial for the cell cycle. Based on our results we propose a novel regulatory mechanism for septin filament formation and dissociation. DOI: http://dx.doi.org/10.7554/eLife.01085.001.

The role of Cdc42 and Gic1 in the regulation of septin filament formation and dissociation.

Tripartite Tc toxin complexes of bacterial pathogens perforate the host membrane and translocate toxic enzymes into the host cell, including in humans. The underlying mechanism is complex but poorly understood. Here we report the first, to our knowledge, high-resolution structures of a TcA subunit in its prepore and pore state and of a complete 1.7 megadalton Tc complex. The structures reveal that, in addition to a translocation channel, TcA forms four receptor-binding sites and a neuraminidase-like region, which are important for its host specificity. pH-induced opening of the shell releases an entropic spring that drives the injection of the TcA channel into the membrane. Binding of TcB/TcC to TcA opens a gate formed by a six-bladed β-propeller and results in a continuous protein translocation channel, whose architecture and properties suggest a novel mode of protein unfolding and translocation. Our results allow us to understand key steps of infections involving Tc toxins at the molecular level.

Mechanism of Tc toxin action revealed in molecular detail.

Mega-hemocyanin is a 13.5 MDa oxygen transporter found in the hemolymph of some snails. Similar to typical gastropod hemocyanins, it is composed of 400 kDa building blocks but has additional 550 kDa subunits. Together, they form a large, completely filled cylinder. The structural basis for this highly complex protein packing is not known so far. Here, we report the electron cryomicroscopy (cryo-EM) structure of mega-hemocyanin complexes from two different snail species. The structures reveal that mega-hemocyanin is composed of flexible building blocks that differ in their conformation, but not in their primary structure. Like a protein origami, these flexible blocks are optimally packed, implementing different local symmetries and pseudosymmetries. A comparison between the two structures suggests a surprisingly simple evolutionary mechanism leading to these large oxygen transporters.

Structure of mega-hemocyanin reveals protein origami in snails.

Enzymes of the tubulin tyrosine ligase-like (TTLL) family posttranslationally modify and thereby mark microtubules by glutamylation, generating specific recognition sites for microtubule-interacting proteins. Garnham et al. report the first structure of a TTLL protein alone and in complex with microtubules, elucidating their mechanism of action.

Deciphering the tubulin code.

Excessive aggregation of proteins has a major impact on cell fate and is a hallmark of amyloid diseases in humans. To resolve insoluble deposits and to maintain protein homeostasis, all cells use dedicated protein disaggregation, protein folding and protein degradation factors. Despite intense recent research, the underlying mechanisms controlling this key metabolic event are not well understood. Here, we analyzed how a single factor, the highly conserved serine protease HTRA1, degrades amyloid fibrils in an ATP-independent manner. This PDZ protease solubilizes protein fibrils and disintegrates the fibrillar core structure, allowing productive interaction of aggregated polypeptides with the active site for rapid degradation. The aggregate burden in a cellular model of cytoplasmic tau aggregation is thus reduced. Mechanistic aspects of ATP-independent proteolysis and its implications in amyloid diseases are discussed.

Determinants of amyloid fibril degradation by the PDZ protease HTRA1.

Tc toxins from pathogenic bacteria use a special syringe-like mechanism to perforate the host cell membrane and inject a deadly enzyme into the host cytosol. The molecular mechanism of this unusual injection system is poorly understood. Using electron cryomicroscopy, we determined the structure of TcdA1 from Photorhabdus luminescens embedded in lipid nanodiscs. In our structure, compared with the previous structure of TcdA1 in the prepore state, the transmembrane helices rearrange in the membrane and open the initially closed pore. However, the helices do not span the complete membrane; instead, the loops connecting the helices form the rim of the funnel. Lipid head groups reach into the space between the loops and consequently stabilize the pore conformation. The linker domain is folded and packed into a pocket formed by the other domains of the toxin, thereby considerably contributing to stabilization of the pore state.

Membrane insertion of a Tc toxin in near-atomic detail.

The self-organizational properties of DNA have been used to realize synthetic hosts for protein encapsulation. However, current strategies of DNA-protein conjugation still limit true emulation of natural host-guest systems, whose formation relies on non-covalent bonds between geometrically matching interfaces. Here we report one of the largest DNA-protein complexes of semisynthetic origin held in place exclusively by spatially defined supramolecular interactions. Our approach is based on the decoration of the inner surface of a DNA origami hollow structure with multiple ligands converging to their corresponding binding sites on the protein surface with programmable symmetry and range-of-action. Our results demonstrate specific host-guest recognition in a 1:1 stoichiometry and selectivity for the guest whose size guarantees sufficient molecular diffusion preserving short intermolecular distances. DNA nanocontainers can be thus rationally designed to trap single guest molecules in their native form, mimicking natural strategies of molecular recognition and anticipating a new method of protein caging.

Christos Gatsogiannis

Department of Structural Biochemistry

Max Planck Institute of Molecular Physiology

High-resolution Single Particle Analysis from Electron Cryo-microscopy Images Using SPHIRE

Christos Gatsogiannis

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Structural Biochemistry

Max Planck Institute of Molecular Physiology

High-resolution Single Particle Analysis from Electron Cryo-microscopy Images Using SPHIRE

Department of Structural Biochemistry